Hexan-6-olide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results are reported in vitro in three Ames tests and in studies of mutagenicity and clastogenicity in mammalian cells. An additional study of clastogenicity with the read-across substance adipic acid also gives negative results.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study conducted according to accepted methodology; only very limited details are reported for individual susbtances

Qualifier:

no guideline followed

Principles of method if other than guideline:

Screening study of reverse mutation in S. typhimurium, conducted according to the method of Ames (1975).

GLP compliance:

no

Remarks:

: older, published literature study

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine deficient Salmonella strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix obtained from the livers of male rats induced with Aroclor 1254

Test concentrations with justification for top dose:

10, 100, 500, 1000 and 100000 µg/plate

Vehicle / solvent:

No vehicle reported

Untreated negative controls:

other: screening study involving a large number of compounds

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

other: The screening study reports positive responses to a number of known mutagens

Positive control substance:

other: The screening study reports positive responses to a number of known mutagens

Details on test system and experimental conditions:

Methodological information is not provided in detail, the methods were the standard plate incorporation test according to : Ames BN et al (1975) Mutation Research 33(1): 27-28.

Evaluation criteria:

The test was considered to be negative if there were < 0.01 revertants/nmol.

Statistics:

Not relevant

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

: tested above the limit concentration

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The substance was not mutagenic, there were <0.0008 revertants/nmol.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The substance was not mutagenic, there were < 0.0008 revertants/nmol.

Conclusions:

Interpretation of results (migrated information):
negative with and without S9

The substance was not mutagenic under the conditions of this assay.

Executive summary:

The mutagenic potential of ε-caprolactone was determined in a standard Ames test, however there was minimal methodolodical information provided in the paper. Salmonella typhimurium strains TA98, TA100, TA1537 and TA1535 were tested with and without metabolic activation (S9 -mix) using the plate incorporation method. The number of revertants per nmol of ε-caprolactone was < 0.0008, therefore it was concluded that the substance was not mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study conducted according to accepted scientific methods

Qualifier:

no guideline followed

Principles of method if other than guideline:

Reverse bacterial mutation (Ames test) in S.typhimurium and E. coli

GLP compliance:

no

Remarks:

: published literature study

Type of assay:

bacterial reverse mutation assay

Target gene:

E. coli bacteria were DNA polymerase I-deficient, and S. typhimurium were histidine dependent strains with a gene deletion encompassing the gal chl bio uvrB region.

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: deep rough (rfa)

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: deep rough (rfa)

Species / strain / cell type:

S. typhimurium, other: TA 1530

Additional strain / cell type characteristics:

other: deep rough (rfa)

Species / strain / cell type:

E. coli, other: The strains were either normal (pol A+) or DNA polymerase I-deficient (pol A-)

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction obtained from the livers of uninduced Sprague-Dawley rats

Test concentrations with justification for top dose:

Salmonella assay: 0, 1, 10µl/plate.
E. coli assay: 10, 250 µl/disc

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

other: pre-incubation of agar checked for bacterial contamination

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli assay

Migrated to IUCLID6: 10 µl

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

other: pre-incubation of agar checked for bacterial contamination

Positive controls:

yes

Positive control substance:

other: chloramphenicol 30 µg

Remarks:

E. coli assay

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Salmonella assay

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: ß-propiolactone

Remarks:

Salmonella assay

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Salmonella assay

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: N-acetoxy-N-2-fluorenylacetamide

Remarks:

Salmonella assay

Details on test system and experimental conditions:

Petri dishes received

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli, other: The strains were either normal (pol A+) or DNA polymerase I-deficient (pol A-)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test substance was negative in the Ames test.

Conclusions:

Interpretation of results (migrated information):
negative

e-Caprolactone was negative in the Ames test.

Executive summary:

e-Caprolactone was assessed for mutagenicity in an Ames test with S. typhimurium strains TA 1535 and TA 1538, and a DNA polymerase deficient E. coli strain. S. typhimurium strains were tested in the presence and absence of exogenous metabolic activation (S9 mix). The test substance was negative for mutagenicity in all strains tested.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study, conducted according to accepted scientific methods

Qualifier:

no guideline followed

Principles of method if other than guideline:

Reverse bacterial mutation (Ames test)

GLP compliance:

no

Remarks:

: older, published literature study

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine dependent strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium, other: TA 1536

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of Aroclor 1254 induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

The test substance was tested up to a concentration of 250 µg/plate

Untreated negative controls:

yes

Remarks:

: screening study reporting negative results for some substances

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

: screening study reports positive results fo known mutagens

Positive control substance:

not specified

Details on test system and experimental conditions:

The S. typhimurium strains were obtained from Dr. B.N. Ames, Unviersity of California. All strains were uvrB-deficient, and rfa. In addition TA 98 and TA 100 contained the plasmid pKM101. Overnight cultures of bacteria were grown at 37°C in nutrient broth (Oxoid, CM67). The strains were checked periodically for relevant genetic markers and for rfa crystal violet sensitivity, strains TA 98 and TA 100 were checked for ampicillin resistance. Bacteria were exhibiting logarithmic growth when used in the assays. Cultures in nutrient broth were individually centrifuged and resuspended in 1/4 volume of normal saline. The liquid incubation mix consisted of 0.1 ml resuspended bacteria (~4x10exp8 cells), 0.3 ml S9 mix (where appropriate), 0.6 ml phosphate buffer and 50 mM of a nitrosamine, in a screw top tube. The tube was placed in a roller drum in an incubator at 37°C. At various times 0.1 ml aliquots were removed, added to 2 ml top agar containing 0.5 mM histidine and 0.5 mM biotin, and plated to determine the number of revertant colonies.

Evaluation criteria:

A positive response was defined as a reproducible, dose related increase in the number of revertants.

Statistics:

Not required

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Species / strain:

S. typhimurium, other: TA 1536

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test substance was negative in all strains, in the presence and absence of S9, when tested up to a concentration of 250 µg/plate. The study was a screening assay for a large number of susbtance and therefore only limited data for the substance are reported, however the sensitivity of the assay is demonstrated by responses to the other substances investigated. The study is limited by the low concetrations tested.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

e-Caprolactone was negative in all strains, in the presence and absence of S9, when tested up to a concentration of 250 µg/plate.

Executive summary:

e-Caprolactone was tested for mutagenicity in an Ames test, with S. typhimurium strains TA 1535, TA 1536, TA 1537, TA 1538, TA 98 and TA 100. Tests were conducted both in the presence and absence of an exogenous metabolic activation system (S9 mix). e-Caprolactone was negative in all strains, with and without S9, when tested up to a concentration of 250 µg/plate. The study is limited by the low concentrations tested.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Older published public domain study; limited by the absence of metabolic activation

Qualifier:

no guideline followed

Principles of method if other than guideline:

Assessment of clastogenicity in vitro in cultured CHL cells

GLP compliance:

no

Remarks:

: older, published literature study

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not relevant; chromosome aberration test.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Clonal sub-line of a Chinese hamster fibroblast cell line (CHL) originally established from the lung of a young adult. Cells had been maintained by 5-day passages and grown in a monolayer in petri dishes with Eagle's MEM, supplemented wth calf serum. Their doubling time was estimated as 18.2h at their exponential growth at 37oC in a 5% CO2 atmosphere.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

No information

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Remarks:

: untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

: ethanol 1%

True negative controls:

no

Positive controls:

no

Positive control substance:

not specified

Details on test system and experimental conditions:

Three doses (including the 50% inhibition dose as estimated by the growth inhibition test) were added to 3-day old cell cultures (approximately 10exp5 cells/6cm dish). Chromosome preparations were made at 24 and 48 hours after treatment. Cells were treated with colcemid for 2 hours, and after trypsinisation they were incubated with KCl solution for 15m at 37oC. The cells were fixed with ice cold fixative, which was changed 3 times. A few drops of the suspension were placed on clean dry clides and stained with Giemsa solution. The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases. The incidence of polyploid cells was also calculated.

Evaluation criteria:

The test was deemed negative if less than 4.9% of the aberration was detected even when doses were elevated to sub-lethal amounts where almost no mitosis was observed. (3% is classed as normal).

Statistics:

No information.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

There were les than 2% incidences of polyploidy and chromosomal aberrations in untreated cells and vehicle controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The maximum effective dose was 43.8mg/ml, chromosome aberration (chromatid gaps and chromatid or chromosomal breaks) was 4% at 48 hours.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

e-Caprolactone was negative in the chromosome aberration test without metabolic activation.

Executive summary:

e-Caprolactone was tested in an in vitro chromosome aberration assay using Chinese hamster fibroblasts, without metabolic activation. At a maximum concentration of 43.8 mg/ml an incidence of 4% aberration was observed, indicating a negative response in this assay.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2nd January to 26th June 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Propietary study conducted according to GLP and similar to guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The mutagenesis assay was performed according to a protocol developed from: Hsie, A.W. et al (1981), Mutation Research 86: 193-214 and O'Neill, J.P. et al (1977), Mutation Research 45: 91-101.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4. Cells cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT) then frozen. The freeze lot of cells was tested and found to be free of mycoplasma contamination. Cells used in the mutation assay were within four subpassages from frozen stock in order to assure karyotypic stability.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254-induced rat liver S9

Test concentrations with justification for top dose:

A preliminary toxicity study was conducted, and based on the results the chosen test concentrations for the mutagenic assays were 1000, 2000, 3000, 4000 and 5000µg/ml for the non-activated cultures, and 250, 500, 1000, 2000, 3000, 4000 and 5000µg/ml for the S9-activated cultures.

Vehicle / solvent:

Sterile distilled water was chosen for the solvent, based on the solubility of the test article and compatability with the target cells. The test article was soluble in sterile distilled water at a concentration of 500 mg/ml, the maximum concentration tested.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

historical positive control data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

historical positive control data

Details on test system and experimental conditions:

Initial and independent repeat mutagenesis assays were used to evaluate the mutagenic potential of the test material. Exponentially growing CHO-K1-BH4 cells were seeded in F12FBS5-Hx at a density of 5x105 cells/25cm2 flask and were incubated at 37±1oC in a humidified atmosphere of 5±1% CO2 in air for 18-24 hours. F12FBS5-Hx is Ham's F12 medium without hypoxanthine supplemented with 5% dialyzed FBS, 100 units penicillin/ml, 100µg streptomycin/ml and 2mM L-glutamine/ml.
Treatment was carried out by refeeding the treatment flasks with 5ml F12FBS5-Hx/25cm2 flask for the non-activated study and 4ml F12FBS5-Hx and 1ml S9 reaction mixture/25cm2 flask for the S9-activated study, to which was added 50µl dosing solution of test or control article in solvent or solvent alone. Duplicate flasks of cells were exposed to test material for 5 hours at 37±1oC. After the treatment period, all media were aspirated, the cells washed with Ca++- and Mg++-free Hanks' balanced salt solution (CMF-HBSS) and cultured in F12FBS5-Hx for an additional 18-24 hours at 37±1oC. Cells were then subcultured to assess cytotoxicity and to initiate the phenotypic expression period.

Evaluation criteria:

The cytotoxic effects were expressed relative to the solvent-treated control (relative cloning efficiency). The mutant frequency was calculated by dividing the total of mutant colonies by the number of cells selected, corrected for the cloning efficiency of cells prior to mutant selection, and was expressed as TG-resistant mutants per 10E6 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies were expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses giving ≤10% relative survival were not considered as valid data points.

Statistics:

Relative cloning efficiency (%) relative to solvent control.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at high doses with S9 activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Initial mutagenesis assay: No test article precipitate was observed at any dose level in treatment medium. Relative cloning efficiency was 78% at 5000µg/ml in the non-activated cultures and 91% at 2000µg/ml in the S9-activated cultures (the 3000µg/ml cultures were lost, one to toxicity and one to contamination prior to mutant selection). None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 10exp6 clonable cells.
Independent repeat assay: No test article precipitate was observed at any dose level in treatment medium. Relative cloning efficiency was 103% at 5000µg/ml in the non-activated cultures and 27% at 2100µg/ml in the S9-activated cultures (the culture treated with 2200µg/ml was too toxic to seed for mutant selection). None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 10exp6 clonable cells.

Cytotoxicity was not observed in the non-activated cultures, and was observed in the initial mutagenesis assay at doses ≥3000µg/ml with S9 activation and in the independent repeat assay at doses ≥2100µg/ml with S9 activation.

Historical control data are presented below.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary toxicity assay: CHO cells were exposed to solvent alone and 0.5, 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/ml concentrations of e-Caprolactone, with and without S9 activation for 5 hours. No test article precipitate was observed at any dose. Cloning efficiency relative to the solvent controls was 97% at 5000µg/ml without activation and 0% at 5000µg/ml with S9 activation.

The table below displays the CHO/HGRPT Assay historical control data for 1994 -1996; solvent control (culture medium and distilled water), and positive controls ethyl methanesulphonate (EMS) and benzo(a)pyrene (B(a)P). MF = mutant frequency per 10E6 clonable cells, SD = standard deviation.

	Non-activated		S9-activated
	Solvent Control	0.2µl/ml EMS	Solvent Control	4.0µg/ml B(a)P
Mean MF	7.3	266.0	7.4	165.1
SD	5.7	77.5	7.1	59.4
Maximum	21.5	475.5	24.2	327.5
Minimum	0.0	144.0	0.4	38.1

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

e-Caprolactone was negative in the CHO/HGPRT mutation assay with and without S9-activation.

Executive summary:

e-Caprolactone was tested for mutagenicity in the chinese hamster ovary (CHO) cell mutation assay at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus, with and without rat liver S9 activation. In the initial mutagenesis assay, no positive responses were observed, i.e. there were no treated cultures with >40 mutants per 10E6 clonable cells (laboratory criteria). Toxicity was not observed in the non-activated cultures, but was observed at concentrations of ≥3000µg/ml with S9 activation. In the independent repeat assay, no positive responses were observed, i.e. there were no treated cultures with >40 mutants per 10E6 clonable cells. Toxicity was not observed in the non-activated cultures, and was observed at concentrations of ≥2100 µg/ml with S9 activation. Therefore e-Caprolactone was concluded to be negative in the CHO/HGPRT Mutation Assay.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

A read-across is proposed because adipic acid is structurally similar to Hexan-6-olide (e-Caprolactone).

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

mammalian cell line, other: human fibroblasts

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The negative controls contained two cells with bridges, one of which contained an acentric fragment. The test compound was negative except for one cell which contained a bridge at the high dosage level. The positive controls were within normal limits.

Remarks on result:

other: strain/cell type: embryonic lung cultures

Remarks:

Migrated from field 'Test system'.

Summary data

Treatment Group	Mitotic Index*	No. Cells	% Cells with Acentric Fragments	% Cells with Bridges	% Multipolar cells	% Cells with other Aberrations**	% Cells with Aberrations***
Adipic Acid 2 µg/ml	1	100	0	0	0	0	0
Adipic Acid 20 µg/ml	1	100	0	0	0	0	0
Adipic Acid 200 µg/ml	1	100	0	1	0	0	1
Saline Control	1	100	1	2	0	0	3
Positive Control (TEM)	1	100	3	12	0	0	15

* Percent of cells in mitosis: 200 cells observed/dose level

** Cells that have polyploidy, pulverisation, fragments or greater than 10 aberrations

*** Duplicate aberrations in a single cell will cause this value to be a % less than a summation of the % aberration seen.

Conclusions:

Interpretation of results (migrated information):
negative

No evidence of clastogenicity was seen under the conditions of this assay.

Executive summary:

Adipic acid was tested in an in vitro chromosome aberration assay, with human lung fibroblasts. The cells were cultured with the test material at concentrations of 0 (saline), 2, 20 and 200 µg/ml. Chromosome aberrations were evaluated when the cells were in anaphase. Only one cell (in the 200 µg/ml group) was found to contain a bridge, no other aberrations were seen. Therefore, adipic acid was negative for mutagenic activity in the in vitro mammalian cell chromosome aberration assay.

This information is used in the read-across approach and the assessment of the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo, negative results are reported in a rat bone marrow cytogenicity assay and in a rat dominant lethal assay, both performed with the read-across substance adipic acid.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

A read-across is proposed because adipic acid is structurally similar to Hexan-6-olide (e-Caprolactone).

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Acute study 1 (Table 1): The negative control groups cells contained no aberrations. The compound produced no aberrations except for one cell containing a break in the 6-hour sample of the 37.5 mg/kg bw group. The expected severe chromosomal damage was observed in the positice control group. The mitotic indices were within normal limits.

Subacute study 1 (Table 2): The negative control group and the 3.75 mg/kg bw group contained no aberrations. The 37.5 mg/kg bw group contained one cell with a reunion and one cell with that was polyploidy. The 375 mg/kg group contained three cells with breaks and one fragment. These were considered to be within normal limits of the historical negative controls.

Acute study 2 (Table 3) and subacute study 2 (Table 4): Neither the variety nor the number of chromosomal aberrations differed significantly from the negative controls.

Table 1. Metaphase summary data from the first acute study

Treatment Group	Time of kill after dosing	No. animals	No. cells	Mitotic Index (%)*	% Cells with Breaks	% Cells with Reunion	% Cells other Aberrations**	% Cells with Aberrations ***
Negative (saline) Control	6	3	150	6	0	0	0	0
	24	3	150	4	0	0	0	0
	48	3	150	8	0	0	0	0
Adipic Acid 3.75 mg/kg	6	5	250	7	0	0	0	0
	24	5	250	4	0	0	0	0
	48	5	250	6	0	0	0	0
Adipic Acid 37.5 mg/kg	6	5	250	5	0.4	0	0	0.4
	24	5	250	7	0	0	0	0
	48	5	250	6	0	0	0	0
Adipic Acid 375 mg/kg	6	5	250	5	0	0	0	0
	24	5	250	6	0	0	0	0
	48	5	250	4	0	0	0	0
Positive control TEM		5	250	4	0	23	22(a)	45

* Percent of cells in mitosis: 500 cells observed/animal

**Cells that have polyploidy (p), pulverisation (pp), or greater than 10 aberrations (a)

*** Duplicate aberrations in a single cell will cause this to be a % less than a summation of the % aberration seen.

Table 2. Metaphase summary data from the first subacute study

Treatment Group	No. animals	No. cells	Mitotic Index (%)*	% Cells with Breaks	% Cells with Reunion	% Cells other Aberrations**	% Cells with Aberrations
Negative (saline) Control	3	150	6	0	0	0	0
Adipic Acid 3.75 mg/kg	5	250	5	0	0	0	0
Adipic Acid 37.5 mg/kg	5	250	5	0	0.4	0.4 (p)	0.8
Adipic Acid 375 mg/kg	5	250	4	1.2	0	0.4 (f)	1.6

* Percent of cells in mitosis: 500 cells observed/animal

**Cells that have polyploidy (p), pulverisation (pp), fragments (f), or greater than 10 aberrations (a)

Table 3. Metaphase summary data from the second acute study

Treatment Group	Time of kill after dosing	No. animals	No. cells	Mitotic Index (%)*	% Cells with Breaks**	% Cells with Reunion**	% Cells other Aberrations**+	% Cells with Aberrations
Adipic Acid 5000 mg/kg	6	5	250	5.51	0	0	2p (0.8)	9 (0.8)
	24	5	250	4.03	0	0	1p (0.4)	1 (0.4)
	48	5	250	4.13	0	0	0	0
Negative (saline) Control	6	3	150	7.47	0	0	1p (0.66)	1 (0.66)
	24	3	150	4.50	0	0	2p (1.33)	2 (1.33)
	48	3	150	4.50	0	0	1p (0.66)	1 (0.66)
Positive Control (TEM)	24	5	250	1.62	4 (1.6)	50 (20.0)	>27 (10.8); 9f (3.6); 1p (0.4)	86 (34.4)

* Percent of cells in mitosis: 500 cells observed/animal

**Numbers in parentheses are % aberrations per total cells counted

+ Symbols: > = greater than 10 aberrations per cell, polyploidy (p), fragments (f)

Table 4. Metaphase summary data from the second subacute study

Treatment Group	No. animals	No. cells	Mitotic Index (%)*	% Cells with Breaks**	% Cells with Reunion**	% Cells other Aberrations**	% Cells with Aberrations
Adipic Acid 2500 mg/kg	5	218	2.98	0	0	1p (0.46)	1 (0.46)
Negative (saline) Control	3	150	5.33	0	0	1p (0.66)	1 (0.66)

* Percent of cells in mitosis: 500 cells observed/animal

**Numbers in parentheses are % aberrations per total cells counted

polyploidy (p)

Conclusions:

Interpretation of results (migrated information): negative
No evidence of clastogencitiy was seen under the conditions of this study.

Executive summary:

The cytogenic potential of adipic acid was evaluated in an in vivo chromosome aberration assay (the study was conducted in two separate experiments). The test substance was administered to male rats by gavage at single doses of 0, 3.75, 37.5, 375 and 5000 mg/kg bw, or at five consecutive daily doses of 0, 3.75, 37.5, 375 and 2500 mg/kg bw. Negative controls (saline) and positive controls (triethylene melamine) were included. Rats were sacrificed at varying intervals after dosing, and bone marrow cells were investigated microscopically for chromosome aberrations. There were no increases, relative to negative controls, in the incidence of chromosome aberrations, and all values were within normal limits. No evidence of clastogencitiy was seen under the conditions of this study.

This information is used in the read-across approach and the assessment of the target substance.

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

A read-across is proposed because adipic acid is structurally similar to Hexan-6-olide (e-Caprolactone).

Reason / purpose for cross-reference:

read-across source

Type of assay:

rodent dominant lethal assay

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information): negative
The data revealed no dose-response or time-trend patterns, therefore it was concluded that adipic acid does not induce dominant lethal mutations.

Executive summary:

A rodent dominant lethal study was conducted with Sprague-Dawley rats, in two separate experiments. The test substance adipic acid was administered to male rats by gavage at single doses of 0, 3.75, 37.5, 375 and 5000 mg/kg bw, or at five consecutive daily doses of 0, 3.75, 37.5, 375 and 2500 mg/kg bw. Negative controls (saline) and positive controls (triethylene melamine) were included. The male rats were mated to virgin untreated females for a period of 7 -8 weeks. The females were sacrificed 2 weeks after mating and the uterine contents examined for the number of corpora lutea, implantations and losses. The data revealed no dose-response or time-trend patterns, therefore it was concluded that adipic acid does not induce dominant lethal mutations.

This information is used in the read-across approach and the assessment of the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Bacterial mutation

The mutagenicity of the substance has been investigated in three separate screening studies reporting results for a large number of substances. Individually, the studies are of limited reliability as the level of reporting for individual substance is inevitably of limited detail; the individual studies also have some methodological deficiencies. Nevertheless, it is concluded based on a weight of evidence approach that consistent negative results in a large number of strains of S. typhimurium and E. coli at concentrations of up to 100 mg/plate show that the substance is not mutagenic in this test system.

Simmon (1975) reports neagtive results in S. typhimurium strains TA 1535, TA 1536, TA 1537, TA 1538, TA 98 and TA 100 in the presence and absence of a metabolic activation system at concentrations of 250 µg/plate. McCann et al (1975) report negative results in S. typhimurium strains TA98, TA100, TA1537 and TA1535 with and without metabolic acitvation as concentrations of up to 10 mg/plate. Rosenkranz & Poirier (1979) report negative results in S. typhimurium strains TA 1535 and TA 1538, and a DNA polymerase deficient E. coli in the presence and absence of metabolic activation.

Mutagenicity in mammalian cells

E-Caprolactone was tested for mutagenicity in the CHO cell mutation assay at HGPRT locus, with and without rat liver S9 activation (San & Clarke; 1997). In the initial mutagenesis assay, no positive responses were observed, i.e. there were no treated cultures with >40 mutants per 10E6 clonable cells (laboratory criteria). Toxicity was not observed in the non-activated cultures, but was observed at concentrations of ≥3000µg/ml with S9 activation. In the independent repeat assay, no positive responses were observed, i.e. there were no treated cultures with >40 mutants per 10E6clonable cells. Toxicity was not observed in the non-activated cultures, and was observed at concentrations of ≥2100 µg/ml with S9 activation.

Clastogenicity

E-Caprolactone also was tested in an in vitro chromosome aberration assay using Chinese hamster fibroblasts, without metabolic activation (Ishidate and Odashima, 1977). e-Caprolactone was negative in the test at a maximum concentration of 43.8mg/ml; an incidence of 4% aberration was observed. The read-across substance adipic acid was tested in a chromosome aberration assay, in a study commissioned by the FDA (Litton Bionetics, 1974).

Human lung fibroblasts were cultured with the test material at concentrations of 0 (saline), 2, 20 and 200 µg/ml. Chromosome aberrations were evaluated when the cells were in anaphase. Only one cell (in the 200 µg/ml group) was found to contain a bridge, no other aberrations were seen. Therefore, it was concluded that adipic acid was negative for mutagenic activity in the mammalian cell chromosome aberration assay (Litton Bionetics, 1971).

Addtional data

The OECD SIDS document (2005) summarises an addtional study conducted by Slesinski et al (1981). The authors conducted a gene mutation assay and a sister chromatid exchange assay with CHO cells, and an unscheduled DNA synthesis study with rat hepatocytes. e-Caprolactone produced three statistically significant increases in the frequency of genetic mutations without metabolic activation, but there was no dose-related effect nor was there a significant effect on mutant frequency with S9 activation. No statistically significant increase in the frequency of sister chromatid exchange was obtained at any concentration tested with or without metabolic activation. Three e-Caprolactone concentrations tested produced statistically significant increases in the amount of unscheduled DNA synthesis, and all six concentrations produced numerical increases compared to the solvent control. However, there was no dose-related effect in the amount of UDS. The results reported here are from the OECD SIDS document, the original study is not available and has therefore not been summarised in the endpoint study records.

Genetic toxicity in vivo

Two in vivo studies, commissioned by the FDA, were conducted by Litton Bionetics (1971) to investigate the genetic toxicity of the read-across substance adipic acid.

Adipic acid was administered to male rats by gavage at single doses of 0, 3.75, 37.5, 375 and 5000 mg/kg bw, or at five consecutive daily doses of 0, 3.75, 37.5, 375 and 2500 mg/kg (Litton Bionetics, 1971). Rats were sacrificed at varying intervals after dosing, and bone marrow cells were evaluated for chromosome aberrations. There were no increases, relative to negative controls, in the incidence of chromosome aberrations, and all values were within normal limits. No evidence of clastogencitiy was seen under the conditions of this study. A rodent dominant lethal study was conducted with Sprague-Dawley rats, in two separate experiments. The test substance was administered to male rats by gavage at single doses of 0, 3.75, 37.5, 375 and 5000 mg/kg bw, or at five consecutive daily doses of 0, 3.75, 37.5, 375 and 2500 mg/kg bw. Negative controls (saline) and positive controls (triethylene melamine) were included. The male rats were mated to virgin untreated females for a period of 7 -8 weeks. The females were sacrificed 2 weeks after mating and the uterine contents examined for the number of corpora lutea, implantations and losses. The data revealed no dose-response or time-trend patterns, therefore it was concluded that adipic acid does not induce dominant lethal mutations (Litton Bionetics, 1971).

Additional data

An in vivo mouse micronucleus study conducted by Gudi & Ritter (1997) is summarised in the OECD SIDS (2005) document. e-Caprolactone did not cause a significant increase in micronucleated polychromatic erythrocytes in the bone marrow of exposed groups compared to controls, and was therefore concluded to be negative in the micronucleus assay. The results reported here are from the OECD SIDS document, the original study is not available and has therefore not been summarised in the endpoint study records.

Short description of key information:
Negative results are reported in vitro in three Ames tests and in two studies of clastogenicity. An additional study of clastogenicity with the read-across substance adipic acid also gives a negative results. In vivo, negative results are reported in a rat bone marrow cytogenicity assay and in a rat dominant lethal assay, both performed with the read-across substance adipic acid.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All studies in vitro and in vivo report negative results and therefore do not indicate that e-caprolactone requires classification according to Regulation (EC) No 1272/2008 (the CLP Regulation).