Cyclooctapentylose

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

A micronucleus test (in vivo), an Ames test and an in vitro chromosome aberration study were performed with .gamma.-cyclodextrin. An in vitro studies (HPRT) was performed with the read-across substance .beta.-cyclodextrin. All four studies were evaluated as negative for the corresponding endpoint.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult female and male Swiss mice (Charles River CD-1 strain) were obtained from a colony maintained under SPF conditions at Charles River Wiga GmbH, Sulzfeld, F.R. Germany. The animals arrived on November 27, 1990. Upon arrival the animals were checked for overt signs of ill health and anomalies. Following an initial quarantaine and acclimatization period of 6 days, the animals were distributed by computer randomization over 3 groups: a vehicle control group, a test group and a positive control group of 15 males and 15 females each. Each group was fitted with a letter code and a colour code. Within each group the animals were identified individually by earmark and computer reference number. The females were odd numbered and the males even. The remaining mice (6/sex) were kept in reserve. At the start of the study the body weights varied from 25.4 to 33.0 g for males and from 21.4 to 28.4 g for females.

Housing and care
The mice were housed in Makrolon, sterilized cages with a grid cover of stainless steel and with a bedding of sterilized softwood chips. Males were housed individually and females up to five per cage. The room was ventilated with about 10 air changes per hour and maintained at 21.0 - 21.5 °C. Relative humidity was between 62 and 68 percent. Lighting was artificial with a sequence of 12 hours light, 12 hours dark. The mice had free access to the Institute's cereal based, open formula diet for rats and mice, except during a period of 2 to 4 h just prior to treatment, when food was withheld. Tap water was freely available at all times. Diet and tap water are analyzed periodically for nutrients and contaminants.

Route of administration:

oral: gavage

Vehicle:

saline

Details on exposure:

On day 0 the animals of the test group were treated orally by gavage with one single dose of 15 g of the test material in 20 ml demineralised water per kg body weight. The negative control group was treated in an identical way with demineralised water. The positive control group was treated once intraperitoneally with the well-known mutagen mitomycin C in an amount of 1.5 mg in 20 ml saline/kg body weight. The suspension was freshly prepared just prior to use. The animals were weighed on day 0, just prior to gavage. They were inspected at 1 and 4 hours after treatment for signs of ill health or reactions to treatment, and once every day thereafter till the end of the experimental period.

Duration of treatment / exposure:

72h

Frequency of treatment:

once

Remarks:

Doses / Concentrations:
15 g/20 ml water/kg body weight
Basis:
nominal in water

No. of animals per sex per dose:

Test animals: 15 per sex
Control group : 15 per sex
Positive control group: 15 per sex

Control animals:

yes, concurrent vehicle

Positive control(s):

A positive control group (15 males, 15 females) was treated once intraperitoneally with the mutagen mitomycin C in an amount of 1.5 mg per kg body weight.

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

Microscopic examination of the bone marrow smears

The slides to be used for the microscopic examination were randomly coded by a person not involved in the study. Care was taken to ensure that no original slide identifications remained visible. The incidence of MPE (= micronucleated polychromatic erythrocytes) and MNE (= micronucleated normochromatic erythrocytes) and the total numbers of poly- and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of at least 2000 and maximally 3000 erythrocytes per animal in such a way that a minimum of 1000 PE was observed, if feasible. Slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.

Statistics:

Statistical analysis was carried out in two stages. In the first step of the procedure overall significance tests were carried out as follows: the fraction of MPE, MNE and ME per counted number of PE, NE, and E, respectively, were analysed with a generalized linear model using a binomial error-distribution, and the numbers of PE per 1000 erythrocytes were analyzed with linear regression techniques. Both methods assess the influence of sex (male, female), treatment (vehicle control, test groups), exposure time (24 h, 48 h, 72 h) and the various interactions on the total variation in the data. As a second stage, a posteriori comparisons of treatment groups with the negative control group were carried out with asymptotic t-tests if either the main effect of treatment or the treatment by sex interaction was significant.
For all calculations involved, the Genstat 5 statistical package was used.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The incidences of MPE, MNE and ME per 1000 PE, NE and E, respectively, in control and test animals were mutually fully comparable at each of the three sacrifice times.

The mean number of PE per 1000 E was fully comparable between control and the .gamma.-cyclodextrin treated group, indicating that treatment with .gamma.-cyclodextrin did not result in cytotoxicity for the bone marrow cells. The considerable increase in the incidence of micronucleated erythrocytes (MPE, MNE, ME) and the decrease in the PE counts per 1000 E in positive control animals, demonstrated the sensitivity of the test system.

Conclusions:

Interpretation of results (migrated information): negative
The results of the present micronucleus test did not provide any evidence of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated orally with a high dose of .gamma.-cyclodextrin.

The reference mutagen, mitomycin C, was clearly positive in this test.

Executive summary:

.gamma.-cyclodextrin was examined for its potential to induce micronuclei in bone marrow of mice in compliance with EEC-protocol B.12, and OECD guideline 474. Test animals (15 males, 15 females) were treated orally with one single dose of 15 g/20 ml water/kg body weight. The control group (15 males, 15, females) was treated in a similar way with the vehicle. A positive control group (15 males, 15 females) was treated once intraperitoneally with the mutagen mitomycin C in an amount of 1.5 mg per kg body weight. At 24, 48 and 72 hours after treatment, 10 controls (5/sex), 10 test animals (5/sex) and 10 positive controls (5/sex) were sacrificed. The incidence of micronucleated poly- and normochromatic erythrocytes (abbreviated MPE and MNE, respectively), and the numbers of poly- and normochromatic erythrocytes (PE and NE) were recorded in a total of at least 2000 and maximally 3000 erythrocytes (E) for each animal, in such a way that a minimum of 1000 PE was observed.

Treatment with the test substance did not induce any apparent signs of intoxication.

The incidence of MPE and MNE in mice treated with .gamma.-cyclodextrin, was fully comparable to that found in the concomitant negative controls.

The number of PE per 1000 E at any of the three sacrifice times after treatment of animals with the test substance was mutually fully comparable.

Animals treated with the mutagen mitomycin C showed an increased incidence of MPE and MNE, and a decrease in the number of PE per 1000 E.

It was concluded that the results of the micronucleus test did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated orally with a high dose of .gamma.-cyclodextrin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

IN VIVO .gamma.-cyclodextrin was examined for its potential to induce micronuclei in the bone marrow of mice according to OECD guideline 474. Treatment with the test substance did not induce any signs of intoxication. The incidence of MPE and MNE in mice treated with .gamma.-cyclodextrin, was fully comparable to that found in the negative controls. The number of PE per 1000 E at any of the three sacrifice times after treatment of animals with the test substance was fully comparable. It can be concluded that the results of the micronucleus test did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated orally with a high dose (15.000 mg/kg bw) of .gamma.-cyclodextrin.

IN VITRO .gamma.-Cyclodextrin was examined for its mutagenic activity in an Ames test using the histidine-requiring Salmonella typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (S-9 mix), in compliance with OECD guideline 471. It can be concluded that .gamma.-cyclodextrin did not show mutagenic activity in Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100, neither in the absence nor in the presence of the S-9 mix. The test substance .gamma.-cyclodextrin was examined for its potential to induce structural chromosome aberrations in cultured human lymphocytes, in both the absence and presence of a metabolic activation system (S-9 mix) and according to OECD guideline 473. .Gamma.-cyclodextrin induced no biologically relevant and statistically significant increases in the percentage of cells with structural chromosome aberrations at none of the concentrations and time points analyzed, when compared with the vehicle control values. It can be concluded that .gamma.-cyclodextrin is not clastogenic under the conditions oft he study.

A study equivalent to OECD 476 was performed to investigate the potential of the read-across substance .beta.-cyclodextrin to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. It can be stated that under the experimental conditions reported .beta.-cyclodextrin is considered to be non-mutagenic in the HPRT assay.

Justification for selection of genetic toxicity endpoint
OECD Guidline study (in vivo)

Justification for classification or non-classification

.gamma.-cyclodextrin and the read-across substance .beta.-cyclodextrin did not show any mutagenic or clastogenic potential in any of the tests performed. The data are conclusive but not sufficient for classification.