Cyclohexapentylose

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

A micronucleus test (in vivo) and an Ames test were performed with .alpha.-cyclodextrin. Two in vitro studies (chromosome aberration, HPRT) were 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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to 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, Germany. The animals arrived on November 13, 1990. 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. At the start of the study the body weights varied from 20.1 to 34.9 g for males and from 17.5 to 31.3 g for females.

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 20.5 - 21.5°C. Relative humidity was between 63 and 70 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:

Test group: One single dose of 10 g of the test material in 20 ml water per kg body weight

Negative control group: Treated in an identical way with water

Positive control group: treated once intraperitoneally with the well-known mutagen mitomycin C (Sigma, lot no.: 119F-0744) in an amount of 1.5 mg in 20 ml saline/kg body weight

Details on exposure:

On day 0 the animals of the test group were treated orally by gavage with one single dose of 10 g of the test material in 20 ml water per kg body weight. The negative control group was treated in an identical way with water. The positive control group was treated once intraperitoneally with the well-known mutagen mitomycin C (Sigma, lot no.: 119F-0744) in an amount of 1.5 mg in 20 ml saline/kg body weight. The suspension/dilution 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:

Single dose; 24, 48, 72 hours exposure period before bone marrow sampling

Frequency of treatment:

Day 0: single dose

Post exposure period:

24, 48, 72 hours

Remarks:

Doses / Concentrations:
10000 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

15 animals per sex per group (vehicle control group, test group, positive control group)

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control group was treated once intraperitoneally with the well-known mutagen mitomycin C (Sigma, lot no.: 119F-0744) in an amount of 1.5 mg in 20 ml saline/kg body weight. 15 animals per sex per group (vehicle control group, test group, positive control group)

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

At 24, 48 and 72 hr after treatment, 5 males and 5 females of the negative and positive control group and the test group, were killed by cervical dislocation. The femurs were dissected, and bone marrow was flushed from the femurs with foetal calf serum (Flow Laboratories, Scotland, lot no.: 9130062) using a syringe and needle. The collected cells were mixed gently with the serum and the cell suspension was centrifuged at ca. 1000 rpm for five minutes. After removal of the excess serum the cell pellets were resuspended in the remaining fluid and processed into glass-drawn smears according to the method described by Schmid (1976). Two bone marrow smears were prepared from each animal. The smears were air-dried, fixed in methanol, stained according to May-Grünwald Giemsa and mounted with a coverslipping mounting medium.

The slides to be used for the microscopic examination were randomly coded by a person not involved in the study. Subsequently, it was taken of 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.

Evaluation criteria:

The incidences of MPE, MNE and ME per 1000 PE, NE and E, respectively, in control and test animals were fully comparable at each of the three sacrifice times.
The mean number of PE per 1000 E was fully comparable between control and the .alpha.-cyclodextrin treated group, indicating that treatment with .alpha.-cyclodextrin did not result in cytotoxicity for the bone marrow cells.
The considerable increase of micronucleated erythrocytes (MPE, MNE, ME) and the decrease in the PE counts per 1000 E in positive control animals, demon-strated the sensitivity of the test system.

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 (McCullagh and Nelder, 1983), and the numbers of PE per 1000 erythrocytes were analyzed with linear regression techniques (e.g. Draper and Smith, 1981). 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.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

sluggishness, piloerection and half closed eyes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

One hour after treatment all male and female animals of the 10 g/kg dose group showed slight signs of sluggishness, piloerection and half closed eyes. The next day still four female animals (B38, B42, B56 and B60) showed signs of piloerection and half closed eyes.
Increased frequency of breathing was also seen in two of these four animals (B38 and B60). Forty eight hours after treatment the animals looked normal again.
One female animal of the 10 g/kg dose group looked ill immediately after treatment and died the next day. This animal showed signs of sluggishness, piloerection, closed eyes, increased frequency of breathing and a low body temperature. At autopsy slightly swollen, dark-red lungs were observed. This animal is most probably intubated in the lungs instead of the stomach.

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 .alpha.-cyclodextrin.

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

Executive summary:

.alpha.-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 10 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.

Clinical signs related to treatment with the test substance included sluggishness, piloerection and half closed eyes on the day of treatment. Forty eight hours after treatment all animals looked normal again. One female animal of the 10 g/kg dose group died 24 hours after treatment probably due to a technical mistake.

The incidence of MPE and MNE in mice treated with .alpha.-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 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 result 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 .alpha.-cyclodextrin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

IN VIVO .alpha.-cyclodextrin was examined for its potential to induce micronuclei in the bone marrow of mice in compliance with EEC-protocol B.12, and OECD guideline 474. Clinical signs related to treatment with the test substance included sluggishness, piloerection and half closed eyes on the day of treatment. 48 Hours after treatment all animals looked normal again. It was concluded that the result 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 (10.000 mg/kg bw) of .alpha.-cyclodextrin.

IN VITRO .alpha.-cyclodextrin was examined for its mutagenic potential in the 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. .alpha.-cyclodextrin did not show mutagenic activity in the Salmonella typhimurium strains mentioned above, neither in the absence nor in the presence of the S-9 mix.

The read across substance .beta.-cyclodextrin was assessed in an in vitro Chromosome Aberration assay with human lymphocytes similar to the OECD guideline 473. The test substance does not show clastogenic activity in the in vitro human lymphocyte metaphase analysis test with and without metabolic activation. 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

.alpha.-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.