trans-(5RS,6SR)-6-amino-2,2-dimethyl-1,3-dioxepan-5-ol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1993 to July 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 May 1983

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

trans-(5RS,6SR)-6-amino-2,2-dimethyl-1,3-dioxepan-5-ol

EC Number:

419-050-3

EC Name:

trans-(5RS,6SR)-6-amino-2,2-dimethyl-1,3-dioxepan-5-ol

Cas Number:

79944-37-9

Molecular formula:

C7H15NO3

IUPAC Name:

6-amino-2,2-dimethyl-1,3-dioxepan-5-ol

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse was chosen as test animal since a wide range of experience with regard to micronucleus tests exists and since the mouse has been recommended by national and international authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzheim, FRG
- Age at study initiation: adult, 8-9 weeks
- Weight at study initiation: 25-35 and 25-29 g for males and females, respectively
- Fasting period before study: 17-21 h
- Housing: housed individually in Macrolon type ll cages containing wood-chip bedding.
- Diet (e.g. ad libitum): ad libitum, (Altromin®R; Altromin, Lage, FRG)
- Water (e.g. ad libitum): ad libitum, mildly acidified demineralized water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 46-50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used:physiological saline
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL

Duration of treatment / exposure:

treated once and killed after 24 and 48 h

Frequency of treatment:

once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

10-13 per treatement and 10 in the negative control group and 5 in the positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): Cyclophosphamide, a known mutagen, was used as a positive control.
- Route of administration: intraperitoneal
- Doses / concentrations: 30 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow from femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: lt was assumed from an acute toxicity study in male mice that 2 g/kg would be a dose level at which toxic effects might be noted (at 2g/kg all animals showed moderate apathy).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
5 males and 5 females from each of the negative control and the treatment groups were killed by carbon dioxide asphyxiation ca. 24 or 48 hours after treatment (the positive control animals were killed 24 hours after treatment) and both femurs were dissected out from each animal.

DETAILS OF SLIDE PREPARATION:
The bone marrow was flushed/aspirated into fetal calf serum. The resulting cell suspensions were centrifuged and smears were prepared from drops of the cell pellets which had been resuspended in a few drops of serum. The slides were air-dried and stained using May-Gruenwald and Giemsa solutions.

METHOD OF ANALYSIS:
The slides were coded and analysed "blind" in random order. The stained smears were examined using oil immersion high power magnification in regions where cells were well-spread and stained. The slides were examined for the incidence of micronucleated cells per 2000 polychromatic (POE) and 1000 normochromatic (NCE) erythrocytes per animal. The ratio of polychromatic to normochromatic erythrocytes was calculated on the basis of 1000 NCE scored.

Statistics:

The statistical analysis was conducted for each of the following variables:
p1: proportion of micronucleated PCE
p2: proportion of micronucleated NOE
p3: ratio of PCE/NCE
For investigation of treatment differences the variables p1 and p2- were arc sin transformed. The analyses were conducted separately for the sample times. Regarding the first sample time one-sided t-tests were performed to assess the difference between positive and negative controls with pooled values for both sexes; the positive control group was then excluded from further analysis. Thereafter in a two-factorial ANOVA (factors "sex", treatment") for each sample time it was investigated as to whether any treatment effect was present. In case of significant interactions, comparisons between the control and each of the treatment groups were conducted separately for each sex. Where no significant interactions occurred but a global treatment effect, comparisons were performed with values pooled for both sexes. The pair-wise comparisons were performed with one-sided t-tests (increase of p1 and D2, decrease of p3), using the error estimate of the ANOVA table. The test levels were always or = 0.05 (least significant differences test-LSD). In addition to the non-transformed individual data the arithmetic means and standard deviations were included.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

in the high dose group

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

After application of the high dose only 1 male and 2 females out of 13 males and 13 females survived the first day. Therefore, only two dose groups could be evaluated. Signs of slight toxicity could be observed in 4 out of 10 males and 4 out of 10 females in the mid dose group.
Regarding micronucleated PCE and NCE counts as well as the ratio POE/NCE, there were no biologically relevant or statistically significant differences (p > 0.05) at any sample time. The positive control group showed a significant increase in micronucleated POE and NCE counts (p < 0.05).

 

MICRONUCLEUS ASSAY RESULTS

MICRONUCLEI (IN PER MILL) SCORED PER TWO THOUSAND POLYCHROMATIC AND ONE THOUSAND ERYTHROCYTES WITH RATIO PCE/NCE

Negative Control 10 mL/kg bw
24 h post application				48 h post application
No.	PCE (M)	NCE (M)	Ratio PCE/NCE	No.	PCE (M)	NCE (M)	Ratio PCE/NCE
Male				Male
1	1.00	1.00	0.97	6	1.00	1.00	0.92
2	0.50	1.00	0.96	7	0.00	1.00	1.14
3	1.00	1.00	0.96	8	1.00	1.00	1.12
4	0.50	0.00	0.98	9	1.50	1.00	1.02
5	1.50	1.00	0.90	10	1.50	1.00	1.11
MV=	0.90	0.80	0.95	MV=	1.00	1.00	1.06
SD=	0.42	0.45	0.03	SD=	0.61	0.00	0.09
N=	5	5	5	N=	5	5	5
Female				Female
1	0.50	0.00	0.95	6	1.00	2.00	0.94
2	1.00	1.00	1.05	7	0.50	0.00	1.02
3	1.00	1.00	0.87	8	0.50	1.00	1.06
4	0.50	0.00	0.95	9	1.00	1.00	1.01
5	0.50	1.00	1.10	10	1.00	0.00	0.88
MV=	0.70	0.60	0.98	MV=	0.80	0.80	0.98
SD=	0.27	0.55	0.09	SD=	0.27	0.84	0.07
N=	5	5	5	N=	5	5	5
Male + Female				Male + Female
MV=	0.80	0.70	0.97	MV=	0.90	0.90	1.02
SD=	0.35	0.48	0.07	SD=	0.46	0.57	0.09
N=	10	10	10	N=	10	10	10
0.5 g/kg bw
24 h post application				48 h post application
No.	PCE (M)	NCE (M)	Ratio PCE/NCE	No.	PCE (M)	NCE (M)	Ratio PCE/NCE
Male				Male
1	1.00	2.00	0.96	6	1.00	1.00	1.07
2	0.50	0.00	0.96	7	0.50	1.00	1.06
3	0.50	0.00	1.15	8	0.50	0.00	1.14
4	1.00	1.00	1.00	9	1.00	1.00	1.04
5	1.50	1.00	1.09	10	1.00	0.00	0.92
MV=	0.90	0.80	1.03	MV=	0.80	0.60	1.05
SD=	0.42	0.84	0.08	SD=	0.27	0.55	0.08
N=	5	5	5	N=	5	5	5
Female				Female
1	0.50	2.00	0.98	6	1.00	1.00	1.10
2	1.00	0.00	1.00	7	0.50	0.00	0.91
3	0.50	1.00	0.94	8	0.50	1.00	1.06
4	1.00	1.00	0.96	9	0.50	1.00	0.94
5	0.50	0.00	0.90	10	0.50	1.00	1.13
MV=	0.70	0.80	0.96	MV=	0.60	0.80	1.03
SD=	0.27	0.84	0.04	SD=	0.22	0.45	0.10
N=	5	5	5	N=	5	5	5
Male + Female				Male + Female
MV=	0.80	0.80	0.99	MV=	0.70	0.70	1.04
SD=	0.35	0.79	0.07	SD=	0.26	0.48	0.09
N=	10	10	10	N=	10	10	10
1.0 g/kg bw
24 h post application				48 h post application
No.	PCE (M)	NCE (M)	Ratio PCE/NCE	No.	PCE (M)	NCE (M)	Ratio PCE/NCE
Male				Male
1	1.00	0.00	0.96	6	1.00	1.00	1.10
2	1.00	1.00	1.03	7	0.50	1.00	1.04
3	1.00	1.00	1.06	8	0.50	0.00	0.97
4	1.00	0.00	0.95	9	1.00	1.00	0.99
5	1.00	1.00	0.83	10	1.00	0.00	0.92
MV=	1.00	0.60	0.96	MV=	0.80	0.60	1.00
SD=	0.00	0.55	0.09	SD=	0.27	0.55	0.07
N=	5	5	5	N=	5	5	5
Female				Female
1	0.50	0.00	1.00	6	1.00	2.00	1.06
2	0.50	1.00	1.05	7	0.50	1.00	1.10
3	0.50	1.00	1.03	8	1.00	1.00	1.05
4	1.00	1.00	0.90	9	0.50	0.00	1.07
5	0.50	1.00	1.01	10	0.50	0.00	1.07
MV=	0.60	0.80	1.00	MV=	0.70	0.80	1.07
SD=	0.22	0.45	0.06	SD=	0.27	0.84	0.02
N=	5	5	5	N=	5	5	5
Male + Female				Male + Female
MV=	0.80	0.70	0.98	MV=	0.75	0.70	1.04
SD=	0.26	0.48	0.07	SD=	0.26	0.67	0.06
N=	10	10	10	N=	10	10	10
Cyclophosphamide 30 mg /kg bw
24 h post application
No.	PCE (M)	NCE (M)	Ratio PCE/NCE
Male
1	9.50	2.00	0.95
2	11.00	1.00	0.95
3	13.50	2.00	0.89
4	15.00	1.00	1.00
5	15.00	1.00	0.96
MV=	12.80	1.40	0.95
SD=	2.46	0.55	0.04
N=	5	5	5
Female
1	10.00	2.00	0.98
2	7.50	1.00	0.89
3	10.50	3.00	0.87
4	11.00	1.00	0.81
5	9.50	1.00	0.96
MV=	9.70	1.60	0.90
SD=	1.35	0.89	0.07
N=	5	5	5
Male + Female
MV=
SD=
N=	10	10	10
PCB = POLYCHROMATIC ERYTHROCYTES NCE = NORMOCHROMATIC ERYTHROCYTES PCE(M) = MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES (IN PER MILL) NCE(H) = MICRONUCLEATED NORMOCHROMATIC ERYTHROCYTES (IN PER HILL) MV: MEAN VALUE SD: STANDARD DEVIATION N: NUMBER 0F ANIMALS PER GROUP

 

Applicant's summary and conclusion

Conclusions:

Evaluation of the data showed that Aminodioxepan has no evidence of mutagenic potential, when administered intraperitoneally up to the toxic dose level of 1.0 g/kg in the mouse micronucleus test.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay equivalent to OECD test guideline 474, (5 animals/sex/dose) were treated intraperitoneally with Aminodioxepan (100 % a.i.) at doses of 0, 500, 1000 and 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 h post-treatment.  The vehicle was physiological saline.

There were signs of toxicity present during the study. After application of the high dose only 1 male and 2 females out of 13 males and 13 females survived the first day. Therefore, only two dose groups could be evaluated. Signs of slight toxicity could be observed in 4 out of 10 males and 4 out of 10 females in the mid dose group.

Regarding micronucleated PCE and NCE counts as well as the ratio POE/NCE, there were no biologically relevant or statistically significant differences (p > 0.05) at any sample time. The positive control group showed a significant increase in micronucleated POE and NCE counts (p < 0.05). Aminodioxepan was tested at an adequate dose (based on an acute toxicity study in mice). There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.