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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

12 May 1981

GLP compliance:

yes

Limit test:

no

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:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected as the test species because it is a standard laboratory animal and the species of choice for systemic toxicity studies with new drugs or chemicals in rodents. Moreover, ample historical control data are available on the parameters recorded in this study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Schering AG, FRG
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: males: 7 weeks; females 9 weeks
- Housing: individually under conventional conditions in Makrolon® cage type lll with wire-mesh bottom
- Diet (e.g. ad libitum): ad libitum; Altromin® R, pulverized
- Water (e.g. ad libitum): ad libitum; sterile filtered, demineralised water acidified with hydrochloric acid (pH 2-3)
- Acclimation period: ≥ 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 52-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1993-01-13 To: 1993-02-18

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

physiological saline

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions with a nominal content of 10, 30 and 100 mg test item/mL in 0.9% (w/v) aqueous sodium chloride, pH 9.2-9.5.
The formulations were prepared fresh daily and kept in brown glass bottles at room
temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test compound was a colourless crystalline solid of 100% purity. Due to the hygroscopic nature of the compound a water content of approx. 10% was found by Karl Fischer titration at various determinations ‘performed during and alter this study. Accordingly the actual doses administered to the experimental animals deviate by approximately -10% from the nominal doses required by the protocol. For reasons of consistency in the documentation for this study and ofeasier legibility of this report, the results will be described on the basis of the nominal dose levels. The actual doses, however, are mentioned in the summary and conclusion sections.
Furthermore, the re-analysis of the test compound after completion of the study revealed that the compound did not conform to the specification anymore with regard to the content of N-acetate (0.13% found instead of < 0.1% required) and tocolour (absorption 0.09 found instead of <0.05 required). These slight deviations are not considered to have affected the quality of this study with regard to risk assessment of the test item.
HPLC analysis of formulations with a nominal concentration of 10 mg test item/mL that were prepared on days 1 and 31 of the study revealed a content of 87+/-1% and 85+/-2%, respectively (KIAN 3628 and 3646). Taking into account the above-mentioned water content of the test compound these data are in good agreement with the expected values and confirm the exactness of the preparation of the solutions used in this study. Furthermore, solutions containing 2-200 mg/mL were found to be stable over 24 hours at room temperature using HPLC analysis (see KIAN 3744, 3745, 3746, 3747 and 3749).

Duration of treatment / exposure:

at least 28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on acute toxicity testing
- Fasting period before blood sampling for clinical biochemistry: Blood samples for laboratory studies were obtained from the jugular veins of the animals. No food was offered to the animals for more than approximately 16 hours prior to blood and urine sampling.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded weekly and the weight gain from days 1 to 28 was calculated for statistical evaluation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
The amount of food consumed was recorded weekly. The mean food consumption per day
between days 1 and 28 was evaluated statistically.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The quantity of water consumed by the individual animal was determined weekly. The mean consumption of drinking water per day between days 1 and 28 was evaluated statistically.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were carried out on day 25 in 5M and 5F animals per group. The pupils of all animals were dilated using a mydriaticum (Mydriaticum-Roche®, 0.5%) and the eyes examined with the naked eye, an ophthalmoscope (Oculus-Visuskop, W. Oculus) and a portable slit-lamp microscope, Kowa SL-5 (Kowa Company).
- Dose groups that were examined:all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 24
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all groups
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 24 h after treatment and at day 25
- Animals fasted: Yes
- How many animals: 5 males and females each group

PLASMA/SERUM HORMONES/LIPIDS: Yes / No / Not specified
- Time of blood sample collection:
- Animals fasted: Yes / No / Not specified
- How many animals:

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The Dunnett-test was used to assess the statistical significance of differences between the control and the treatment groups.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

After treatment on day 1 slight reddening and slight to severe swelling of the lower and upper limbs were observed in all animals of the high dose group. The findings persisted in part until day 2 of the study. Additionally, such findings were observed in some animals at the mouth, the external auditory meatus, at the nose (swelling only) and at the tail (reddening only). These findings had completely disappeared on day-3. Moreover, slight to marked sialorrhea was observed before and after treatment in the mid dose group (on days 19-31 in a total of 2M and 3F) and in the high dose group (on days 8-31 in a total of 9M and 10F). The finding was dose-dependent with regard to time of onset and frequency. All further findings such as alterations of the skin (alopecia, thinning of fur, wet fur, scab formation), apathy, hyperactivity, hematomas and palpable mass were not regarded as being compound-related because they occurred without dose-dependence or only sporadically.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal (no. 64M, group 4) died during week 3 of the study, most likely as a consequence of misapplication.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was slightly reduced at the high dose of 1000 mg/kg in male (p < 0.05) and female rats (statistically not significant). The decrease was 17% in relation to controls for both sexes.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

A higher consumption of drinking water was noted in male animals from 300 mg/kg onwards (p < 0.05; p < 0.01) and in females at 1000 mg/kg (p < 0.01). The increase in relation to the values of the control animals was 17-20% in the males and 41% in the females.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A slight increase in the leucocyte count was observed in male rats of the mid (n.s.) and high (p < 0.05) dose groups. This change was due to a slight to moderate increase in neutrophils (n.s. to p < 0.01). Likewise, a slight statistically significant (p < 0.01) decrease in platelet count in male and female animals at the top dose was considered as being compound-related.
Moreover, the slight and partially significant decreases in erythrocyte count (n.s.), hemoglobin (n.s.) and hematocrit (p < 0.05) in male rats of the high dose group are suspected of being compound-related. These indications of a slight anemia were associated with a slight reactive increase in relative and absolute reticulocyte counts (p < 0.05 or n.s., respectively).
None of the other statistically significant differences to the control group are regarded as compound-related, because the values are within the range of historical reference values and the differences to the control group are too small to be of biological relevance. Furthermore, the non-significant increase in blood lymphocytes of male animals in all three treatment groups was considered a chance finding, since it showed no dose-dependence and all individual values remained within the historical reference range.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

An increase in serum GPT was observed in the mid and high dose groups on days 2 and 24 in male (p < 0.01 in each case) as well as in female animals (n.s. or p < 0.01). This change was slight at 300 mg/kg and moderate at 1000 mg/kg. On day 24 it was associated with an increase in serum alkaline phosphatase at 1000 mg/kg in both sexes (p<0.01). A slight increase in serum glucose (p < 0.01) and an increase in serum cholesterol (p < 0.01) were noted at the high dose on day 24 in male and female rats. Furthermore, a very slight decrease in serum total protein was observed only in male animals of the high dose group on day 24 (p<0.05).
All other statistically significant differences to the control group are not considered as being of biological relevance either because of lack of dose- and/or time relationship or because the values remained within the historical reference range or within the minimum-maximum values of the reference range.

Endocrine findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The increase in absolute and relative splenic weight in both sexes in the high dose group is
considered to be compound-related. In addition, the absolute and relative increase of ovary weight in the same group is regarded as a compound effect. The increase in relative liver weight at the high dose in both sexes is also suspected to be compound-related. Moreover, the decrease in absolute and relative thymic weights in males and females of this group is suspected to be compound-related. However, the decrease in absolute weight of this organ at 100 and 300 mg/kg and the decrease in relative weight at 100 mg/kg noted in female animals are not considered to be compound-related. The absolute or relative thymus weights of the respective animals lie well within the 90% reference range of this laboratory for control rats of the same body weight range. On the other hand, in the high dose group one female animal (no. 75) did not lie within this range for absolute weights and three females fell below the range for relative weights. The slight increase in relative heart weight in females of the mid dose group and the relative increase in lung weight in females of the low and high dose group are not considered to be compound-related due to the absence of a dose-response relationship. In addition, the individual values lie well within the 90% reference range calculated for historical control animals. Finally, microscopic examination did not reveal a morphological substrate of these organ weight changes. The slight relative increase in weights of pituitary gland, adrenal gland; brain and submandibular salivary glands in males at 1000 mg test item/kg is not considered to be compound-related, because the absolute organ weights did not show a statistically significant alteration. Therefore, the relative increases of these organ weights are probably related to the decrease in body weight. In addition, the weights of these organs lie-within the 90% range of the historical reference values. Finally, no corresponding change could be determined histologically. Nor was the slight relative increase in weights of kidney, pancreas and submandibular salivary glands in females of the high dose group regarded as compound-related, because the absolute organ weights were not changed when compared to the control group and all weights were within the 90%. Again, histological examination did not show any correlate to the increased weights of these organs.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlargement of the right ovary in one animal of the high dose group (no. 77) is considered to be compound-related (see organ weights and microscopic examination). All other findings are not regarded as treatment-related due to their spontaneous and isolated occurrence.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Vacuolation of macrophages and increased hematopoiesis in red pulp could be observed with higher incidence and severity in the high dose group. Focal cell degeneration and megakaryocytosis in red pulpwere observed only in this group at low incidence. Increased number of corpora lutea in the ovaries was observed with increasing incidence and mean severity inthe mid and high dose groups. Focal granulomatous inflammation could be detected with slightly higher mean severity in the high dose group compared to the control group. Although this change was only slight in degree, it is considered to be compound-related because of the corresponding changes in biochemical parameters (SGPT, SAP).
All other findings listed below which were observed in both control and treated animals or which were observed only in treated animals are not considered to be compound-related because of their isolated character, lack of dose-dependence or as they were findings which can be observed regularly in Han: WIST (SPF) rats from control groups:
Kidneys: interstitial nephritis (1 animal group 1;1 animal group 4)
hydronephrosis (1 animal group 1)
focal tubular hyperplasia (2 animals group.4)
pelvic dilatation (2 animals group 4)
tubular dilatation (1 animal group 4) A
focal round cell infiltration (1 animal group 4)
passive hyperemia (2 animals group 1; 3 animals group 4)
unilateral blood cyst (1 animal group 1)
focal vacuolaticn of proximal tubules (2 animals group 4)
Liver: diffuse clear cell change, probably glycogen (1 animal group 4)
microvesicular focal/diffuse vacuolaticn, probably lipid (6 animals group 1; 1 animal group 2; 1 animal group 3; 3 animals group 4)
passive hyperemia (1 animal group 4)
Lungs: focal alveolar emphysema (1 animal group 1)
round cell infiltration (4 animals group 1; 1 animal group 4)
passive hyperemia with focal hemoptoe (1 animal group 4)
focal interstitial edema (1 animal group 4)
Trachea: foamy cells in submucosal glands (1 animal group 4)
focal lymphocytic infiltration in submucosa (1 animal group 1)
Thymus: cortical lymphocytic pyknosis (17 animals group 1; 18 animals group 4)
focal acute hemorrhage (1 animal group 1)
Spleen: focal capsular fibrosis (1 animal group 4)
passive hyperemia (1 animal group 4)
Tongue: glossitis cystica (1 animal group 1)
GI. submandibularis: focal degeneration (2 animals group 4)
Esophagus: focal necrosis in T. muscularis (2 animals group 1)
Stomach: focal granulocytic infiltration in L. propria (1 animal group 1; 2 animals group 2)
acute steatitis of abdominal fat tissue (1 animal group 4)
focal acute erythrodiapedesis (1 animal group 4)
Colon: focal granulocytic infiltration in L. propria (1 animal group 1)
Rectum: single cell necrosis (1 animal group 4)
acute, focal subserosal hemorrhages (2 animals group 4)
Pancreas: focal hyperplasia (1 animal group 4)
Ovaries: single follicular cyst (2 animals group 4)
Adrenal glands: accessory cortical tissue (1 animal group 1; 2 animals group 4)
diffuse vacuolaticn in zona fasciculoreticularis (13 animals group 1; 14 animals group 4)
passive hyperemia (1 animal group 4)
acute degeneration of cells in zona fasciculata (6 animals group 1; 8 animals group 4)
focal hypertrophy of zona glomerulosa/zona fasciculata with focal degeneraion (1 animal group 1; 1 animal group 4)
Mesenterial lymph node: focal vacuolaticn of cortical cells (1 animal group 4)
sinus histiocytosis (1 animal group 1; 2 animals group 4)
lliac lymph node: focal vacuolaticn of cortical cells (1 animal group 4)
acute focal blood resorption (1 animal group 1; 1 animal group 4)
acute cortical single cell necrosis (1 animal group 4)
Mandibular lymph nodes: acute focal blood resorption (2 animals group 1; 2 animals group 4)
focal vacuolaticn of cortical cells (1 animal group 4)
focal single cell necrosis (1 animal group 4)

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

female reproductive system

Organ:

other: increase in corpus lutea

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Hematological parameters showing statistically significant differences to the control group which are not considered to be compound-related effects

Parameter	Change	Group	Sex	Day	p-value	Reason
MCHC	↑	2	M	24	p<0.05	R, D
Reticulocyte count (rel + abs)	↑	4	F	24	p<0.05	R
Leucocyte count	↑	3	F	24	p<0.05	R, D
Lymphocyte count	↑	3	F	24	p<0.05	R, D
R: values within the historical-reference range D: lack of dose-dependence ↑: increase ↓: decrease

Biochemical parameters showing statistically significant differences between control and treatment groups that were not considered compound-related

Parameter	Change	Group	Sex	Day	p-value	Reason
Urea nitrogen	↑	3	M	24	p<0.05	R, D
Glucose	↑	3	M	24	p<0.05	R
Albumin, rel.	↓	4	F	24	p<0.05	R
Albumin, abs.	↓ ↓	4 4	M F	24 24	p<0.05 p<0.05	R
Total β-globin, rel.	↑	4	F	24	p<0.05	R
Total γ-globin, abs.	↓	4	F	24	p<0.05	R
R: values within the historical-reference range D: lack of dose-dependence ↑: increase ↓: decrease

Macroscopic findings observed in controls and ZK 38.199-treated animals which were sacrificed at study termination

Organ/Finding	Group 1		Group 2		Group 3		Group 4
	Control		100 mg/kg		300 mg/kg		1000 mg/kg
	10M	10F	10M	10F	10M	10F	10M	10F
Kidneys
- cysts, watery		1
- pelvic dilatation	1
- reduced in size		1
Ovaries
- cystic dilatation				1
- enlarged								1
Skin
- areas with alopecia				1		1
- thinning of fur								2
Prostate
- diminished in size							1
Seminal vesicles
- diminished in size							1
Spleen
- thickened								1
Body cavities
- nodules				1

Changes in absolute and relative organ weights in animals treated with ZK 38.199 in comparison to the controls

Organ weight	Group 2 100 mg/kg		Group 3 300 mg/kg		Group 4 1000 mg/kg
	M	F	M	F	M	F
Terminal body weight					↓*	↓ n.s.
Liver (absolute) Liver(relative)					↑**	↑ n.s.   ↑**
Kidneys (relative)						↑*
Heart (relative)				↑*
Lungs (relative)		↑*				↑**
Spleen (absolute) Spleen (relative)					↑ n.s.   ↑**	↑**   ↑**
Thymus (absolute) Thymus (relative)		↓   ↓		↓	↓   ↓	↓   ↓
Pituitary gland (relative)					↑*
Adrenal glands (relative)					↑*
Brain (relative)					↑**
Pancreas (relative)						↑*
Submandibular gland (relative)					↑*	↑*
Ovaries (absolute) Ovaries (relative)						↑ n.s.   ↑**
↑: increase ↓: decrease * =  significantly different from the control group with p < 0.05 ** = significantly different from the control group with p < 0.01 n.s. = not significant

Microscopic findings in sacrificed animals that are considered as clear or suspected effects of the test item

Organ/Finding		Group 1 control		Group 2 100 mg/kg		Group 3 300 mg/kg		Group 4 1000 mg/kg
	Grade	M n=10	F  n=10	M n=10	F  n=10	M n=10	F  n=10	M n=10	F  n=10
Spleen vacuolation of macrophages in red pulp	1	2	1		2	2	2	2	3
	2	1		1			1	3	2
	3								2
- hemopoiesis increased in red pulp	1	1					2	1
	2	1	1		1			2	3
- focal cell degeneration in red pulp	1							3
	2								2
- megakaryocytosis in red pulp	2							1	1
	3								1
Ovaries
- increased number of corpora lutea	2		1		3		5		5
	3		2				1		3
	4								2
Liver
- focal granulomatous inflammation, partly centrilobular	1	7	7	4	6	3	5	5	5
	2	3	2	3	2	4	3	4	5
	3			1		2		1
Grading: 1 = minimal; 2 = slight; 3 = moderate; 4 = marked

 

 

Applicant's summary and conclusion

Conclusions:

In a study conducted according to OECD test guideline 407 (1981) male and female Wistar rats were administered Aminodioxepan at doses of 100, 300, and 1000 mg/kg bw for at least 28 days. Several changes in hematology, blood biochemistry, organ weights and gross necropsy findings occurred which are difficult to adress. However, adverse effects are considered to be seen at 300 mg/kg bw upwards and thus the NOAEL was determined at 100 mg/kg bw.

Executive summary:

In a subcacute toxicity study according to OECD test guideline 407 (1981) Aminodioxepan (100% a.i.)] was administered to 10 Wistar rats /sex/dose by gavage at dose levels of 0, 100, 300, and 1000 mg/kg bw/d.

 

Several changes were noted during this subacute toxicity study, e.g.: reddening and swelling of the skin following the first treatment with 1000 mg test item, increase of the spleen, occurrence of a megakaryocytosis in three animals of the high dose group, hematological findings of slight anemia and elevated reticulocyte counts in male animals at 1000 mg/kg, also focal vacuolation of macrophages in the red pulp of the spleen could be detected with increasing incidence. Corpora lutea were increased in the 300 mg/kg and the 1000 mg/kg treatment group, however, alterations in other female genital organs were not observes thus indicating that the estrous cycle is not affected. Furthermore, focal inflammatory infiltration in the liver with granulomatous appearance was detected with high incidence in all groups. Considering the slightly elevated mean severity of this finding in the high dose group and the corresponding change in serum enzyme levels (GPT, AP) as well as in biochemical parameters indicatinga disturbance of the metabolic functions of the liver (serum glucose, cholesterol and total protein), this change was considered to be a compound-related effect but to be a non-specific change in the animals due to high loading of the organism with a foreign compound.

A compound-related decrease in thymus weight in both sexes of the high dose group was not correlated with a microscopic finding. Lymphocytic degeneration, although apparent in 18 animals of the high dose group could be observed in the control group with approximately the same frequency (17 animals). Even the mean severity did not differ between the two groups. Thus, the relevance of this organ weight change remains doubtful.

Based on the present results a NOAEL of 100 mg/kg bw/d is assumed for Aminodioxepan in male and female Wistar rats.