4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane

Endpoint summary

Description of key information

Study conducted according to OECD test guideline 407; 6 female/male Wistar rats were administered Trioxabicyclooctane at doses of 0, 40, 200 and 1000 mg/kg bw/d for 28 consecutive days, result: NOAEL= 40 mg/kg bw/d

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr. to May 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 25 July 1995

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han: WIST (SPF)

Details on species / strain selection:

Rats were selected as test species because they are standard laboratory animals and the species of choice for rodent toxicology studies to evaluate the potential of new drugs or chemicals. In addition, background 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
- Weight at study initiation: 167 - 190 g male and 125 - 166 g female animals
- Fasting period before study: not specified
- Housing: conventionally, 1 animal per cage; Makrolon® type Ill with embedding
- Diet (e.g. ad libitum): pulverized Aaromin® R ad libitum 24 hours per day, in metal boxes
- Water (e.g. ad libitum): demineralized, acidified water, pH 2-3, in water bottles ad libitum 24 hours per day
- Acclimation period: ≥ 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-24
- Humidity (%): 50-58
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

physiological saline

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in vehicle consisting of 0.9 g sodium chloride per 100 mL bidistilled water. The solution for the dose group receiving 1000 mg test item/kg was formulated at a concentration of 100 mg test item/mL vehicle freshly every day before application. The solution for the dose group receiving 200 mg test item/kg was formulated by diluting the freshly prepared formulation with 100 mg test item/mL in the ratio 1:5 resulting in a concentration of 20 mg test item/mL. This formulation was further diluted in a ratio of 1 :5 to produce a formulation with the concentration of 4 mg test item/mL for the group receiving 40 mg test item/kg.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.9% NaCl in bidistilled water
- Concentration in vehicle: 100, 20 and 4 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

no

Remarks:

The accuracy of preparing formulations could not be analytically confirmed, since unexplained peaks appeared in the chromatogrammes, presumably representing the impurities or degradation products.

Details on analytical verification of doses or concentrations:

The accuracy of preparing formulations could not be analytically confirmed, since unexplained peaks appeared in the chromatogrammes, presumably representing the impurities or degradation products. However, the validity of the study is not comprimized, since the formulation was prepared freshly prior to each application according to established procedures and at the used pH (approx. 6) the hydrolysis half-life is assumed to be > 1 day extrapolated from the hydrolysis study, where T1/2 was < 0.01 d and 22 days for pH 4 and 7 respectively.

Duration of treatment / exposure:

28-29 days

Frequency of treatment:

daily

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

200 mg/kg bw (total dose)

Dose / conc.:

40 mg/kg bw (total dose)

Dose / conc.:

0 mg/kg bw (total dose)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As in an acute toxicity test a dose of 2000 mg test item/kg was tolerated with only minor signs of intoxication (slight apathy, ruffled fur) (Scantox Lab. No. 11449), in the present study 1000 mg test item/kg were selected as the maximum dose because this dose is recommended as top dose by OECD guideline 407, adopted 27 Jul. 95.
- Fasting period before blood sampling for clinical biochemistry: 18 h

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked regularly during the application in the morning and thereafter in the afternoon. On weekends a second checking was done just before the technicians left the laboratory.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded weekly and evaluated until day 27 and body weight gain was calculated over this period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The amount of food consumed was recorded weekly and evaluated until day 27.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Yes
- Time schedule for examinations: The quantity of water consumed by the individual animal was recorded weekly and evaluated until day 27.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were carried out on day 27 in all animals. The pupils of all animals were dilated using a mydriaticum (Mydriaticum Stulln®, 0.5%) and the eyes examined with the naked eye (magnifying glass), an ophthalmoscope (Oculus-Visuskop, W. Okulus) and a hand-slit-lamp microscope (Kowa SL 5, Kowa Company, Japan).
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 28
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: glutamic-pyruvic-transaminase (GPT}, alkaline phosphatase (ALP) on days 2 and 28, total cholesterol, glucose, urea nitrogen, sodium, potassium, calcium, chloride, total protein and serum protein electrophoresis on day 28.
- Animals fasted: Yes on day 28
- How many animals: all

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: The following parameters were determined in spontaneous urine samples collected over a period of 24 hours from 6 male and 5 female animals per group on day 23: urinary volume, pH, protein, glucose, blood, ketones, urobilinogen and bilirubin using the Combur-9-RL-test (Boehringer), specific gravity using a densitymeter (DMA35) and sediment analysis using a microscope.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The functional tests of the nervous system were done on day -2 (prevalues) and on days 19 to 20. The function of the nervous system was tested by stimulation of certain effector-organs and the following reactions were evaluated:
pupillary light reflex, consensual pupillary light reflex, cornea:1 and palpebral reflex, flexor reflex, placing reflex, righting reflex, balancing test, grasping reflex, sensitivity of skin and head shaking reaction.
- Dose groups that were examined: all

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

Optional endpoint(s):

Optional endpoints: No

Statistics:

The Dunnett-test was used to assess differences between group 1 (control) and the treatment groups. Group mean values which differ significantly from the control group are marked by *{p < 0.05) or **(p < 0.01 ).
Because of coagulation of blood samples, the number of female animals of group 4 on days 26-30 was not sufficient for statistical evaluation. Thus, the results of these animals were interpreted by comparison of the individual values to control values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the group receiving a dose of 1000 mg test item/kg, the following findings occurred, which were considered to be compound related: flat-footed gait (2 males), diminished muscular tone (4 male, 3 female) and slightly increased sialorrhea (2 males, 1 female).
All other findings such as changes in the skin (thinning or ruffled fur, scab formation of necrosis) were not regarded as being compound-related because there was no dose-relationship, the findings were sporadic or occurred also in the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a compound-related decrease in body weight gain from a dose of 200 mg test item/kg onwards in males (non-significant to p < 0.01) and in females (p < 0.05 to p < 0.01).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A compound-related signiticant (p < 0.01) decrease in mean food consumption (27-32%) was observed in group 4 in both sexes.

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

effects observed, treatment-related

Description (incidence and severity):

A non-significant increase in mean drinking water consumption was found in both sexes of group 4.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No clear compound-related effects were observed.
The following changes, observed on day 28:
• a decrease in erythrocyte count (p < 0.05) in female rats of group 4
• an increase in relative reticulocyte count (p < 0.05 to p < 0.01) ,in female animals of groups 3 and 4
• an increase in reticulocytes absolute (p < 0.01) in fema,le animals of group 4, were regarded as being of minor biological relevance at the doses tested, as the values remained within the 90% reference range (reticulocytes) or in the minimum/maximum range (erythrocytes).
Except for shortening of thrombin time in male (p < 0.01) and female animals (visual comparison to control group due to low number of samples) and activated partial thromboplastin time in male animals (p < 0.01 ), observed only in the high dose group on days 26-30, no other compound~related effects were observed. The statistically significant shortening (p < 0.01.) of thromboplastin time observed in male animals of the mid dose group on days 26-30 is not considered to be compound-related because of a lack of dose-dependence.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Expect for a slight increase in serum calcium (p < 0.01) in the female animals of group 4, and slight changes in serum protein-electrophoresis such as slight decreases in a-globulins associated with a slight increase in a 1 lbumin from group 3 onward!, in male animals and group 4 with an additionally slight decrease in y-globulins in female animals, no other compound-related effects were observed after treatment with ZK 39294 over 28 days in rats.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Except for a slight significant increase in urinary volume (p < 0.01) with a slight decrease in specific gravity (p < 0.05) observed in female animals of the high dose group on day 23, no other compound-related effects were observed. The statistically significant increase of pH-value in female animals of group 4 on day 23 was not considered as being compound-related, because the values remained within the minimum/maximum reference range .. None of the other findings in urine or urinary sediment observed in animals of the treatment groups were considered to be compound-related, because similar findings were also seen in the control animals.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In the balancing test, both sexes in the group receiving a dose of 1000 mg test item/kg showed a non-significant to significant (p < 0.05) reduction in time of movement on the rotating rod. One male (41 M) also failed in another balancing test. Additionally, 4 males (37M, 39M, 40M and 42M) showed failure in a placing reaction test {raising of hindlegs onto the table when hanging by forelegs). These changes were regarded as compound-related.
Lack of single attitude reactions in single animals of different groups (group 2: 24F, group 3: 28M, 36F) or of a single sensitivity reaction in one male in group 3 (26M) was not regarded as compound-related, but as by a chance finding because of the lack of dose-dependence.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Only in animals of the high dose group were treatment-related changes in body and organ weights observed. Dramatic decreases in body weights (males and females p < 0.01), absolute spleen weights (males and females with p < 0.01) and absolute thymus weights (males with p < 0.01 and females with p < 0.05) were considered as being treatment-related effects. Moreover, due to the treatment-related drastically retarded growth in all high dosed animals (reduction in body weights after exsanguination of males ca: 33% and of females ca. 23% when compared to controls), a decrease in absolute weights of the testes (p < 0.05), seminal vesicles (p < 0.01) and epididymides (p < 0.01) was found (histologically these findings corresponded to a remarkable maturation arrest in these organs). Although the female genital organs were histologically unaffected, the statistically significant decrease in absolute uterus weights was also considered as being treatment-related. Additionally, a treatment-related reduction, in absolute weights of the adrenal glands (p < 0.05) was observed in females. Moreover, decreases in absolute and relative weights of the pituitary gland were more pronounced in females (p < 0.05 to p < 0.01) than 1 in males (p < 0.05). The increases in relative weights of kidneys (in males and females with p < 0.01 ), liver (in males and females with p < 0.01 ), lung (in males with p < 0.05; in females with p < 0.01 ), brain (in males and females with p < 0.01 ), pancreas (only in females with p < 0.01 ), testes (in males with p < 0.01 ), thyroid gland and heart (only in males with p < 0.05 respectively) as well as adrenal glands (in males with p < 0.01; in females n.s.) were also considered as being indirect effects, due to the decreased body weights in both genders.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Only in high-dosed animals were treatment-related microscopic findings observed.
Maturation arrest with hypospermatogenesis in the testes of 6/6 male animals associated with oligospermia and the presence of cellular debris in the epididymides of 6/6 male animals and combined with a reduced secretory activity in the seminal vesicles in 2/6 males was considered as being a treatment-related indirect effect, which was interpreted as a consequence of the dramatically reduced body growth. Furthermore, an increase in incidence and severity of thymic atrophy in 5/6 males and 2/5 females combined with an increase in incidence and severity of the pyknosis and phagocytosis of lymphocytes (6/6 males and 6/6 females) associated with a slight lymphoid atrophy in 1/6 males and 1/6 females in the spleen was considered as being treatment-related. These findings did not reflect a direct lymphotoxic effect of the compound but represented clearly stress-related changes.

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

organ weights and organ / body weight ratios

sperm measures

water consumption and compound intake

Key result

Critical effects observed:

not specified

Conclusions:

The intragastric application of Trioxabicyclooctan at a daily dose of 40 mg/kg body weight was well tolerated over a period of 4 weeks without any compound-related findings.

Executive summary:

In an oral repeated dose toxicity study over an experimental period of 28 days according to OECD test guideline 407 (1995) 6 female/male Wistar rats were administered Trioxabicyclooctane at doses of 0, 40, 200 and 1000 mg/kg bw/d for 28 consecutive days.

From a dose of 200 mg test substance/kg onwards slight adverse effects occurred. A non-significant to significant dose-dependent decrease in body weight gain was observed in both sexes, but it was more pronounced in females. There was also a slightly significant decrease in α-globulins (total α-globulins and α-1-globulins) associated with a slightly non-significant to significant increase in albumin and albumin/globulin-quotient in male animals at the end of the study. However, effects on alpha-globulins (typically in relation to nephropathy) are known to be specific for male rats and not relevant for humans. Since the serum findings in this study were only observed in male rats it is assumed to be not relevant as well.

In the group receiving a dose of 1000 mg test substance/kg, different clinical signs of the deteriorated general condition of the animals were found: significantly reduced food consumption, diminished muscular ,tone, flat-footed gait, slightly increases sialorrhea, significant reduction in time of movement on the rotating rod in a balancing test, failures in another balancing test or in a placing test.

Signs of drastically retarded growth and distress of the animals were the significantly lower body weights at the end of the study and the significant decrease in absolute spleen and thymus weights (histologically represented by an atrophy combined with a lymphocytic pyknosis) as well as in absolute weight of the pituitary and adrenal glands (F). Due to the retarded growth, a spermatogenic maturation arrest was found in male rats indicated by changes such as hypospermatogenesis and oligospermia associated with a significant decrease in absolute weights of the testes, seminal vesicles and epididymides. Correspondingly, in female rats, the absolute weight of the uterus was significantly reduced. Besides the retarded growth and the signs of distress and reduced general condition, some minor changes were found such as increased water consumption and increased urinary volume, impairment in blood coagulation and increase in serum calcium, but as no specific organ-toxic effects were observed, definite target organs could not be identified.
The determination of creatinine in the serum was omitted. However, the evaluation of the biochemical data did not reveal significant changes that point to renal injury subsequently to administration of the test item. In case of severe renal injury an increase in urea nitrogen would be expected. Additionally, the urinalysis did not reveal clinical findings that indicate severe renal damage. The slight increase in urinary volume and the slight decrease in specific gravity of the urine of female animals of the high dose group on day 23 are considered to be of minor biological relevance and are not indicative for severe kidney damage. Futhermore, the microscopic examination of the kidneys did not reveal any histopathological changes. Therefore, the lack of creatinine analysis does not invalidate the study. 

Based on these results the NOAEL for Trioxabicyclooctane is 40 mg/kg bw/da in female and male Wistar rats under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

Guideline study, high reliability

System:

other: body weight, body weight gain, food and water consumption, organ weights, neurobehavior

Organ:

adrenal glands

liver

pituitary gland

seminiferous tubules

spleen

testes

thymus

uterus

other: neurobehavior

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an oral repeated dose toxicity study over an experimental period of 28 days according to OECD test guideline 407 (1995) 6 female/male Wistar rats were administered Trioxabicyclooctane at doses of 0, 40, 200 and 1000 mg/kg bw/d for 28 consecutive days.

From a dose of 200 mg test substance/kg onwards slight adverse effects occurred. A non-significant to significant dose-dependent decrease in body weight gain was observed in both sexes, but it was more pronounced in females. There was also a slightly significant decrease in α-globulins (total α-globulins and α-1-globulins) associated with a slightly non-significant to significant increase in albumin and albumin/globulin-quotient in male animals at the end of the study. This is assumed to be a male rat specific effect, less relevant for humans.
In the group receiving a dose of 1000 mg test substance/kg, different clinical signs of the deteriorated general condition of the animals were found: significantly reduced food consumption, diminished muscular ,tone, flat-footed gait, slightly increases sialorrhea, significant reduction in time of movement on the rotating rod in a balancing test, failures in another balancing test or in a placing test.

Signs of drastically retarded growth and distress of the animals were the significantly lower body weights at the end of the study and the significant decrease in absolute spleen and thymus weights (histologically represented by an atrophy combined with a lymphocytic pyknosis) as well as in absolute weight of the pituitary and adrenal glands (F). Due to the retarded growth, a spermatogenic maturation arrest was found in male rats indicated by changes such as hypospermatogenesis and oligospermia associated with a significant decrease in absolute weights of the testes, seminal vesicles and epididymides. Correspondingly, in female rats, the absolute weight of the uterus was significantly reduced. Besides the retarded growth and the signs of distress and reduced general condition, some minor changes were found such as increased water consumption and increased urinary volume, impairment in blood coagulation and increase in serum calcium, but as no specific organ-toxic effects were observed, definite target organs could not be identified.
The determination of creatinine in the serum was omitted. However, the evaluation of the biochemical data did not reveal significant changes that point to renal injury subsequently to administration of the test item. In case of severe renal injury an increase in urea nitrogen would be expected. Additionally, the urinalysis did not reveal clinical findings that indicate severe renal damage. The slight increase in urinary volume and the slight decrease in specific gravity of the urine of female animals of the high dose group on day 23 are considered to be of minor biological relevance and are not indicative for severe kidney damage. Futhermore, the microscopic examination of the kidneys did not reveal any histopathological changes. Therefore, the lack of creatinine analysis does not invalidate the study. 

 

Justification for classification or non-classification

Based on these results the NO(A)EL for Trioxabicyclooctane is 40 mg/kg bw/da in female and male Wistar rats under the conditions of the test. However, no specific target organs could be identified and the NO(A)EL is based on serum protein changes (a-globulin) in 200 mg/kg group in males only which are considered to be male rat-specific and less relevant for humans.

In absence of any histopathological findings in 40 & 200 mg/kg groups, no STOT-RE classification according to Regulation (EC) No. 1272/2008 (CLP) is warranted.