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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991-10-10 to 1991-11-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study; GLP study; scientifically well performed study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF-Zucht
- Age at study initiation: ca. 6 months
- Housing: In groups of five in Makrolon type-4 cages, in air-conditioned room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Duration of treatment / exposure:
28 days
Frequency of treatment:
once per day
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 62.5, 250, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during working days. Once daily during weekends or holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice twice daily during working days. Once daily during weekends or holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100g body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Once weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once weekly.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of the test.
- Anaesthetic used for blood collection: Yes (Ketanest)
- Animals fasted: Yes
- How many animals: All
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, MCV, MCH, MCHC, Leukocyte count, Differential leukocyte count, Reticulocyte count, Heinz bodies, Prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after the collection of blood.
- Animals fasted: Yes
- How many animals: All.
- Parameters checked: Sodium, Potassium, Phosphorus, Uric acid, Total bilirubin, Direct bilirubin, Creatinine, Serum glucose, Uric nitrogen, Calcium, Chloride, ASAT, ALAT, AP, CGT, Total protein, Albumin.

URINALYSIS: Yes
- Time schedule for collection of urine: On the night of test day 26/27.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Appearance, color, pH, Haemoglobin, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Density, Sediment.

NEUROBEHAVIOURAL EXAMINATION: no data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Reduced throbocytes in females at 1000 mg/kg bw
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
reduced alkaline phosphatase in males and females at 1000 mg/kg bw
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver weight increas for females at 1000 mg/kg bw; reduced tested and reduced thymus in males at 1000 mg/kg bw
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
findings in testes and thymus at 1000 mg/kg bw
Details on results:
BODY WEIGHT AND WEIGHT GAIN
At the beginning of the test, significant difference of body weight was observed in female animals at 62.5 mg/kg bw. No difference was observed in other groups. This is not considered to be treatment related because there is no dose-effect relationship.

HAEMATOLOGY
The clotting time was decreased in male animals of all treated groups without dose-effect relationship. The thrombocyte counts were slightly decreased in female animals at 1000 mg/kg bw.

CLINICAL CHEMISTRY
At 1000 mg/kg bw, the AP activity was significantly decreased, and the Creatine values were increased. In male animals at 62.5 mg/kg bw, the phosphorus values were signficantly increased. In male animals at 250 mg/kg bw, the sodium values were decreased. In male animals of 62.5 and 250 mg/kg bw , the calcium levels were increased and the urea levels were decreased. All the effect showed no dose-response relationship.

ORGAN WEIGHTS
In male and female animals at 1000 mg/kg bw, the thymus weights were decreased. In males the tested weight was decreased as well.

HISTOPATHOLOGY: NON-NEOPLASTIC
Hign dose group:
In the thymus of 4 males and 2 females, narrowed and loosend cortex with high reduction of lymphocytes were observed. In testes of 2 males, degradation of germinal epithelium and increased single cell necrosis were found. The number of matured sperm cells was significantly reduced.

Effect levels

Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: clinical chemistry; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Justification of use of the reported study as key study for repeated dose toxicity:

The reported study is qualified to be used as key study for the repeated dose toxicity based on the metabolism, kinectis and mode of action considerations.

It has been shown that tetraglyme induces toxicity via systemic exposure to toxic metabolites 2-methoxy ethanol and 2-methoxy acetic acid, indicating that the repeated dose toxicity of tetraglyme is the result of systemic exposure to steady state toxic metabolites. Considering that tetragylme undergoes extensive metabolism and its toxic metabolites are highly hydrophilic, it can be derived that the 28-day exposure is likely to reflect the steady state toxic metabolites and thus the subchronic/chronic toxicity.

The steady-state toxic metabolites premises no metabolic overload. According to the reported 28 -day study, tetraglyme did not induce any effects at dose levels of 62,5 and 250 mg/kg bw and it is not likely that a metabolic overload could occur upon prolonged exposure. The NOEL obtained in this study is considered to reflect the NOEL of subchronic/chronic toxicity.

In conclusion, the available data (physico-chemical data, metabolism/kinetic data, mode of toxicity, 28-day toxicity study) supports that the results obtained the 28-day study on tetraglyme can be used as key study for the endpoint repeated dose toxicity.

Applicant's summary and conclusion

Conclusions:
The no observed effect level (NOEL) after 28-day exposure of rats is determined to be 250 mg/kg bw/day. The NOEL obtained in this study is considered to be valid for subchronic/chronic toxicity in combination of data on metabolism, kinetics, and mode of action of glymes.
Executive summary:

Tetraglyme was administereddaily by oral gavage to Wistar rats of male and female at dose levels of 62.5, 250 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle - water only.

In all the groups, the behavior, clinical sign, body weight, food consumption and water consumption were recorded. The Haematology and clinical chemistry analyses, and urinalysis tests were performed at the end of the treatment. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues.

All animals survived during the treatment period. No clinical signs, body weight, food and water consumption changes were noted. Haematological analysis showed that the thrombocyte counts were slightly decreased in females of high dose group. The clinical chemistry test showed that the decreased AP activity and elevated creatinine were found in both males and females at 1000 mg/kg bw. The urinalysis showed no compound related toxicity. In male and female animals of high dose group, the thymus weights were decreased.

Histopothology investigation showed several treatment related effect in animals of high dose group. In the thymus of 4 males and 2 females, narrowed and loosened cortex with high reduction of lymphocytes were observed. In testes of 2 males, degradation of germinal epithelium and increased single cell necrosis were found. The number of matured sperm cells was significantly reduced.

Based on the effects of clinical chemistry, organ weight and histopathology, the no observed effect level (NOEL) of the test item is 250 mg/kg bw/day for male and female rats.

The reported 28 -day study is considered to be qualified to be used as key study for the repeated dose toxicity together with the data on metabolism, kinectics and mode of action of glymes. It can reasonably assumed that the NOEL obtained in this 28-day reflect the steady state of toxic metabolites and thus the NOEL of subchronic/chronic toxicity.