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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP; guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: 84/449/EWG, B.7; OECD 407
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): triethylene glycol divinyl ether (alternatively named TEGDVE)
- Physical state: clear light brown liquid
- Lot/batch No.: Lot 87/1 - 87/2 - 87/3
- Storage condition of test material: ambient temperature

Test animals

Species:
rat
Strain:
other: Charles River Crl : CD (SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Kent, UK
- Age at study initiation: 28 ± 1 days
- Weight range at study initiation: 65 to 85 g
- Housing: 5
- Diet: Labsure LAD 1 Diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean maximum of 21.4 °C and a mean minimum of 18.9 °C.
- Humidity (%): 71.1 %
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 h/ 12h


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: SCMC (Sodium carboxymethylcellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The high dosage concentration of TEGDVE was prepared daily as a 7.35% v/v suspension in 0.5% w/v aq. SCMC (Sodium carboxymethylcellulose). The intermediate (2.1% v/v) and low (0.6% v/v) dosage concentrations were prepared by serial dilution of the high dosage concentration with the vehicle.


VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HRfC Department of Analytical Chemistry prior to the start of
Concentration analyses of suspensions prepared for administration on Day 1 were similarly conducted by HRC Department of Analytical Chemistry.
The homogeneity, physical and chemical stability of suspensions of TEGDVE in 0.5% w/v aqueous sodium carboxymethylcellulose (aq. SCMC) were
assessed by HRC Department of Analytical Chemistry prior to the start of treatment.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 60, 210, 735 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
other: yes, 0.5 % w/v aq. SCMC
Details on study design:
- Dose selection rationale: The dosage levels of the test substance were selected on the basis of acute oral toxicity data (HRC Report No. 87978D/GFC 2/AC).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for dead or moribund animals, allowing a post mortem examination at the same day. At weekends the final check was carried out at mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes, ill health, behavioural changes or toxicosis.
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Two days before the start of treatment and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood: prior to termination (week 4), from the orbital sinus
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, overnight
- How many animals: All animals
- The following parameters were estimated by Ortho ELT-1500 analyser, using standard Ortho methodology:
• Packed cell volume (PCV): %
• Haemoglobin (Hb): g/dL
• Red blood cell count (RBC): x10e6/mm³
• Platelet count (Plts) x10³/mm³
• Absolute indices:
- Mean corpuscular haemoglobin concentration (MCHC), Calculated: Hb (g/dl) x 100 + PCV (%): %
- Mean corpuscular volume (MCV), Calculated: PCV (%) x 10 + RBC (x 10 e6/mm³): fl
• Total white blood cell count (WBC) x10³/mm³
• Differential white cell count (Diff) - by standard microscopy of a blood smear stained with modified Wriqht's stain counting 100 cells:
- Neutrophils (N), (x10³/mm³)
- Lymphocytes (L), (x10³/mm³)
- Eosinophils (E), (x10³/mm³)
- Basophils (B), (x10³/mm³)
- Monocytes (M), (x10³/mm³)

The percentage distribution of each cell type was determined by microscopy. These values were then converted to absolute values by computer. This
inevitably involved a "rounding off' in a proportion of the results and for this reason the measured total WBC may differ slightly from the total of the different cell types.

Cell morphology: No abnormal cells were observed when examining the stained slides.
The following parameter was also estimated:
Thrombotest (TT) - Method of Owren, P.A. (Lancet, 1959, II, 754)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination (week 4), from the orbital sinus
- Animals fasted: Yes, overnight
- How many animals: all animals

- The following parameters were examined by Roche Cobas centrifugal analyser:
• Glucose - using BCL Test Kit (hexokinase mediated): (mg/dL)
• Alkaline phosphatase (AP) - using BCL Test Kit Reaction temperature 30°C: (mU/mL)
• Glutamic-pyruvic transaminase (GPT), also known as 'alanine aminotransferase' - using BCL Test Kit, Reaction temperature 30°C: (mU/mL)
• Glutamic-oxaloacetic transaminase (GOT), also known as 'aspartate aminotransferase' - using BCL Test Kit, Reaction temperature 30°C: (mU/mL)
• Total bilirubin - using BCL Test Kit (mg/dL)
• Cholesterol (Chol) - using BCL Enzymatic Test Kit (mg/dL)

- The following pararneters were estimated by Technicon SMA 12/60 using standard Technicon SMA methodology:
• Urea nitrogen (mg/dL)
• Total protein (g/dL)
• Albumin (g/dL)
• Globulin (g/dL) - by subtraction: Total protein (g/dL) minus Albumin (g/dL).
• Albumin/Globulin ratio (A/G) - by calculation from Total protein und Albumin concentrations.
• Sodium (Na) (mEq/L)
• Potassium (K) (mEq/L)
• Calcium (Ca) (mEq/L)
• Chloride (Cl) (mEq/L)
• Inorganic phosphorus (P) (mEq/L)
• Creatinine (mg/db)

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data


Sacrifice and pathology:
GROSS PATHOLOGY: Yes,
one animal was found dead on day 6 and subjected to a detailed macroscopic examination. The heart was subjected to histological examination. The other major organs were missing and were presumed to have been cannibalised.
After 28 days of treatment (day 29) all remaining animals were randomly killed by carbon dioxide asphyxiation and a complete autopsy undertaken. The macroscopic appearance of the tissues was recorded.

The following organs from each animal killed after four weeks were dissected free of fat and weighed:
liver, adrenals, ovaries, kidneys, testes (with epididymides)

HISTOPATHOLOGY: Yes,
- Samples of the following tissues from all rats in the control und high dosage groups were preserved in 10% buffered formalin for subsequent histological examination:
adrenals, heart, kidneys, liver, spleen any other macroscopically abnormal tissue.

- Fixed-tissue samples required for microscopic examination were embedded in paraffin wax (m.p. 56°C), sections cut at 4 ~m and stained vieh
haernatoxylin and eosin.
Microscopic examinations were carried out for: adrenals, heart, kidneys, liver, spleen any other macroscopically abnormal tissue.

Statistics:
All statistical analyses were carried out separately for males and females.

Body weight data were analysed using weight gains.

The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:
(i) If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%>, the proportion of values different from the mode was analysed by appropriate methods.
Otherwise:
(ii) Bartlett's test (Proc. Roy. Soc. A., 160: 268-282, 1937) was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
(iii) lf no significant heterogeneity was detected, a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks (J. Amer. Statist. Ass., 47: 583-621 and 48: 907-912, 1952/53) was used.
(iv) Analyses of variance were followed by Student's t-test and Williams' test (Biometrics, 27: 103-117 and 28: 519-531, 1971/72) for a dose-related response, although only the one thought more appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the t-test and Williams' test (Shirley's test, (Biometrics, 33: 386-389).
Where appropriate, for organ weight data, analysis of covariance was used in place of analysis of variance in the above sequence. The final bodyweight was used as covariate in an attempt to allow for differences in bodyweight which might have influenced the organ weights.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- No clinical abnormalities were observed for any rat during the four-week treatment period.
- One male rat of the mid dose group (210 mg/kg/day) was found dead on Day 6. No abnormal clinical signs were observed for the animal prior to death. Post mortem examination revealed partial cannibalization. No macroscopic abnormality of the remaining tissues was observed. Similarly, microscopic examination, which was limited to the heart tissue only, revealed no abnormalities. The cause of death was not established; however, it is considered unlikely that the death was related to the administration of TEGDVE.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gains for rats receiving TEGDVE were similar to those of the controls throughout the study poriod.

FOOD CONSUMPTION AND COMPOUND INTAKE
No changes in food consumption were observed.

HAEMATOLOGY
No toxicological relevant changes releated to treatment with TEGDVE were observed.

CLINICAL CHEMISTRY
- For females statistically significantly higher cholesterol levels were observed in the high dose group. The changes may be related to the treatment.
- Higher globulin levels, resulting in higher total protein levels and lower albumin/globulin (A/G) ratios were recorded for males of the mid and high dose group. In the absence of any histological changes in the livers of male high-dosed rats, the observed changes were considered unlikely to be of toxicological importance.
- Statistically significant lower creatinine levels recorded in the high-dosed males were considered not to be of toxicological importance, due to the small magnitude of the shift in this parameter.

ORGAN WEIGHTS
No changes were observed.

GROSS PATHOLOGY
No macroscopic abnormalities were observed, that were considered to be related to the treatment with TEGDVE.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related findings were detected.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
210 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: elevated cholesterol values were observed in females
Dose descriptor:
NOEL
Effect level:
210 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: elevated cholesterol values were observed in females

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

Triethyleneglycol divinylether was administered to rats by intragastric intubation, daily, for twenty-eight consecutive days at dosage levels of 60, 210 and 735 mg/kg bw/day. In comparison with control animals, statistically significantly higher blood cholesterol levels were recorded in the high dose group during week 4 for female rats. Cholesterol levels for several individual female rats in this high dosage group were in the region of, or above the upper limit of the expected parameter range both on initial and on repeat analyses. Therefore, these changes in cholesterol levels may be related to treatment with TEGDVE. No other changes in biochemical parameters were recorded. In all other respects, including general health, bodyweight gains, food consumption, haematology, organ weights, macroscopic and microscopic pathology, rats receiving the test substance were similar to those receiving the vehicle.