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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
(RCC Ltd.)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,3,5-Trioxane
- Physical state: white solid
- Analytical purity: 99 %
- Lot/batch No.: 12.12.01 1300
- Expiration date of the lot/batch: 12 June 2002
- Stability under test conditions: stable under storage conditions
- Storage condition of test material: in refrigerator (at 2-8 °C) in the original container, away from direct sunlight

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Biotechnology & Animal Breeding Division, Switzerland
- Weight at acclimatization: males 150 g (±20%), females 125 g (±20%)
- Housing: 5 per cage
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433
- Water (e.g. ad libitum): Community tap-water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly.
1, 3, 5-Trioxane was weighed into a glass beaker on a tared Mettler balance and the vehicle (bi-distilled water) added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
15 for 0 and 300 mg/kg bw dose groups (incl. 5 for recovery groups);
10 for 30 and 100 mg/kg bw dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 4 weeks (0 and 300 mg/kg bw dose groups; 5 animals/sex/group)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before and weekly thereafter
- Parameters: clinical signs, appearance, motor activity, behavior, respiration, reflexes


FOB: Yes, during week 13 and 17 (grip strength and locomotor activity)


BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during pretest, treatment and recovery periods


FOOD CONSUMPTION:
- Food consumption for each animal determined Yes, weekly


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 13/17 week
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 13/17 week
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin, concentration, Platelet count, Reticulocyte count, Reticulocyte tluorescence ratios, Nucleated erythrocytes (normoblasts), Heinz bodies, Methemoglobin, Total leukocyte count, Differential leukocyte count, Red blood cell morphology, Thromboplastin time (=prothrombin time), Activated partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 13/17 week
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin, total Cholesterol, total Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl transferase, Calcium, Phosphorus, Sodium, Potassium, Chloride, total Protein, Protein electrophoresis, Globulin, Albumin/Globulin ratio.


URINALYSIS: Yes
- Time schedule for collection of urine: 13/17 week
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Volume (18-hour), Specific gravity, Osmolality, Color, Appearance, pH, Protein, Glucose, Ketone, Urobilinogen, Bilirubin, Blood and Sediment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Samples of the tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution for microscopic examination. The organ weights were recorded for spleen, testes, epididymides, ovaries, thymus, kidneys, adrenals, uterus, brain, heart, liver, thyroids w/parathyroids. The organs to terminal body weight ratios as well as organ to brain weight ratios were determined.
Statistics:
The Dunnett-test was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Student's t-test was applied to grip strength and locomotor activity.
Fisher's exact-test was applied to the macroscopic findings.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY:
All animals survived until scheduled necropsy. No test item-related clinical signs of toxicological relevance were noted at any dose level.


FOB:
No test item-related changes were noted during the functional observational battery (week 13 and 17).


BODY WEIGHT AND WEIGHT GAIN:
From treatment day 29 onwards, the mean body weights of the male rats treated with 300 mg/kg bw/day was slightly less than that of the control males. Although the mean body weights were slightly lower from days 29 to 85, the reduction noted on day 91 attained statistical significance (p<0.05). The lower body weights were also noted during the recovery period; the reduction noted on day 8 of recovery also attained statistical significance (p<0.05). The reduction in body weight in the 300 mg/kg bw dose group on day 91 or on day 8 of recovery period was below 7% in comparison to controls.
From treatment day 15 onwards, the mean body weight gain of the male rats treated with 300 mg/kg bw/day was slightly lower than that of the control males. From treatment day 71-91, the differences attained statistical significance (p<0.05) (9-11 % lower). The reduced mean body weight gain continued during the recovery period (8-11 % lower).
The mean daily body weights and the mean body weight gain of the test item-treated females compared favorably with those of the controls during treatment and recovery.


FOOD CONSUMPTION:
The mean daily food consumption and the mean relative food consumption of the test item treated rats were similar to those of the controls during the treatment and recovery periods.


OPHTHALMOSCOPIC EXAMINATION:
A small number of typical findings were noted in control and test item-treated rats. None were considered to be related to the treatment with the test item.


HAEMATOLOGY:
After 13 weeks treatment, a shift in the reticulocyte maturity indices (towards older, low fluorescent reticulocytes) was noted in males treated with 300 mg/kg bw/day (Table 1). These differences were accompanied by slightly elevated but statistically insignificant differences in absolute and relative reticulocyte counts. The high and mid reticulocyte fluorescence ratios were -38 and -25 %, respectively, and the low reticulocyte fluorescence ratio was +22 % in males of the 300 mg/kg bw dose group. These findings were considered to be test item related.
No test item related changes were seen in the females after the treatment or recovery periods.
The differences seen in the males treated with 300 mg/kg bw/day were reversible after 4 weeks recovery, and no further differences were noted in any other parameter.


CLINICAL CHEMISTRY:
Marginally higher gamma glutamyltransferase activity levels were seen in males treated with 300 mg/kg bw/day after 13 weeks treatment. This finding was reversible after the 4-week recovery period, and was considered to be due to metabolic adaptation. No further test item-related changes were noted after the treatment and recovery periods.


URINALYSIS:
No test item-related differences were noted in the urinalysis parameters after 13 weeks treatment and 4 weeks recovery.


ORGAN WEIGHTS:
There was a significant decrease in the mean absolute spleen weight (-18 %) and the mean spleen-to-brain weight ratio (-18 %) of males treated with 300 mg/kg bw/day after the 13-week treatment period (Table 2). Though the mean spleen-to-body weight ratio was not statistical significant in comparison to control, there was 12% decrease. These changes coincided with microscopical changes noted in the spleen and were considered to be test item-related.
The mean kidney-to-brain weight ratio of the test item-treated males was also significantly lower (p<0.05) in males (9 % decrease) treated with 300 mg/kg/day after 13 weeks treatment. Insofar as the kidney-to-body weight ratio of these males compared favorably with those of the control males, these changes were considered to be incidental.
All other organ weights and organ weight ratios compared favorably with those of the respective controls.


GROSS PATHOLOGY:
All macroscopical changes noted after 13 weeks treatment and 4 weeks recovery were considered to be typical background changes commonly seen at necropsy in rats of this strain and age and therefore incidental.


HISTOPATHOLOGY: NON-NEOPLASTIC:
After 13 weeks of treatment, evidence of a possible test item-related effect was noted in the spleen. Minimal brown (hemosiderin) pigment was recorded in four of ten males dosed at 300 mg/kg bw/day. There was no evidence of an effect of treatment in females. After the 4-week recovery period, there was no evidence of an effect of treatment.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no adverse effects in highest dose level tested
Dose descriptor:
NOEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: reversible decrease of spleen weight

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Reticolocyte count in males

Dose

(mg/kg bw)

RETIC. %

RETIC. T/L

HFR %

MFR %

LFR %

NEN/100 WBC

HEINZ BOD. (0/00)

After 13 weeks

0

2.92

0.2564

12.5

30.8

56.7

1.3

0

30

3.08

0.2669

12.0

28.5

59.5

1.5

0

100

3.12

0.2704

11.4

28.2

60.4

1.5

0

300

3.46

0.3157

7.7**

23.1**

69.2**

1.3

0

After 17 weeks

0

2.78

0.2453

14.8

25.9

59.3

2.2

0

300

2.92

0.2528

15.3

24.0

60.6

3.0

0

RETIC. %: Reticulocyte count (rel.)     

RETIC. T/L: Reticulocyte count (abs.) 

HFR: Reticulocyte fluorescence ratios, high

MFR: Reticulocyte fluorescence ratios, meddle

LFR: Reticulocyte fluorescence ratios, low

NEN: Nucleated erythrocytes (normoblasts)

HEINZ BOD.: Heinz bodies

**: significance at 1% level

 

Table 2. Spleen weight (Mean±SD) in males

Dose

(mg/kg bw)

Absolute weight (g)

Organ/body weight ratio

Organ/brain weight ratio

0

0.739±0.135

0.185±0.037

36.309±6.417

30

0.736±0.082

0.185±0.023

37.113±2.798

100

0.739±0.133

0.183±0.025

35.828±6.571

300

0.607±0.072*

0.162±0.021

29.779±3.203*

*: significance at 5% level

Applicant's summary and conclusion