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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (according to OECD 422 and GLP)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: particulates

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Atsugi Breeding center)
- Age at study initiation: 8 weeks
- Weight at study initiation: males 309-355 g, females: 206 to 232 g
- Housing: individually; mating period: 1 male, 1 female; 1 litter during lactation
- Diet: autoclaved pellet diet for laboratory animals (CRF-1, Oriental Yeast Industry Co., LTD), ad libitum
- Water: sterile tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % sodium-carboxymethylcellulose (CMC-NA) aqueous solution containing 0.1 % Tween 80
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of test substance : once or twice a week
- Storage temperature of suspension: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the first preparation, the dosing formulations were analyzed by HPLC and it was confirmed that the concentrations were within the target value +/-10%.
Duration of treatment / exposure:
males: from 14 days before mating and throughout the mating period until the day before necroscopy for a total of 37 days
females: from 14 days before mating, through the mating and gestation periods and delivery until day 4 of lactation for a total of 42 to 47 days. Non-delivering females, however, were treated until the day before necropsy.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: highest dose is the upper limit dose level stipulated in the OECD guideline
- Rationale for animal assignment (if not random): stratified-by-weight randomization method on the basis of the body weights to get almost the same mean body weights for each group.
Positive control:
none

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before and after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: days 0,3,7 and 14 during the pre-mating period and once a week thereafter, days 0,7,14 and 20 of gestationand days 0 and 4 of lactation

FOOD CONSUMPTION: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to killing
- Anaesthetic used for blood collection: Yes (thiopental sodium)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count, platelet count, prothrombin time, activated partial thromboplastin time, white blood cell count, differential leukocyte ratio and count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just priot to killing
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: ASAT, ALAT, gamma GT, ALP, total bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, triglyeride, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium, potassium, chlorine


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On the day after the final dosing, all surviving males and females were euthanized by exsanguination from the abdominal aorta after blood sampling, and then were subjected to necropsy.

HISTOPATHOLOGY: Yes
The following organs/tissues of all animals were removed, and fixed and preserved in 10% neutral phosphate-buffered formalin. The testis and epididymis were fixed in Bouin's solution and preserved in 10% neutral phosphate-buffered formalin.
Brain, pituitary, thymus, lymphnode (mandibular and mesenteric), trachea, lung, stomach, intestines (duodenum, jejunum, ileum, cecum, colon, rectum), thyroid and parathyroid, heart, liver, spieen, kidney, adrenal, urinary bladder, testis, epididymis, seminal vesicle (including coagulating gland), ventral prostate, ovary, uterus, vagina, bone marrow (femur), sciatic nerve, spinal cord
As for the histopathological examination, the above-mentioned organs/tissues of all males and females in the control and 1000 mg/kg groups, ovary of non-pregnant females, and any gross lesions of all animals were stained with hematoxylin and eosin by standard methods, and were examined microscopically. However, both sides of the parathyroid could not be examined in 1 male in the control group, and only one side was examined in 4 males in the 1000 mg,/kg group because of loss of the specimen during the preparation.
Statistics:
Multiple comparison test:
Body weight, body weight gain, food consumption, hematology, blood chemistry, organ weight, number of corpora lutea, number of implantations, number of offspring

Kruskal-Wallis test and Dunnett-type multiple comparison test:
Days until copulation, number of estrus stages without copulation, mean estrous cycle length, gestation length, implantation index, delivery index, live birth index, incidence of offspring with external anomaly, viability index an Day 4

Chi-square test: Histopathological findings

Fisher's exact probability test:
Incidence of females with irregular estrous cycles, copulation index, fertility index, gestation index, sex ratio (rnale/fernale), incidence of dams with externally abnormal offspring

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 examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths were found in males or females in any group.
Reddish stool of a similar color to the test substance was observed every day from the day after the start of dosing in all males and females in the treated groups. Besides, transient loose stool was seen only an Day 22 of dosing in 2 males in the 1000 mg/kg group. However, there were no abnormal findings considered to be toxic changes in males or females in any group.

BODY WEIGHT AND WEIGHT GAIN
No significant differences were found in body weights or body weight gain of males or females between the control and treated groups throughout the observation period.

FOOD CONSUMPTION
No significant differences in food intake were found in males or females between the control and treated groups throughout the observation period.

HAEMATOLOGY
In males, a significant shortening of prothrombin time was observed in the 100 mg/kg group. However, it was judged to be incidental, because no change was detected in the 300 or 1000 mg/kg group.
In females, no significant differences were observed in any parameter between the control and treated groups.

CLINICAL CHEMISTRY
In males, A/G ratio in the 1000 mg/kg group was significantly higher than that in the control group. However, since it was a slight change without changes in total protein or albumin, this change was considered to be within physiological variations. Therefore, it was judged not treatment-related.
In females, ASAT (GOT) in the 100 mg/kg group was significantly higher than that in the control group. However, it was judged to be incidental, because no change was found in the 300 or 1000 mg/kg group.

ORGAN WEIGHTS
In males, significant increases in relative liver weights were noted in the 1000 mg/kg group. Besides, significantly lower absolute spleen weights were observed in the 300 mg/kg group, as were significantly higher relative adrenal weights in the 100 and 300 mg/kg groups. However, these changes were judged to be incidental, because no changes in their organs were found in the 1000 mg/kg group.
In females, significant increases in absolute and relative liver weights were noted in the 1000 mg/kg group. There were no changes in any Organs in the 100 or 300 mg/kg group.

GROSS PATHOLOGY
Changes considered to be attributable to the test substance were noted in the gastrointestinal tract.
Reddish-colored contents of a similar color to the test substance was observed in the stomach of 3 males in the 1000 mg/kg group, and the small and large intestines of all mal es and 2 females in the 100 mg/kg group and all males and females in the 300 and 1000 mg/kg groups. Besides, the following findings were observed; however they were judged to be incidental and not treatment-related, because there was no constant trend in their incidences: focal hemorrhage, incomplete contraction and white patch in the lung, tar-like contents in the stomach, white patch in the liver, dilatation of the renal pelvis, distention of the uterus and vagina, and dilatation of the cerebral ventricle.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicological adverse effects observed up to highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

There were no treatment related findings on reproduction. Details are presented in section 7.8.

Data on liver weight are presented in the following table:

 

Dose group (mg/kg bw/d)

Body weight (g)

Absolute liver weight (g)

Relative liver weight (g/100 g b.w.)

Animals per dose group

Females

0

309.9 +/- 20.9

10.486 +/_ 0.886

3.388 +/- 0.228

12

100

305.8 +/- 16.5

10.539 +/- 0.693

3.445 +/- 0.129

11

300

308.0 +/- 12.1

10.956 +/- 0.890

3.562 +/- 0.330

10

1000

306.1 +/- 25.5

11.414 +/- 0.775

3.743 +/- 0.271

12

Males

0

451.7 +/- 32.4

11.628 +/- 1.530

2.566 +/- 0.199

12

100

439.3 +/- 27.4

11.293 +/- 0.901

2.573 +/- 0.146

12

300

440.5 +/- 37.5

11.533 +/- 1.195

2.618 +/- 0.140

12

1000

458.0 +/- 33.5

12.650 +/- 1.429

2.759 +/- 0.179

12

data are presented as mean +/-SD

As the effects on liver weight were only minimal (less than 10%) and neither any changes in clinical chemistry indicative for liver damage nor any histopathological findings were observed in the liver, these effects on liver weight were regarded not to be adverse.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test the only toxic effect observed was an increase in liver weights in males and females in the highest dose group (1000 mg/kg bw/d). As these effects were only minimal (less than 10% weight changes) and not accompanied by histopathological or biochemical changes they were judged not to be adverse. Therefore the no-observed-adverse-effect level (NOAEL) for repeated dose toxicity was 1000 mg/kg in both sexes, under the conditions of this study. There were no effects on reproduction or development.
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according to OECD TG 422. The test item was administered to Sprague Dawley rats (12/sex/dose) by gavage at dose levels of 0, 100, 300 and 1000 mg/kg/day for a total of 37 days (males) and through mating, gestation and delivery until day 4 of lactation (females). No toxic effects were observed in this study except increased liver weights in males and females of the highest dose group. As the effects on liver weight were minimal (less than 10% weight changes) and not accompanied by any histopathological or biochemical changes these effects were judged not to be adverse. The no-observed-adverse-effect level (NOAEL) for repeated dose toxicity was 1000 mg/kg under the conditions of this study. There were no effects on reproduction or development.