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Toxicological information

Acute Toxicity: oral

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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 April -11 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No 8147, April 2011, including the most recent partial revisions.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): OS 2600

Test animals

Species:
rat
Strain:
other: Wistar strain, Crl:WI (Han)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 8 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (control group: 152 - 178 grams; 1000 mg/kg group: 147 - 177 grams ; 2000 mg/kg group: 153 - 179 grams).
- Fasting period before study: Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test substance. Water was available ad libitum.
- Housing: Group housing of 3 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 12 April 2012 to 11 May 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
GAVAGE METHOD: plastic feeding tubes.

Frequency: single dosage, on Day 1.

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg (10 mL/kg) body weight.
Dose volume calculated as dose level (g/kg) / density (g/mL).

DOSAGE PREPARATION: The test substance was dosed undiluted as delivered by the sponsor. No correction for purity was made

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Based on anticipated mortality at the next higher dose.
Doses:
1000 mg/kg body weight
2000 mg/kg body weight


No. of animals per sex per dose:
1000 mg/kg: 6 (2 groups of three females in a stepwise manner)
2000 mg/kg: 3
Control animals:
yes
Details on study design:
The toxicity of the test substance was assessed by stepwise treatment of groups of 3 females. The first group was treated at a dose level of 1000 mg/kg. The absence or presence of mortality of animals dosed at one step determined the next step, based on the test procedure defined in the guidelines. The onset, duration and severity of the signs of toxicity were taken into account for determination of the time interval between the dose groups. A concurrent control group was added to the first two treated groups. Thereafter no concurrent control was added.

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality/Viability: Twice daily. The time of death was recorded as precisely as possible.
Body weights: Days 1 (pre-administration), 8 and 15 and at death (if found dead after Day 1).
Clinical signs: At periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15.
- Necropsy of survivors performed: on Day 15
- Other examinations performed: bloodsampling on Day 5 and 15 (red blood cell count, reticutolcyte count, hemoglobin level and met-hemoglobin level, organ weights (spleen, kidney and liver) at necropsy, hostopathological examination of the spleen
Statistics:
No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 1 000 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred at 1000 mg/kg and control groups. Two females at 2000 mg/kg (2/3) were found dead on Days 1 or 4.
Clinical signs:
No clinical signs were noted in the control animals.
Animals at 1000 mg/kg showed lethargy, hunched posture, uncoordinated movements, piloerection, ptosis, slow breathing, and/or watery discharge from the eye on Day 1.
Animals at 2000 mg/kg showed lethargy, flat and/or hunched posture, uncoordinated movements, slow breathing, piloerection, hypothermia and/or ptosis until death (two animals) or on Days 1-3 (surviving animal).
Body weight:
The body weight gain shown by the surviving animals over the study period was considered to be similar to the control animals.
Gross pathology:
Necropsy did not reveal any toxicologically relevant alterations.
A control animal showed pelvic dilation and black nodules in the liver, which are considered incidental findings not related to the test substance. One animal at 2000 mg/kg showed advanced autolysis and the skin of the left region of the head was missing due to cannibalism.
Other findings:
The animals at 1000 mg/kg showed lower red blood cell counts in combination with higher percentage of reticulocytes, compared to the control animals, on Day 5 and 15. A lower level of total hemoglobin was noted on Day 5, which recovered on Day 15. The slight higher met-hemoglobin levels on Day 5 and/or 15 (as compared to the controls) are within the range to be expected for rats of this age and strain.
The surviving animal treated at 2000 mg/kg showed comparable trends in all four parameters.

Liver, spleen and kidney weights of treated animals were considered to be similar to those of control animals.

There were no treatment-related findings in the spleens of the 1000 mg/kg treated rats.
No direct cause of death at 2000 mg/kg could be established from the examined sections of the spleen.
There was a treatment related microscopic finding in the 2000 mg/kg treated rats. Atrophy of the white and red pulp of the spleen was noted at minimal degree in the one surviving female and at moderate degree in one female (found dead on Day 4).
The remainder of the recorded microscopic findings in the spleen were all within the normal range of background alterations encountered in rats of this age and strain and therefore not regarded as test article related.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
In an acute oral toxicity study with female rats, the LD50 was determined to be between 1000 and 2000 mg/kg bw.
Executive summary:

The acute oral toxicity study was performed in accordance with OECD Guideline 423 and EU Method B1 tris. Initially, OS 2600 was administered by oral gavage to 3 female Wistar rats at 1000 mg/kg body weight. In a stepwise procedure additional groups of females were dosed at 1000 and 2000 mg/kg body weight. Concurrent control groups were added in the first two treatment groups. All animals were subjected to daily observations and weekly determination of body weight. Blood samples were taken from all surviving animals on Day 5 and prior to necropsy. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15). Histopathology examination was performed on the spleen. No mortality occurred at 1000 mg/kg and control groups. Two females at 2000 mg/kg (2/3) were found dead on Days 1 or 4 and therefore, the oral LD50 value of OS 2600 in Wistar rats was established to be within the range of 1000-2000 mg/kg body weight. Haematology parameters were affected by treatment at a dose level of 1000 mg/kg, and the same trend was noted at 2000 mg/kg. The following findings were included: lower red blood cell count, higher reticulocyte percentage and lower haemoglobin levels. These findings were not accompanied by treatment related morphological changes in the spleen at 1000 mg/kg. Two animals at 2000 mg/kg (one surviving and one which died on Day 4) showed atrophy of the white and red pulp of the spleen at microscopic examination.