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Acute Toxicity: oral

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acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 21 to October 25, 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:

Test material

Test material form:
solid: particulate/powder

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Japan Hino Rearing Center
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks old
- Weight at study initiation: 184.2 to 188.0 g (first dose group) and 186.2 to 194.6 g (second dose group)
- Fasting period before study: yes, approximately 17.5 hours starting on the day prior to administration and for approximately 4 hours after administration.
- Housing: Stainless steel wire-bottom cages were used
- Diet (e.g. ad libitum): ad libitum (MF, Lot number 10034, Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): ad libitum (chlorinated Hita municipal tap water)
- Acclimation period: yes, at least 6 days

- Temperature (°C): 23.0 to 24.5°C
- Humidity (%): 52.4 to 62.5 %
- Air changes (per hr): ventilation frequency of 10 to 15 times/hour
- Photoperiod (hrs dark / hrs light): light/dark cycle of 12-hour intervals (lights on at 7:00 and off at 19:00)

IN-LIFE DATES: From: July 27 to August 20, 2010

Administration / exposure

Route of administration:
oral: gavage
olive oil
Details on oral exposure:
- Concentration in vehicle: 25.0 w/v% concentration. The "Homogeneity, stability, and concentration confirmation study on a test solution of 6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol " (study code: X02-0238) conducted prior to the start of administration confirmed the homogeneity of the test solution of 0.0100 and 25.0 w/v% and the stability of the test solution when stored in a cool place using high speed liquid chromatography (HPLC).
- Amount of vehicle (if gavage): 10 mL/kg
- Justification for choice of vehicle: the test substance did not dissolve in olive oil at a 25.0 w/v% concentration, but was evenly suspended.
- Lot/batch no. (if required): 050ORS


DOSAGE PREPARATION: The test solution was stored in a cool place and confirmed to be stable 2 days after preparation (stable for 15 days in the end), so the test solution was prepared the day before administration. The test substance was weighted accurately and suspended in a mortar while adding olive oil. Then, while adding olive oil, a constant volume was created and the test solution (suspension) thus prepared. The prepared test solution was place in a lidded plastic container and stored in a cold place (actual value 4 to 9°C).

- Rationale for the selection of the starting dose: In a "Chromosomal aberration study of 6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol using mammalian cultivated cells" conducted at the study facility (study code: K06-1358), the 50% maximal inhibitory concentration (IC50) was calculated to be at least 34 µg/ml. Based on a correlation equation of cytotoxicity IC50 and acute toxicity LD50 proposed by ICCVAM*, rodent LD50 was calculated to be 550 mg/kg. But because it was predicted that no deaths would occur even with a single dose of 2,000 mg/kg based on the above results, 2,000 mg/kg was selected as the initial dose (step 1). The dose for step 2 was selected in accordance with "OECD Test Guidelines 423".

*In Vitro Cytotoxicity Test Methods for Estimating Acute Oral Systemic Toxicity. NIH Publication No. 07-4518, November 2006.
2000 mg/kg bw
No. of animals per sex per dose:
3 per group
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: General status was observed, including death. On the day of administration, all animals were observed immediately after administration, 5, 10, and 30 minutes and 1, 2, and 4 hours after administration. Detailed observations were collected, including the time of onset of symptoms. Furthermore, in the first dose group, symptoms were observed on the day of administration and up to 5 days after administration, so observations were taken in the afternoons during this period. Symptoms disappeared 6 days after administration, so a single observation was taken in the mornings between day 6 and day 14.
In the second dose group, symptoms were observed on the day of administration and up to 2 days after administration, so observations were taken in the afternoons during this period. Symptoms disappeared 3 days after administration, so single observations were taken in the mornings between day 3 and day 14.
The animals were weighed prior to administration and 1, 7, and 14 days after administration.
- Necropsy of survivors performed: yes, gross observations were taken and included the body surface, orifices, subcutis, intracranial cavity, thoracic cavity, abdominal cavity, and pelvic cavity.
Not applicable

Results and discussion

Effect levels
Key result
Dose descriptor:
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol did not cause mortality at a dose level of 2000 mg/kg bw.
Clinical signs:
Reduced spontaneous movement appeared 2 hours after administration and was present in all 6 animals 4 hours after administration. Additionally, 5 animals had reduced respiratory frequency and 3 had incomplete eyelid opening. These symptoms disappeared in the morning of the day following administration, but one animal had reduced respiratory frequency and incomplete eyelid opening on the day after administration and reduced spontaneous movement for 5 days after administration. Also, loose stool was observed in 4 animals, mucous stool in 3 animals, and lower abdominal soiling in 2 animals starting the day after administration. Twodays after administration, diarrhea was observed in all 6 animals and 1 of these had reduced fecal quantity. These stool abnormalities disappeared by the afternoon of the second day after administration. No abnormalities were observed from 6 days after administration and until 14 days after administration and deaths did not occur.
Body weight:
Suppression of weight increase or reduction in weight was observed 1 day after administration, appropriate weight gain was observed 7 days and 14 days after administration.
Gross pathology:
No macroscopic observations were seen in animals dosed at 2000 mg/kg bw terminated on Day 14.

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
Under the conditions of this study, the acute oral LD50 value of the test item 6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol was found to be above 2000 mg/kg bw in female Crl:CD(SD) rats.
Executive summary:

The single-dose oral toxicity of 6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol was studied according to the acute toxic class method (OECD 423 in Crl:CD(SD) rats. Two groups of 3 female Sprague-Dawley rats were treated with the test item at a dose level of 2000 mg/kg bw (Group 1 and Group 2).

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 4 hours after the treatment.The test item was formulated in olive oil at a concentration of 200 mg/mL at a dose volume of 10 mL/kg bw.

Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw. As no mortality was observed, a confirmatory group (Group 2) was treated at the same dose level. Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (day 14).

No mortality occurred. Clinical signs were reduced spontaneous movement, reduced respiratotry frequence, incomplete eyelid opening, loose stool, mucous stool, lower abdominal soiling, diarrhea, and reduced fecal volume. Although suppression of weight increase or reduction in weight was observed 1 day after administration, appropriate weight gain was observed 7 days and 14 days after administration. No abnormalities were found at macroscopic post mortem examination of the animals. The oral LD50 value of the test substance in Sprague-Dawley rats was established to exceed 2000 mg/kg body weight and therefore the substance is not classified for acute oral toxicity according to the EC/1272/2008 CLP criteria.