Estra-4,6-dien-3-one, 17-hydroxy-, (17.beta.)-

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Dec 2011-27 Dec 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to internationally accepted guideline and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions.

GLP compliance:

yes

Test type:

acute toxic class method

Test material

Test material information

Constituent 1

Reference substance name:

(1S,3aS,3bR,9aR,9bS,11aS)-1-hydroxy-11a-methyl-1H,2H,3H,3aH,3bH,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-one

EC Number:

700-803-2

Cas Number:

14531-84-1

Molecular formula:

C18H24O2

IUPAC Name:

(1S,3aS,3bR,9aR,9bS,11aS)-1-hydroxy-11a-methyl-1H,2H,3H,3aH,3bH,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-one

Constituent 2

Reference substance name:

Δ6-nandrolone

IUPAC Name:

Δ6-nandrolone

Details on test material:

Molecular formula
C18H24O2
Molecular weight
272.39
CAS Number
14531-84-1
Description
White crystalline powder (determined at NOTOX)
Batch
PPEMC0010 (taken from label)
Purity
Not indicated by the sponsor; treated as 100% pure
Test substance storage
At room temperature protected from light
Stability under storage conditions
Stable
Expiry date
30 April 2012
Vehicle Corn oil (Nieuwerkerk a/d IJssel, The Netherlands) (specific gravity 0.92).
Rationale The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.
Preparation The formulations (w/w) were prepared within 4 hours prior to dosing. Homogeneity was accomplished to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for purity of the test substance.
The concentration of the test substance in vehicle was varied to allow constant dosage volume in terms of mL/kg body weight.

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Species Rat, Wistar strain Crl:WI (Han) (outbred, SPF-Quality). Recognized by international guidelines as the recommended test system (e.g. OECD, EC).
Source: Charles River Deutschland, Sulzfeld, Germany.
Number of animals 9 Females (nulliparous and non-pregnant). Each dose group consisted of 3 animals.
Age and body weight Young adult animals (animals 1-6 approx. 9-10 weeks old, animals 7-9 approx. 12 weeks old) were selected. Body weight variation did not exceed +/- 20% of the sex mean.
Identification Ear- and tailmark.

Health inspection A health inspection was performed prior to commencement of treatment, to ensure that the animals were in a good state of health.
Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 19.7 – 21.7ºC), a relative humidity of 40-70% (actual range: 40 - 56%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Accommodation
Group housing of 3 animals per cage in labeled Makrolon cages (MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
Acclimatization period was at least 5 days before start of treatment under laboratory conditions.
Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water
Free access to tap water.
Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Method Oral gavage, using plastic feeding tubes.
Fasting 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.
Frequency Single dosage on Day 1.

Doses:

Dose level (volume) 2000 mg/kg (10 mL/kg) body weight.
300 mg/kg (10 mL/kg) body weight

No. of animals per sex per dose:

3 females at 2000 mg/kg, two sets of 3 females at 300 mg/kg

Control animals:

no

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 2000 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.

Observations
Mortality/Viability Twice daily.
Body weights Days 1 (pre-administration), 8 and 15 and at death (if found dead after Day 1).
- Page 9 -
Clinical signs At periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 4: grading slight (1) to very severe (4)
Maximum grade 3: grading slight (1) to severe (3)
Maximum grade 1: presence is scored (1).
Necropsy The animals surviving to the end of the observation period were sacrificed by oxygen/carbon dioxide procedure. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities recorded.

Statistics:

The oral LD50 value of the test substance was ranked within the following ranges: 0-5, 5-50, 50-300 or 300-2000 mg/kg b.w. or as exceeding 2000 mg/kg b.w. The LD50 cut-off value was established based on OECD guideline 423. No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations and Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures.

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

LD50

Effect level:

1 000 mg/kg bw

Based on:

test mat.

Mortality:

Two females at 2000 mg/kg were found dead on Days 1 or 2. No further mortality occurred.

Clinical signs:

other: Clinical signs observed during the study period were as follows: Dose level Clinical signs 2000 mg/kg General tremor, flat and/or hunched posture, shallow or quick breathing, piloerection and/or restless behavior were noted among all three animals. The su

Gross pathology:

Beginning of autolyse or mucous content of the jejunum and reddish discolouration of the thymus were found among the animals that died during the study, at macroscopic post mortem examination. Macroscopic post mortem examination of the other animals that died during the study and of the surviving animals at termination did not reveal any abnormalities.

Applicant's summary and conclusion

Interpretation of results:

Toxicity Category IV

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The oral LD50 value of delta-6-nandrolone in Wistar rats was established to be within the range of 300-2000 mg/kg body weight.
According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 1000 mg/kg body weight.
Based on these results:
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), Δ6-nandrolone should be classified as: harmful if swallowed (Category 4) for acute toxicity by the oral route.
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, Δ6-nandrolone should be classified as Category 4 and should be labeled as H302: Harmful if swallowed.

Executive summary:

Assessment of acute oral toxicity with delta-6-nandrolone in the rat (Acute Toxic Class Method). The study was carried out based on the guidelines described in: OECD No.423 (2001) "Acute Oral Toxicity, Acute Toxic Class Method" Commission Regulation (EC) No 440/2008, B1 tris: "Acute Oral Toxicity, Acute Toxic Class Method" EPA, OPPTS 870.1100 (2002), "Acute Oral Toxicity" JMAFF guidelines (2000) including the most recent partial revisions. Initially, delta-6-NANDROLONE was administered by oral gavage to three female Wistar rats at 2000 mg/kg body weight. In a stepwise procedure additional groups of females were dosed at 300 and 300 mg/kg body weight. All animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15). Two females at 2000 mg/kg were found dead on Days 1 or 2. No further mortality occurred. Clinical signs observed during the study period were as follows: Dose level Clinical signs 2000 mg/kg General tremor, flat and/or hunched posture, shallow or quick breathing, piloerection and/or restless behavior were noted among all three animals. The surviving animal recovered from these symptoms on Day 10. 300 mg/kg hunched posture, piloerection and/or chromodacryorrhoea were noted in all animals on Days 1 and/or 2. The body weight gain shown by the animals at 300 mg/kg over the study period was considered to be similar to that expected of normal untreated animals of the same age and strain. The surviving animal at 2000 mg/kg showed body weight loss between Days 1-8 and recovered between Days 8-15. Beginning of autolyse or mucous content of the jejunum and reddish discolouration of the thymus were found among the animals that died during the study, at macroscopic post mortem examination. Macroscopic post mortem examination of the other animals that died during the study and of the surviving animals at termination did not reveal any abnormalities. The oral LD50 value of detla-6-nandrolone in Wistar rats was established to be within the range of 300-2000 mg/kg body weight. According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 1000 mg/kg body weight. Based on these results: - according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), delta-6-nandrolone should be classified as: harmful if swallowed (Category 4) for acute toxicity by the oral route. - according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, Δ6-nandrolone should be classified as Category 4 and should be labeled as H302: Harmful if swallowed.