Iron(1+), chloro[dimethyl 9,9-dihydroxy-3-methyl-2,4-di(2-pyridinyl-kN)-7-[(2-pyridinyl-kN)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate-kN3,kN7]-, chloride

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 4 December 2003 to 17 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 17th December 2001

Deviations:

yes

Remarks:

A further dose of 200 mg/kg was required in order to adapt the test to Eu regulatory classifications

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,5-dimethyl 3-methyl-9-oxo-2,4-bis(pyridin-2-yl)-7-[(pyridin-2-yl)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate dichloroiron hydrate

EC Number:

610-388-9

Cas Number:

478945-46-9

Molecular formula:

C28H31N5O6FeCl2

IUPAC Name:

1,5-dimethyl 3-methyl-9-oxo-2,4-bis(pyridin-2-yl)-7-[(pyridin-2-yl)methyl]-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate dichloroiron hydrate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SEAC sample number S2539801
- Expiration date of the lot/batch: 1 January 2005
- Purity test date: The supporting data for purity of the test item was not made available at the time of issuing this report and hence this information has been excluded from statement of compliance. However, sponsor has adressed this in a GLP compliant study, SEAC Study Reference Number AC030449

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (range of 20±3 deg celsius), protected from light
- Stability under test conditions:stability of the dosing solution was adressed by ensuring fresh solutions were made immediately prior to dosing.
- Solubility and stability of the test substance in the solvent/vehicle: A solubility trial was carried out to determine the choice of vehicle.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanBrl: Wist (SPF)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, laboratory Animal Services
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 157.3 to 177.1 g
- Fasting period before study: yes
- Housing:In groups of 3 in Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433 rat/mousemaintenance diet, batch nos. 54/03 and 78/03 ad libitum
- Water (e.g. ad libitum): community tap water ad libitum
- Acclimation period: under laboratory conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 deg celsius
- Humidity (%): 30-70% relative humidity
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark, music during the daytime light period

IN-LIFE DATES From: : 5 December 2003 To: 5 February 2004

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0.03, 0.02 or 0.005 g/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Justification for choice of vehicle: purified water was found to be a suitable vehicle
- Lot/batch no. (if required): not specified
- Purity: not specified

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION (if unusual): The dose formulation were made shortly before each dosing occasion using a magnetic stirrer and spatula as homogeneizers. The test item was weighed into a tared glass beaker on a suitable precision balance and the vahicle added (w/v). Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:no justification was provided

Doses:

300, 200 and 50 mg/kg bw

No. of animals per sex per dose:

3 animals per group was used

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Daily for mortality, daily for clinical signs during day 1 to 15 and 1, 2, 3 and 5 hours after administration on day 1. Once daily on days 2 to 15. Body weight was measured at day 1, 8 and 15
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, macroscopic examinations

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 200 - < 300 mg/kg bw

Based on:

test mat.

Mortality:

Three 300 mg/kg bw treated animals were found dead approximately 15 to 20 minutes after test item administration and one animal of this dose group had to be killed in extremis for ethical reasons immediately after 2 hours reading. One animal treated at 200 mg/kg was found dead at the 1 hour reading. All the 50 mg/kg bw animals and the remaining animals of the other two dose groups survived until the end of the study period.

Clinical signs:

other: Sedation, ventral recumbency and slight to moderate convulsions were observed in five animals of 300mg/kg bw dose groupat the 0 hour reading and, except sedation, persisted in one animal up to the 1 hour reading. Lateral recumbency was noted in one animal

Gross pathology:

One 300 mg/kg treated animal was observed with a hear reduced in size and one animal treated at 200 mg/kg was noted with liquid contents in its stomach. No macroscopic findings were recorded in the remaining animals of these two dose groups and in all 50 mg/kg treated animals at scheduled and unscheduled necropsies.

Applicant's summary and conclusion

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

According the results of the study, the LD50 was defined in the range of 200 mg/kg and 300 mg/kg. Hence, according CLP and GHS critera, the registered substance was classified as Category 3 for Acute Toxicity Hazard.

Executive summary:

The GLP compliant study was performed to assess the potential acute toxicity of the registered substance E-700-2003 in rats by oral gavage in a Toxic class method (according to OECD guideline 423).

Six groups, each of three females HanBrl: WIST were treated with the test item by oral gavage at dosages of 300, 200 and 50 mg/kg bodyweight, diluted in purified water and at 10 mL/kg. The animal were examined for 14 days post administration for mortality, clinical signs. Body weight was recorded too.

The following animals were treated and percentage of mortality was observed :

6 females treated at 300 mg/kg bodyweight : 69%

6 females treated at 200 mg/kg bodyweight : 17%

6 females treated at 50 mg/kg bodyweight : 0%

Sedation, ventral recumbency and slight to moderate convulsions were observed in five animals of 300mg/kg bw dose groupat the 0 hour reading and, except sedation, persisted in one animal up to the 1 hour reading. Lateral recumbency was noted in one animal and bradypnea in three animals at the 0 hours reading before death occured. One animal of this dose group was in a moribound state at the 2 hour reading before it was killed. Slight ataxia was observed in two 200 mg/kg treated animals from 0- or 1- to the 5-hour reading and hunched posture from 2- to the 3-hour reading before death occured. Sedation was noted in two animals at the 0- hour reading  and also at the 1-hour reading for one of the animals. Ventral recumbency and slight convulsions were also observed at the 0 hour reading in two animals. One animal of this dose group was seen with bradypnea at the 0-hour reading before being found dead at the 1 hour- reading. No clinical signs were observed in three 200 mg/kg in all 50 mg/kg treated animals during the course of the study.

According the results of the study, the LD50 was defined in the range of 200 mg/kg and 300 mg/kg. Hence, according CLP and GHS critera, the registered substance was classified as Category 3 for Acute Toxicity Hazard.