1,1'-{(phenylmethanediyl)bis[(3-substituted benzene-4,1-diyl)diazene-2,1-diyl{1-[3-(dimethylamino)propyl]-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridine-5,3-diyl}]}dipyridinium chloride lactate salts

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study, conducted according to internationally accepted technical guidelines and in compliance with GLP in recognized contract research organization.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

of 2001

Deviations:

yes

Remarks:

Temporarily (during 3 hours) the relative humidity in the animal room was lower than the guideline specified lower target limit of 30% r.h. (minimum 18% r.h.). This minor deviation did not compromise the quality, integrity or outcome of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2008

Deviations:

yes

Remarks:

Temporarily (during 3 hours) the relative humidity in the animal room was lower than the guideline specified lower target limit of 30% r.h. (minimum 18% r.h.). This minor deviation did not compromise the quality, integrity or outcome of the study.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

of 2002

Deviations:

yes

Remarks:

During 3 h rel. humidity in the animal room was lower than 30% r.h. (recorded minimum 18% r.h.). In addition, animals were housed in groups of 3 rather than individually. These minor deviations did not compromise the outcome of the study.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Wistar rats, strain: Crl:WI (Han) (outbred, SPF-Quality), with appropriate range of bodyweight at study start.
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation (day of dosing): Approx. 8-10 weeks.
- Weight at study initiation( day of dosing): Mean: xxx g, minimum 147 g, maximum 196 g.
- Fasting period: Overnight prior to dosing until 3-4 hours post administration. Water was available.
- Housing: Group housing with 3 animals/M IV type cage.
- Diet (ad libitum): Commercially available rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days before dosing under laboratory conditions.


ENVIRONMENTAL CONDITIONS

Animal housing and environmental conditions were appropriate for acute toxicity testing in the rat: Controlled environment with approximately 15 airchanges per hour, 12 hours artificial fluorescent light and 12 hours darkness per day and 19 – 21ºC. The relative humidity during the study period was 34 – 69%, except for three hours, during which the relative humidity dropped to a minimum of 18% due to a short term failure of the air humidifying system. This transient decline to low relative humidity did not compromise the quality, integrity or outcome of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Vehicle: Water (Elix, Millipore S.A.S., Molsheim, France).
- Concentration in vehicle: The concentration of the test substance in vehicle varied between dose groups thus allowing constant dosage volume in terms of ml/kg bw.
- Amount (dose volume by gavage): 10 ml/kg bw
- Justification for choice of vehicle:
Trial formulations at the testing laboratory and test substance data supplied by the sponsor led to the choice of water as a suitable vehicle. (The test substance is well soluble in water and stable for at least 96 hours in water and no adverse side effects of this vehicle on the animals are to be expected)
DOSAGE PREPARATION:
All doses listed in the present study are expressed as water- and minor impurity-free test substance. This was accounted for during dosage preparation by the application of factor 1.16 to all test substance concentrations and all test substance doses as follows: Test substance concentration and test substance dose administered = 1.16 times the test substance received at the testing laboratory. The formulations (w/w) were prepared within 4 hours prior to dosing. Homogeneity was accomplished to a visually acceptable level.

ACUTE TOXIC CLASS METHOD
Rationale for the selection of the starting dose:
The starting dose was 2000 mg/kg bw, because initially test substance related mortality was considered to be unlikely.

Doses:

Doses (expressed as water- and minor impurity-free test substance):
2000 mg/kg bw (3 females),
300 mg/kg bw (3 + 3 females),
50 mg/kg bw (3 + 3 females).

No. of animals per sex per dose:

Only female animals were dosed in the present study. For the numbers of animals treated per dose see "Doses".

Control animals:

no

Details on study design:

Post-dosing observation period was 14 days during which mortality/survival and clinical signs were recorded at: 0, 2 and 4 hours after dosing on day 1 and twice daily (mortality/survival) or once daily (clinical signs) thereafter until day 15. Body weight was recorded on day 1 (prior to dosing) for each animal and on days 8 and 15 for each survivor. In addition, animals found dead after day 1 were weighed post mortem. Premature deaths and survivors killed at the end of the 14-day post-dosing observation period (day 15) were necropsied and macroscopic pathology findings recorded.

Statistics:

Not applicable: The acute toxic class method is not intended for the calculation of a precise LD50 value.

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

discriminating dose

Effect level:

50 mg/kg bw

Remarks on result:

other: Dose expressed as water- and minor impurity-free test substance

Mortality:

Dose level Mortality Date of treatment
50 mg/kg 0/3 (f) 13 January 2009
50 mg/kg 0/3 (f) 15 January 2009
300 mg/kg 1/3 (f) 07 January 2009
300 mg/kg 3/3 (f) 09 January 2009
2000 mg/kg 3/3 (f) 06 January 2009
All but one premature death happened within 2 or 4 hours after dosing. The latter decedent animal was dosed at 300 mg/kg and was found dead on the day after dosing (day 2).

Clinical signs:

50 mg/kg: Hunched posture, piloerection, rales, shallow respiration and salivation were noted among the animals on Day 1.
300 mg/kg: Lethargy, hunched posture, piloerection, rales and red faeces were noted among the animals on Days 1 and/or 2.
2000 mg/kg: No clinical signs were noted at 0 hour post treatment. By the next recording of clinical signs 2 h post treatment all animals had died.

Body weight:

Adverse effects on body weight were not evident in the surviving animals.

Gross pathology:

All animals which survived during the 14-day post-dosing observation period and the 300 mg/kg decedent animal found dead on the day after dosing (day 2) were free from macroscopic pathology findings. In all other decedent animals (found dead 2 or 4 hours after dosing) reddish discoloration of the stomach, duodenum and jejunum was evident. In addition, several reddish foci on both sides of the thymus were recorded in one 300 mg/kg decedent animal on the day of dosing. Discolouration of the gastrointestinal tract organs was attributed to staining properties of the test substance and therefore not considered to be toxicologically relevant. Reddish foci on both sides of the thymus is occasionally seen in rats of this strain and age regardless of their treatment and therefore the single incidence in the present study was not attributed to treatment with the test substance.

Applicant's summary and conclusion

Interpretation of results:

harmful

Remarks:

Migrated information if swallowed Criteria used for interpretation of results: EU

Conclusions:

In view of the discriminating oral dose of 50 mg/kg bw attained in the present study and the LD50 cut-off value considered to be 300 mg/kg bw, the substance was classified as "harmful (Xn)" and "harmful if swallowed (R22)" according to EU classification rules [DIRECTIVE 67/548/EEC], and as "Category 3 (Danger, Toxic if swallowed) according to EU-GHS classification rules [REGULATION (EC) 1272/2008].