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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 03 - March 03, 2009

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 other 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 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

of 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050 (28-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/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 treatment start: Approx. 6 weeks.
- Weight treatment start: Mean (males): 179 g, minimum 171 g, maximum 189 g.
Mean (females): 141 g, minimum 133 g, maximum 146 g.
- Housing: Group housing (5 animals/sex) in M IV type cages.
During overnight motor activity recording individual housing in M III type cages.
- Bedding material: Sterilized saw dust. Paper as cage enrichment.
During overnight motor activity recording no cage enrichment.
- 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 treatment start under laboratory conditions.


ENVIRONMENTAL CONDITIONS

Animal housing and environmental conditions were appropriate for subacute 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 18.9 – 21.7ºC. The relative humidity during the study period was 34 – 86%. Temporary fluctuation from the light/dark cycle with a maximum of 1 hour occurred because of the conduct of functional observations in the same room.

IN-LIFE DATES:

Animal allocation: 19-JAN-2009
Treatment: 03-FEB to 02-MAR-2009
Necropsy: 03-MAR-2009

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): 5 ml/kg bw. Actual dose volumes were calculated weekly accounting for the latest body weight.
- 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)
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations foreseen for treatment were prepared daily weight by weight (w/w) within six hours prior to dosing, and were homogenized to visually acceptable levels. All dose levels listed in the present report are expressed as water- and minor impurity-free test substance. Therefore, a multiplication factor of 1.16 was applied to calculate the amount of test substance required for preparation of the test substance formulations, e.g. in order to attain a dose level of 100 mg/kg bw of water- and minor impurity-free the test substance, 116 mg/kg bw of the test substance as received at the testing laboratory were administered and the formulations were prepared accordingly.


CONCENTRATION OF TEST SUBSTANCE IN VEHICLE (expressed as water and minor impurity-free Orange HAS 2-1177/KL2-RW):
Group 1 (Vehicle control, 0 mg/kg/day): 0.0%
Group 2 (Low Dose, 10 mg/kg/day): 0.2% (weight/weight)
Group 3 ( Mid Dose, 30 mg/kg/day): 0.6% (weight/weight)
Group 4 (High Dose, 100 mg/kg/day): 2.0% (weight/weight)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A validated high performance liquid chromatography method with spectrophotometric detection (HPLC/UV) was adopted for analysis. Samples of test substance formulations foreseen for treatment were analyzed on a single occasion during the in-life phase of the study for accuracy of preparation (all concentrations) and for homogeneity (highest and lowest concentration). Stability in vehicle over 6 hours at room temperature under protection from light was also determined (highest and lowest concentration). Preparation accuracy and homogeneity of the samples, as well as stability of the test substance in the vehicle over 6 hours at the above conditions were confirmed, i.e. the mean recovery in accuracy samples was within 90 to 110%, and the coefficient of variation for homogeneity samples as well as the change in concentration over the storage period were ≤ 10%.

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 30, 100 mg/kg bw/day (mf)
Basis:
actual ingested

No. of animals per sex per dose:

5 (no satellite groups)

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

Dose selection in the 28-day oral toxicity study was based on an acute oral (gavage) toxicity study (Notox Project 489783) and on a repeat dose range-finding oral (gavage) toxicity study. In the acute study, all of 6 female rats survived the discriminating dose of 50 mg/kg, whilst the next higher dose of 300 mg/kg caused the death of 4 of 6 female rats, and 2000 mg/kg killed all of 3 female rats. In the repeat dose range-finding study, dose levels of 100, 300, 600 and 1000 mg/kg/day were administered to 3 female rats/dose group over 11, 8, 2 and 1 days, respectively. In the latter study, all animals treated with 300, 600 or 1000 mg/kg/day were found dead or killed in extremis, whilst at 100 mg/kg/day all animals survived their 11-day treatment period. Overt signs of toxicity were not evident at 100 mg/kg/day. Liver weights appeared to be increased. Histopathology was not performed.

Recovery animals and a post-treatment observation period for reversibility check were not included in the present 28-day oral toxicity (gavage) study.
Remark: Doses and dose leves are expressed as water and minor impurity-free the test substance.

Positive control:

Not applicable.

Examinations

Observations and examinations performed and frequency:

The following observations and examinations were performed on all animals:

Mortality/viability: Twice daily
Clinical observations: Once daily
Clinical signs in standard arena outside the home cage: Once prior to treatment start and once weekly during treatment.
Functional observations (hearing ability, papillary reflex, static righting reflex, grip strength, motor activity): During treatment week 4.
Body weights: Weekly
Food consumption: Weekly
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Haematology/Clinical chemistry: at study end.

Sacrifice and pathology:

All surviving animals were killed on Day 29 after 28 consecutive days of oral (gavage) treatment.

Organ weights and organ to body weight ratios determined: Adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, uterus (including cervix), prostate, seminal vesicles including coagulating glands, ovaries

Organs/tissues examined at necropsy from both, premature deaths and all animals surviving until the scheduled necropsy:
Adrenal glands; (Aorta); Brain [cerebellum, mid-brain, cortex]; Caecum; Cervix; (Clitoral gland); Colon; Duodenum; Epididymides; Eyes (including optic nerve and Harderian gland); (Female mammary gland area); Femur including joint; Heart; Ileum; Jejunum; Kidneys; (Larynx); (Lacrimal gland, exorbital); Liver; Lung, infused with formalin; Lymph nodes – mandibular, mesenteric; (Nasopharynx); (Oesophagus); Ovaries; (Pancreas);Peyer's patches [jejunum, ileum], if detectable; (Pituitary gland); (Preputial gland); Prostate gland; Rectum; (Salivary glands - mandibular, sublingual); Sciatic nerve; Seminal vesicles including coagulating gland; Skeletal muscle; (Skin); Spinal cord -cervical, midthoracic, lumbar; Spleen; Sternum with bone marrow; Stomach; Testes; Thymus; Thyroid including parathyroid [if detectable]; (Tongue); Trachea; Urinary bladder; Uterus; Vagina; all gross lesions. From animals of the vehicle control and 100 mg/kg/day groups, all of these tissues/organs except those (in parentheses), were also examined microscopically by a pathologist. In addition, kidneys of the 10 and 30 mg/kg/day dose groups were microscopically examined. Tissues/organs mentioned (in parentheses) were not microscopically examined, since no signs of toxicity were noted at macroscopic examination.

Other examinations:

No

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups with the control groups for each sex.
The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
A total of three animals (one male, two females) at 100 mg/kg/day were found dead during the observation period. Apart from piloerection in one animal, no clinical signs of toxicity were observed in these animals prior to death and no cause of death could be established at necropsy and after histopathological examination. However, a relation to treatment with the test substance was considered likely since these deaths occurred at the highest dose tested. No clinical signs of toxicity were noted among the surviving animals, and functional observation tests revealed no abnormalities.

Orange faeces were noted at 100 mg/kg/day from Treatment Week 2 onwards. This finding was attributed to staining properties of the test substance and therefore considered to be of no toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
At 100 mg/kg/day, male body weight gain was statistically significantly lower than concurrent controls throughout the treatment period leading to absolute male body weights significantly lower than controls on treatment day 28. A trend similar to this, but statistically not significant, was seen in female animals at this dose level.

WATER CONSUMPTION
Water consumption was not measured. However, subjective appraisal of water consumption during the conduct of the study did not reveal any overt test substance related effect on water consumption.

HAEMATOLOGY
At 100 mg/kg/day activated partial tromboplastin time (APTT) was slightly lower in female animals than in concurrent controls and in one of these treated females red blood cell count (RBC) haemoglobin and haematocrit levels appeared slightly lower than in concurrent controls. Hematology findings attributable to treatment with the test substance were not evident in treated male animals.

CLINICAL CHEMISTRY
The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from concurrent controls:
-Higher cholesterol levels in females at 10, 30 and 100 mg/kg/day (see Table 1 in Remarks on results; no clear dose response; at 100 mg/kg/day in
two females slightly higher than the upper limit of the historical reference range for female rats of this age and strain),
-Lower calcium levels in females at 100 mg/kg/day (in one female of this group slightly lower than the lower limit of the historical reference range for female rats of this age and strain),
-Higher sodium levels for males at 100 mg/kg/day (in two males of this group slightly higher than the upper limit of the historical reference range fo r male rats of this age and strain).

ORGAN WEIGHTS
At 100 mg/kg/day, terminal body weight was statistically significantly lower in male animals than in concurrent controls. At 100 mg/kg/day, absolute kidney weights and kidney to bodyweight ratios were considerably higher than concurrent controls in one male and one female animals, contributing to kidney to body weight ratios statistically significantly higher in both sexes of this group than in concurrent controls. In both animals with high kidney weights, increased severity of tubular basophilia in the kidneys was recorded.

GROSS PATHOLOGY
Orange contents of the stomach and/or jejunum and orange discolouration of the glandular mucosa of the stomach were noted among premature deaths. This was considered to be related to the staining properties of the test substance, and therefore not toxicologically relevant. Absence of those findings among surviving animals was considered to be related to overnight fasting prior to scheduled necropsy and to the time period between the last dose and necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
An increased severity of tubular basophilia in the kidneys to moderate or marked degrees was noted in two male and one female survivor at 100 mg/kg/day. In two of these animals, this finding was associated with increased absolute kidney weights and kidney to body weight ratios.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: overall effects clinical signs and functional observational battery; mortality; body weight; food consumption; food efficiency; water consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology

2

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Clinical Biochemistry Summary  –  Cholesterol
		GROUP 1 CONTROL	GROUP 2 10 MG/KG/DAY	GROUP 3 30 MG/KG/DAY	GROUP 4 100 MG/KG/DAY
END OF TREATMENT
Males	MEAN	1.70	1.75	1.81	1.77
mmol/L	ST.DEV	0.46	0.24	0.30	0.15
	N	5	5	5	4
Females	MEAN	1.18	1.84 *	1.79 *	2.04 **
mmol/L	ST.DEV	0.39	0.13	0.12	0.52
	N	5	5	5	3
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Historical Reference Range  –  Cholesterol
	Unit	Mean ± SD	N	P5	P95
Males	mmol/L	1.73 ± 0.340	170	1.220	2.370
Females	mmol/L	1.47 ± 0.366	170	0.890	2.090

The slight increase in blood cholesterol levels in female rats at 10 and 30 mg/kg/day were the reason for setting the NOAEL for females at 30 mg/kg/day. All individual cholesterol values from these treated females were within the historical reference range for female rats of this strain and age. Therefore, this effect was not adverse.

Applicant's summary and conclusion

Conclusions:

In the present 28-day oral toxicity (gavage) study, the No Observed Adverse Effect Level (NOAEL) for the test substance was set at 30 mg*/kg/day, because of minor increases in blood cholesterol levels in female animals at 10* and 30 mg*/kg/day. At 100 mg/kg/day, a relationship to treatment with the test material could not be discounted for three premature deaths, piloerection, retarded bodyweight gain, slight changes in a number of hematology and clinical biochemistry blood parameters and an increase in the severity of tubular basophilia in kidneys.

According to EU classification rules [DIRECTIVE 67/548/EEC] the results attained in this study would lead to classification and labelling as “Xn” (harmful) and “R48/22” (Danger of serious damage to health by prolonged exposure / route of exposure oral) and according to EU-GHS classification rules [REGULATION (EC) 1272/2008] they would lead to “STOT RE 2” (Specific target organ toxicity — repeated exposure) / “H373” (May cause damage to organs)
________________________________________________________________
* Expressed as water- and minor impurity-free test substance

Executive summary:

the test substance was tested for its subacute toxicity in the rat according to OECD Guideline 407 and the corresponding EC and EPA-OPPTS Technical Guidelines in compliance with GLP. Reliability grade 1 was assigned.

 

Rats (5 males and 5 females/dose group) were treated by oral gavage administration with the test substance at dose levels of 0, 10, 30 or 100 mg*/kg bodyweight/day once daily, seven days/week over 28 days. All animal survivors were killed after 28 treatment days (day 29). At regular intervals during the study, overall effects, mortality, clinical signs, body weights, food consumption and food efficiency were recorded. In addition, a subjective appraisal of water consumption (without quantitative measurement) was maintained during the study. Functional observations (hearing ability, pupillary reflex, static righting reflex and grip strength) and motor activity were recorded during Treatment Week 4. At the end of the study, haematology and clinical biochemistry examinations were performed on all animal survivors. Organ weights were determined and macroscopic pathology was performed on a number of organs/tissues from all animals. Histopathology was performed on a number of organs from animals of the control and high dose groups and on the kidneys from all groups.

 

At 10* and 30 mg*/kg/day, the only findings attributable to treatment with the test substance were blood cholesterol levels slightly higher in females than in concurrent controls. This minor change in female cholesterol was considered not to represent an adverse effect, because all individual cholesterol values attained at these two dose levels were within the historical reference range for rats of this sex, strain and age and the mean value of the concurrent female control group was fairly low (within historical reference range).

 

At 100 mg*/kg/day, a relationship to treatment with the test substance could not be discounted for the following:

– Three premature deaths (one male and two females) preceded by piloerection in one of these

decedents.

– Male bodyweight gain and male absolute bodyweight statistically significantly lower than in

concurrent controls during the treatment period and on treatment day 28, respectively.

– Changes in blood parameters (haematology and clinical biochemistry compared with concurrent

controls) comprising in males: sodium increased and in females: activated partial thromboplastin

time slightly shortened; red blood cell count, haemoglobin and haematocrit reduced (one animal);

cholesterol increased (with the values from two animals slightly above the upper limit of the

respective historical reference range); and calcium reduced.

– Increased severity of tubular basophilia in the kidneys to moderate or marked in degree in two male

and one female survivor concomitant with increased absolute kidney weights and kidney to body

weight ratios in two of these animals.

 

Based on these results the No Observed Adverse Effect Level (NOAEL) for Orange HAS 2-1177/KL2-RW was 30 mg*/kg/day.

 

According to EU classification rules [DIRECTIVE 67/548/EEC] the results attained in this study would lead to classification and labelling as “Xn” (harmful) and “R48/22” (Danger of serious damage to health by prolonged exposure / route of exposure oral) and according to EU-GHS classification rules [REGULATION (EC) 1272/2008] they would lead to “STOT RE 2” (Specific target organ toxicity — repeated exposure) / “H373” (May cause damage to organs).

________________________________________________________________

* Expressed as water- and minor impurity-free test substance