Reaction mass of 5,5'-{(phenylmethanediyl)bis[benzene-4,1-diyl-diazene-2,1-diyl]}bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride and 5,5’-[3,4’-(phenylmethanediyl)diphenylene]bis(diazene-2,1-diyl)bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride

Endpoint summary

Description of key information

Based on an oral (28-d) subacute study with the structural closely related source molecule a NOAEL of 450 mg/kg bw/d is delineated for the registration substance as target molecule using read-across.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007-12-13 to 2008-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations, conducted with the analogue substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age at study initiation: Approx. 6 weeks
- Weight at study initiation: Males: 124.1 to 158.3 g (141.3 ± 8.8 g); Females: 111.3 to 132.3 g (122.9 ± 5.1 g)
- Fasting period before study: not reported
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard rat maintenance diet was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days, under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light/12-hour dark cycle

IN-LIFE DATES: From: 2007-12-14 To: 2008-01-18 (main group), 2008-02-01 (recovery group)

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. Test item was weighed into a glass beaker on a tared Mettler balance and the vehicle, purified water, was added to give the appropriate final concentration of test item in the suspension. The mixtures were prepared using a magnetic stirrer Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

DIET PREPARATION
- not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 5, 15, and 45 mg/l
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): not applicable
- Purity: purified water was used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

General procedures:
On the first treatment day (21-Dec-2007) and once during week 3 (04-Jan-2008), one sample of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. One sample of about 2 g of each concentration was taken 2 hours and 7 days after commencement of dosing (dose formulation of the first treatment day) to confirm 2 hrs and 7 d-stability at room temperature (20 ± 5 °C). The aliquots for analysis of dose formulations were delivered to Dr. D. Flade (RCC Ltd, Itingen / Switzerland) and stored at -20 ± 5 °C until analysis. The dose formulations were delivered under ambient conditions. Analyses were be performed by the Study Scientist, Dr. D. Flade, according to an HPLC analytical method supplied by the Sponsor and previously adapted at RCC Ltd (RCC Study No.
B62032). The test item served as analytical standard. Details of the analytical method were documented in the raw data and in the part report generated by the Study Scientist.


Analytical Procedure

Preparation of Standard Solutions:
Stock solutions of test item in acetonitrile/purified water (1+1 v/v) were prepared for external standard calibration. For example, 25.29 mg of the test item was exactly weighed into a 50 mL volumetric flask and approximately 40 mL of acetonitrile/purified water (1+1 v/v) was added. Then, the mixture was sonicated for 5 minutes and the flask was brought to volume with acetonitrile/purified water (1+1 v/v) to yield a solution with a concentration of 505.8 μg/mL. Aliquots of this stock standard solution were used to prepare five working standard solutions in acetonitrile/purified water (1+1 v/v) with a concentration range of 10.12 to 101.2 μg/mL. Thirteen standard solutions derived from two stock standard solutions were used for calibration.

Analysis of Samples:
The samples received were dissolved in acetonitrile/purified water (1+1 v/v) by sonication for 5 minutes and then diluted to volume with acetonitrile/purified water (1+1 v/v). The sample solutions were further diluted with acetonitrile/purified water (1+1 v/v) into the calibration range.

High Performance Liquid Chromatographic Determination:
Apparatus: Pump: Merck-Hitachi L -7100
Sampling Unit: Merck-Hitachi L-7200
Column Oven: Merck-Hitachi L-7300
UV/VIS Detector: Merck-Hitachi L-7400
Interface: Merck-Hitachi D-7000
Column: Symmetry C18; 150 x 3.9 mm, 5 μm
Pre-Column: Phenomenex C18; 4 x 3 mm, 5 μm
Column Temperature: 40 °C
Eluent A: 0.1% Trifluoro acetic acid in purified water
Eluent B: Acetonitrile
Gradient: Eluent A 65%, Eluent B 35 % (at 0 and 6 min, resp.)
Flow: 1.0 mL/min
Wave Length: 254 nm
Injection Volume: 10 μL

Evaluation of Results
Injected samples were quantified by comparing peak areas of the test item with reference to the calibration curve. The latter was obtained by correlation of the peak areas of the working standards with their corresponding concentrations (μg/mL), using linear regression.

Duration of treatment / exposure:

28 days,

plus 14 days untreated recovery period of control and high dose animals

Frequency of treatment:

7 days/week

Remarks:

Doses / Concentrations:
0 mg/kg bw/d
Basis:
actual ingested

Remarks:

Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested

Remarks:

Doses / Concentrations:
150 mg/kg bw/d
Basis:
actual ingested

Remarks:

Doses / Concentrations:
450 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

Males: 10 animals at 0 mg/kg bw/day; 5 animals at 50 mg/kg bw/day; 5 animals at 150 mg/kg bw/day; 10 animals at 450 mg/kg bw/day.
Females: 10 animals at 0 mg/kg bw/day; 5 animals at 50 mg/kg bw/day; 5 animals at 150 mg/kg bw/day; 10 animals at 450 mg/kg bw/day.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats, RCC Study Number B62032, using dose levels of 100, 250, and 500 mg/kg/day.
- Rationale for animal assignment (if not random): Computer-generated random algorithm.
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for viability / mortality were recorded twice daily. The animals were observed for clinical signs once daily during acclimatization, twice daily on treatment days 1-3 and once daily thereafter including the recovery period.
- Cage side observations checked in table 1, attached below, were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Body Weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The food consumption was recorded once during the acclimatization period and weekly thereafter (however no feeding study)

FOOD EFFICIENCY:
- No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 Weeks: main group and recovery group. After 6 Weeks: recovery group
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all involved in the study
- Parameters checked in table 2, below, were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 Weeks: main group and recovery group. After 6 Weeks: recovery group
- Animals fasted: Yes
- How many animals: all involved in the study
- Parameters checked in table 2, below, were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: After 4 Weeks: all animals. After 6 Weeks: recovery group
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 2, below, were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: all animals examined
- Battery of functions tested: sensory activity / grip strength / motor activity /

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenal glands
Aorta
Bone (sternum, femur including joint)
Bone marrow (femur)
Brain – including section of medulla/pons,
cerebral and cerebellar cortex
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Esophagus
Eyes w/optic nerve (fixed in Davidson's
solution)
Harderian gland (fixed in Davidson's solution)
Heart including auricles
Ileum, with Peyer's patches
Jejunum with Peyer's patches
Kidneys
Larynx
Lacrimal gland, exorbital
Nasal cavity
Ovaries
Pancreas
Pharynx
Pituitary gland
Prostate gland incl. coagulating glands
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland, if possible)
Tongue
Trachea
Liver
Lungs, filled w/formalin at necropsy
Lymph nodes - mesenteric, mandibular and
popliteal
Mammary gland area
Urinary bladder, filled w/formalin at
necropsy
Uterus
Vagina
Gross lesions

HISTOPATHOLOGY: Yes (all organs as stated above)

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, macroscopical findings, organ weights and ratios, as well as clinical laboratory data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality occurred in one male and one female of the 150 mg/kg/d group, and in three males and two females of the 450 mg/kg/d group due to incidental aspiration of test material caused by the oral gavage application. Mortalities are considered not related to systemic toxicity of the test substance.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality occurred in one male and one female of the 150 mg/kg/d group, and in three males and two females of the 450 mg/kg/d group due to incidental aspiration of test material caused by the oral gavage application. Mortalities are considered not related to systemic toxicity of the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

450 mg/kg/day (males)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

450 mg/kg/day (males)

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day: Leukocytosis was recorded in females; in both sexes at 450 mg/kg/day (fully reversible at 150 mg/kg bw/d, partly reversible at 450 mg/kg/day).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day: females cholesterol levels were slightly to moderately increased (partly reversible).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day: marked increases in protein concentration and leukocyte count were noted in both sexes; erythrocyte counts were increased in females (all effects reversible).

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

450 mg/kg/day: Increased liver, kidney and spleen weights recorded in females (reversible).

Gross pathological findings:

no effects observed

Description (incidence and severity):

discoloration effects all reversible

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

150 and 450 mg/kg/day: increase in incidence and mean severity grade (minimal to moderate) of hyaline droplets in the kidneys of males (not classification relevant, as effect to be considered as not relevant for humans).

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on an oral (28-d) subacute study with the structural closely related source molecule a NOAEL of 450 mg/kg bw/d is delineated for the registration substance as target molecule using read-across.

Critical effects observed:

not specified

Conclusions:

Based on the oral (28-d) subacute study with the structural closely related source molecule a NOAEL of 450 mg/kg bw/d is delineated for the registration substance as target molecule using read-across.

Executive summary:

A valid oral (28-day) subacute repeated dose toxicity study was conducted with the analogue substance. The applicability of the read across approach was confirmed by the registrant via an expert statement (refer to IUCLID chapter 13), and is based on very close structural comparability as well as the very close physico-chemical characteristics of target and source molecule.

Findings related to the treatment with the analogue substance consisted of unspecific clinical symptoms like breathing noises and ruffled fur noted in high dose animals, and a slight but significant decrease in body weights of male animals of the high dose group which, however, reversed during the recovery period. Haematology revealed a minor degree of microcytar and hyperchromatic anemia which, however, were not accompanied by histologic lesions in the bone marrow. According to the pathologist, the observed increase in reticulocytes indicated a compensatory effect to changes in the hemoglobin synthesis that was fully reversible during the recovery period. Clinical chemistry revealed slightly to moderately increased cholesterol levels in male and female animals. Additionally, slight changes in urine parameters were also reversible after the recovery period.

With regard to organ weights, slight but significant increased absolute and relative liver, kidney and spleen weights in females of the high dose group were recorded, which were all reversible during recovery. A dose-response relationship was not seen and no histopathological correlate indicative of structural and/or functional changes was identified in any of these organs. No significant organ weight changes were observed in females of the low and mid dose group and in males of any dose group.

Pigment related yellowish-brown discoloration was noted in some organs like trachea, tongue, esophagus and gastrointestinal tract. However, although pigment was observed in tissues like trachea or intestinal mucosa, no degenerative lesions were observed by histopathology in these or any other organ investigated.

Microscopically an increase in incidence and mean severity grade from minimal to moderate of hyaline droplets in the kidneys of mid and high dose male animals was observed. However, no histopathological correlate in the kidneys in the affected animals was found. Moreover, these findings were reversible during recovery and no difference was seen to the animals from the control group. It is scientifically well established, that the male rat is prone to hyaline droplet formation in the kidneys and that this unique male rat-specific kidney effect has no relevance for human health (Flamm and Lehman-McKeeman 1991; Lehmann-McKeeman and Caudill 1992; Swenberg 1993; Hard eta al. 1993).

Based on the study results and in depth pathological evaluations, it was concluded by the study director that all findings are not considered adverse in nature but mainly of functional adaptive character. These effects were all reversible in a subsequent recovery period. The observed mortalities are pathologically primarily related to incidental aspiration of test material and not considered to represent systemic toxicity. Based hereupon it was concluded that the NOAEL in this study can be determined to be 450 mg/kg body weight per day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline-conform study under GLP without deviations, conducted with the analogue substance.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key information: Summary of the results from the oral subacute toxicity study (with analogue substance)

A valid oral (28-day) subacute repeated dose toxicity study was conducted with the analogue substance. The applicability of the read across approach was confirmed by the registrant via an expert statement (refer to IUCLID chapter 13), and is based on very close structural comparability as well as the very close physico-chemical characteristics of target and source molecule.

Findings related to the treatment with the analogue substance consisted ofunspecific clinical symptoms like breathing noises and ruffled fur noted in high dose animals, and a slight but significant decrease in body weights of male animals of the high dose group which, however, reversed during the recovery period. Haematology revealed a minor degree of microcytar and hyperchromatic anemia which, however, were not accompanied by histologic lesions in the bone marrow. According to the pathologist, the observed increase in reticulocytes indicated a compensatory effect to changes in the hemoglobin synthesis that was fully reversible during the recovery period. Clinical chemistry revealed slightly to moderately increased cholesterol levels in male and female animals. Additionally, slight changes in urine parameters were also reversible after the recovery period.

With regard to organ weights, slight but significant increased absolute and relative liver, kidney and spleen weights in females of the high dose group were recorded, which were all reversible during recovery. A dose-response relationship was not seen and no histopathological correlate indicative of structural and/or functional changes was identified in any of these organs. No significant organ weight changes were observed in females of the low and mid dose group and in males of any dose group.

Pigment related yellowish-brown discoloration was noted in some organs like trachea, tongue, esophagus and gastrointestinal tract. However, although pigment was observed in tissues like trachea or intestinal mucosa, no degenerative lesions were observed by histopathology in these or any other organ investigated.

Microscopically an increase in incidence and mean severity grade from minimal to moderate of hyaline droplets in the kidneys of mid and high dose male animals was observed. However, no histopathological correlate in the kidneys in the affected animals was found. Moreover, these findings were reversible during recovery and no difference was seen to the animals from the control group. It is scientifically well established, that the male rat is prone to hyaline droplet formation in the kidneys and that this unique male rat-specific kidney effect has no relevance for human health (Flamm and Lehman-McKeeman 1991; Lehmann-McKeeman and Caudill 1992; Swenberg 1993; Hard eta al. 1993).

Based on the study results and in depth pathological evaluations, it was concluded by the study director that all findings are not considered adverse in nature but mainly of functional adaptive character. These effects were all reversible in a subsequent recovery period. The observed mortalities are pathologically primarily related to incidental aspiration of test material and not considered to represent systemic toxicity. Based hereupon it was concluded that the NOAEL in this study can be determined to be 450 mg/kg body weight per day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The selected study was performed under GLP and in accordance with OECD TG 407. No other studies are available.

Justification for classification or non-classification

Resulting from the oral subacute toxicity study on the analogue substance, and based on the applicability of the proposed read across approach, the substance registered is not to be classified as to its repeated dose toxicity properties. Special target organs and/or effects with toxicological relevance for humans have not been identified.