A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)

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:

1989-03-21 to 1989-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

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:

1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Principles of method if other than guideline:

The following deviations from the original protocol were not considered to have affected the integrity of study results:
1) Paraffin blocks were despatched to EPS (UK) for slide production due to heavy internal workload.
2) Pathology was performed at RCC (UK) by C.J. Springall and not by O. Vogel and J. Wilson at RCC AG, Itingen due to changed procedures.
3) On day 19, animals from group 4 were dosed with group 3 concentration due to technician error.
4) Homogeneity and stabilty of the test substance in vehicle was also determined. This was an omission in the original protocol and these determinations are routinely performed.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)

EC Number:

404-540-1

EC Name:

A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)

Cas Number:

159405-95-5

Molecular formula:

C50H56Cl4N14O6

IUPAC Name:

1'-[3-(dimethylamino)propyl]-5'-(2-{3-[2-(4-{2-[3-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium 1'-[3-(dimethylamino)propyl]-5'-(2-{4-[2-(4-{2-[4-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium tetrahydrochloride tetrachloride

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH Wiga, Sulzfeld, West Germany.
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 186 to 220 grams; females: 128 to 158 grams
- Fasting period before study: not reported
- Housing: Animals were housed 5 to a cage (same sex) in stainless steel suspended cages with wire mesh floors.
- Diet (e.g. ad libitum): free access to standard pelleted laboratory animal diet
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 35 - 80 %
- Air changes (per hr): 7.5
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: the test substance was weighed into a glass flask on an analytical balance and the vehicle (w/w) added.

DIET PREPARATION
not appliacble

VEHICLE
- Concentration in vehicle:
Concentration formulated: 200.1 mg/g; Concentration analysed after 4 h: 207.2 mg/g
- Amount of vehicle (if gavage): 5 ml/kg body weight.
- Purity: prepared yb reverse osmosis

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The detailed report on analytical verification of test item concentrationin vehicle is given as appendix to this orginial study report.

Sampling procedure:
Samples for the stability and accuracy of preparation testing were taken at 50 % height from the glass flask. For the homogeneity testing three samples were taken: one at the top (at 90% height), one at the middle (at 50 % height) and one at the bottom (at 10 % height). All samples were weighed accurately using an analytical balance. All samples were further diluted with Milli-Ro water to give suitable concentrations for analysis.

Quantitative analysis:
Calibration solutions: 2 independently prepared calibration solutions of test substance in Milli-Ro water were used each day of analysis to calibrate the analytical method.
Method of chemical analysis: spectrophotometric method.
Calibration curve: starting from two independently prepared stock solutions, six dilutions in Milli-Q water were prepared in the concentration range of 1 of 43 ug/ml. The absorbance reading was correlated with the concentration test substance, using linear regression analysis.

The concentrations of test substance in water were determined using a spectrophotometric method.

The test substance was determined to be stable for at least 4 hours in water at all concentrations tested. Furthermore, the substance formed a homogeneous suspension in water at all concentrations tested. The accuracy of preparation testing revealed that the concentrations analyzed were in agreement with the concentrations prepared. Concentration formulated: 200.1 mg/g; Concentration analysed after 4 h: 207.2 mg/g.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 10-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day) to provide a basis for selection of dose levels for the 28-day study. No differences of biological significance were observed in clinical appearance, body weight, food consumption, macroscopic appearance or liver weights between the treated groups.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: no data

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily.
- Cage side observations checked in table were included in the original study report.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, however data per group per week only

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination,
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes. The animalswere fasted overnight before blood sampling.
- How many animals: all rats/sex/group
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes /
- Time schedule for collection of blood: immediately prior to post mortem examination,
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes. The animalswere fasted overnight before blood sampling.
- How many animals: all rats/sex/group
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were necropsied and descriptions of all macroscopic abnormalities recorded. All animals surviving to the end of the observation period (day 29) were anaesthetised with pentobarbitone and killed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands
Heart
Kidneys
Liver
Spleen
Stomach
Testes

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded at termination on the scheduled dates of necropsy listed:
Adrenal glands
Kidneys
Liver
Spleen
Testes

HISTOPATHOLOGY
Slides of adrenals, heart, kidneys, liver and spleen, collected at termination from all animals of the control and high dose group and slides of liver and kidneys from the intermediate groups were examined by a pathologist. All abnormalities were described and included in the report.

Statistics:

The following statistical methods were used to analyse the body weight, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
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 and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the
basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Dark faeces and regurgitation of the test substance was noted in males receiving 1000 mg/kg/d.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Although there were no statistically significant differences in the body weights of treated rats compared to controls, the body weight gain by females receiving 1000 mg/kg was consistently statistically significantly decreased compared to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption by females receiving 1000 mg/kg/day was marginally decreased compared with controls. After adjustment for differences in body weight, relative food consumption by females receiving 1000 mg/kg/day was still noted as being marginally low.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no differences that were considered to have arisen as a result of treatment between control and treated rats. Any difference that did achieve a level of statistical significance was considered to have arisen fortuitously as all values were within normal limits as seen in rats of this age and strain.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no significant differences measured between control and treated rats. Minor statistically significant differences occurring were not considered to be related to treatment as all values remained within biologically normal limits.
In particular, statistically significantly high creatinine values noted in males receiving 1000 mg/kg were within the range normally seen in rats of this age and strain, but with the difference from control being exacerbated by one high value of 59 umol/l. Statistically significant differences in urea values between controls and males receiving 50 or 1000 mg/kg/day were, in the absence of a treatment related distribution, considered to have arisen by chance.
Treated females had statistically significantly decreased alanine aminotransferase (ALAT/GPT) levels when compared to controls, but this difference was attributed to a slightly abnormally high control value and of no biological significance.
There were no other significant differences in blood chemistry parameters measured between control and treated rats. Minor statistically significant differences occurring were not considered to be related to treatment as all values remained within biologically normal limits for rats of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights (after adjustment for body weight) of females receiving 1000 mg/kg were statistically significantly greater when compared to control values.
Liver weights of males receiving 1000 mg/kg were also greater but did not achieve a level of statistical significance in comparison with control values.
Organ weights of males and females recelvlng 50 or 200 mg/kg were similar to those of controls before and after allowance for body weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations that were noted at necropsy (eg. pelvic dilation of the kidneys) were not considered related to treatment but within the normal background range for animals of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There was an apparent increase in interstitial nephritis seen in high and intermediate dose males. The increase of this relatively frequent spontaneous background lesion was mainly unilateral and absent in treated females. Consequently, this effect is considered to be of no toxicological significance. No other treatment related findings were noted.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

2

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Animals receiving 1000 mg/kg showed increased liver weights (although not statistically significant in males). However, as there were no changes in serum liver enzyme levelt or pathological evidence to corroborate this, the toxicological significance of this change must therefore be considered doubtful.

The decreased food consumption by females receiving 1000 mg/kg/day noted even after adjustment for body weight suggest a loss of appetite among these females rather than any impairment of metabolic function.

Applicant's summary and conclusion

Conclusions:

Given some slight, but unequivocal, indices of biological effect among animals treated at 1000 mg/kg/day, the no observed effect level (NOEL) was set at 200 mg/kg/day, and the NOAEL at 1000 mg/kg bw.

Executive summary:

A sub-acute toxicity study was run to assess the toxicologic potential of the test substance, when administered to rats by daily oral gavage for a 28 -day period. The study followed the OECD guideline 407.

A 10-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day) to provide a basis for selection of dose levels for the 28-day study. No differences of biological significance were observed in clinical appearance, body weight, food consumption, macroscopic appearance or liver weights between the treated groups.

In the main study, the substance was administered daily by gavage to SPF-bred Sprague-Dawley rats. The study was comprised of 4 dose groups, 0, 50, 200, and 1000 mg/kg/d.

At 50 and 200 mg/kg/d no treatment related findings were being observed.

Following observations were made at 1000 mg/kg/d:

1) dark faeces and regurgitation of test substance.

2) decreased body weight gain in females.

3) decreased food consumption by females.

4) increased liver weight in females only.

Animals receiving 1000 mg/kg showed increased liver weights (although not statistically significant in males). However, as there were no changes in serum liver enzyme levelt or pathological evidence to corroborate this, the toxicological significance of this change must therefore be considered doubtful. The decreased food consumption by females receiving 1000 mg/kg/day noted even after adjustment for body weight suggest a loss of appetite among these females rather than any impairment of metabolic function.

Given these slight, but unequivocal, indices of biological effect among animals treated at 1000 mg/kg/day, a no observed effect level (NOEL) was set at 200 mg/kg/day, and the NOAEL at 1000 mg/kg bw.