1-Octadecanaminium,N,N-Dimethyl-N-Octadecyl-,(Sp-4-2)-[29h,31h-Phthalocyanine2-Sulfonato(3-)-N 29,N 30,N 31,N 32]Cuprate(1-)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study; well documented study report

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)

Deviations:

yes

Remarks:

Stomachs were sampled at termination and preserved due to the changed standard procedures. Determination of accuracy of preparation was carried out in wks 2, 3 & 4 instead of in wk 1 & 4, because analytical method had not been determined in wk 1. These de

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Stomachs were sampled at termination and preserved due to the changed standard procedures. Determination of accuracy of preparation was carried out in wks 2, 3 & 4 instead of in wk 1 & 4, because analytical method had not been determined in wk 1. These de

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US Food and Drug Administration, Bureau of Food, Toxicological Principle for the Safety Assessment of Direct Food Additives And Color Additive Used in Food. (1982).

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: Blue powder
- Lot/batch No.: Ref Mix 51785
- Purity: 100% - Moisture
-Stable under storage conditions
- Storage condition of test material: At room temperature in the dark

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: 166~224 g; Females: 139~166 g.
-Fasting period before study: None
-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 (RMH-B from Hope Farms, The Netherlands)
-Water (e.g. ad libitum): free access to tap-water. Results of chemical and contaminates analyses are archived quarterly.
-Acclimation period: 14 days (7 days pre- and 7 day post randomization) .

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 21± 3 oC
-Humidity (%): 30-70%
-Air changes: 7.5 – 15 air changes per hr
-Photoperiod (12 hrs dark /12 hrs light)

IN-LIFE DATES: From: February 28th, 1989 To: March 28th, 1989

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% Aqueous methyl cellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared at the appropriate concentration as a solution in 1% aqueous methyl cellulose. Analysis of the stability of these formulations showed them to be stable for at least 4 h. Formulations were therefore prepared daily immediately prior to dosing and kept at room temperature.
VEHICLE
- Justification for use and choice of vehicle (if other than water): At all concentration tested, the test substance formed a homogenous suspension in 1% aqueous methyl cellulose, and this preparation was determined to be stable for at least 4 h at room temperature.
- Concentration in vehicle: 10, 40 and 200 mg/g.
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations, homogeneity, stability of the test material in 1% aqueous methyl cellulose, and accuracy of preparation of formulation were determined for the subacute 28-day oral toxicity study in the rat using a spectrophotometer method.

Concentration:
Concentration of the test article in vehicle was determined on days 9, 15 and 22. Results of analysis revealed that the concentrations analyzed were in agreement with the concentration prepared.

Homogeneity: the highest and lowest nominal concentrations prepared in week 2 were tested. Samples were taken: one at the top (~90% height), one at the middle (~50% of height), and one at the bottom (~10% of height).
Stability: the highest and lowest nominal concentration prepared in week 2 and 3 were tested immediately after preparation and 4 h after storage at ambient temperature. Samples were taken at 50% height from the glass flask.

Accuracy of preparation: All formulations prepared in week 2,3,4 were analyzed. Samples were taken at 50% height from the glass flask.

Spectrophotometer: Perkin Elmer Lamda 5 (double beam), band pass 1nm, detection wavelength 675 nm, curettes (quarts).

Conclusions: the test substance formed a homogenous suspension in 1% aqueous methyl cellulose, and this preparation was determined to be stable for at least 4 h at ambient temperature.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, approximately the same time each day, 7 days per week.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day active ingredient (vehicle control)
Basis:
other: nominal via gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg/day active ingredient
Basis:
other: nominal via gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
200 mg/kg/day active ingredient
Basis:
other: nominal via gavage

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg/day active ingredient
Basis:
other: nominal via gavage

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels based upno a preliminary range finding study. The 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 difference of biological significance were observed in clinical appearance, body weight, food consumption, macroscopic appearance or liver weights between the treated groups of the dose range finding study.
- Rationale for animal assignment (if not random): Random
- Section schedule rationale (if not random): Random

Positive control:

Not used

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked in table were included. No.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Day 29
- Dose groups that were examined: vehicle control, 50, 200 and 1000 mg/kg bw/day treated groups.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes (pentobarbitone)
-Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters checked in table 1 were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters checked in table 2 were examined.
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data
OTHER: Organ weight, and histopathology

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see table 3)

Statistics:

The following statistical methods were used to analyze the body weight, food assumption, organ weight, and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
In the variable 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 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 round off before printing. For example, test statistics were calculated on the basis of extract 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

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Dark feces were noted among all rats receiving the test material. the onset of this clinical finding was recorded as being from day 4 for all treated animals with severity being dose-related.
No other clinical signs of a reaction to treatment were noted.
One male receiving 200 mg/kg/day showed an incidental finding of chromodacryorrhea on day 4 only.
Mortality: One animal (a male receiving 2000 mg/kg) was sacrificed on day 4 due to mis-alignment of its incisors which caused wounding of the gum and an inability to eat.
No mortality occurred as a result of treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
The body weights and body weight gains of rats receiving the test material were similar to those of controls at all times.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food intake by treated rats was similar to that of the controls at all times.

FOOD EFFICIENCY
No data.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not applicable.

OPHTHALMOSCOPIC EXAMINATION:
No data.
HAEMATOLOGY
There were no differences that were considered to have arisen as a result of treatment and any difference that attained a level of statistical significance was considered to have arisen fortuitously.

CLINICAL CHEMISTRY
Male receiving 1000 mg/kg/day had a slight, but statistically significant increased levels of alanine aminotransferase (GPT) when compared to controls.
There were no other differences noted between control and treated rats that were considered to be related treatment with the test material. any other value that achieved level of statistical significance was considered to have arisen fortuitously and not to be of toxicological significance.

URINALYSIS
No data.

NEUROBEHAVIOUR
No data.

ORGAN WEIGHTS
Males received 1000 mg/kg/day has statistically scientifically heavier livers and spleens, after adjustment for body weight, when compared to control values.
No differences were noted between males receiving 50 or 200 mg/kg/day or treated females when compared with controls.

GROSS PATHOLOGY:
Two male receiving 1000 mg/kg/day had dark areas in the stomach that were considered to represent small areas of hemorrhage.

HISTOPATHOLOGY: NON-NEOPLASTIC
Male receiving 1000mg/kg/day showed slight increase in splenic haematopoiesis which was not apparent in males receiving 200 or 50 mg/kg/day, or in treated females.
Small foci of hepatic necrosis were slightly more severe in males receiving 1000 mg/kg/day.
Two males and two females receiving 1000 mg/kg/day had slight hyperkeratosis of the forestomach.
There was no pathological evidence for macroscopically observed area of hemorrhage in the stomach of two males receiving 1000 mg/kg/day.

Effect levels

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The discoloration of the feces noted clinically was considered consistent with the coloration of the test material. This observation was not therefore considered of biological significance in the toxicological evaluation of the test material.

A primary effect of the test material related to the liver of males receiving 1000 mg/kg/day, and was manifest as increased alanine aminotransferase levels and increased relative liver weights.

Hyperkeratosis, noted in the stomach of both male and female receiving 1000 mg/kg/day, may be indicative of a local, but mild, irritant effect of the test material.

In the absence of any changes in the hematological cell profile, or corroborative effects in females, the toxicological significance of the increased splenic haematopoisis and increased relative spleen weights in males receiving 1000 mg/kg/day must be considered doubtful.

No evidence of a toxic reaction to treatment with the test material was noted at any of the three treatment levels in any of the females or in males receiving 200 or 50 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material to rats, by gavage, at dose levels of 1000 mg/kg/day caused (1) increased levels of alanine aminotranserase (male only); (2) increased relative liver and spleen weights (male only); 93) increased splenic hematopoiesis (males only), increased severity of small foci of hepatic necrosis (male only), and slight hyperkeratosis of the forestomach (males and females). From the results, the NOEL of 200 mg/kg/day was established.

Executive summary:

The 28-day study was carried out to OECD 407 guidelines. The doses used were 1000, 200 & 50 mg/kg/day. No major findings were observed and 200 mg/kg/day was considered to be the NOEL.

At the top dose minor increases in weight were observed in the liver & spleen of males and there were slightly raised alanine aminotransferase levels in 4/5 males.

The increase in splenic haematopoiesis in the top dose males was very slight and in the absence of effect on the blood parameters and findings in females, is considered of no toxicological significance.

Stomach haemorrhage was observed but only in 2/5 males and hyperkeratosis was noted in stomach of males (2/5) & females (2/5) given 1000 mg/kg/day. These stomach effects could have been caused by the mild irritant effect of the substance when given at the top dose.

There was a slight increase in the severity of small foci of hepatic necrosis in top dose males but this was reported as being limited to a few cells and is known to vary wildly, in rats of the age and strain used.

The faeces of all animals were discoloured in a dose response related manner, indicating the excretion of unchanged material.