Low chlorinated copper, [29H,31Hphthalocyaninato(2-)-N29,N30,N31,N32]-, wherein the number of chlorines is more than or equal to 0 and less than or equal to 5.

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:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

including 14-day recovery period

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Substance type: dark blue powder
- Physical state: solid
- Impurities (identity and concentrations): moisture and water soluble matter < 1 %

Specific details on test material used for the study:

- Name of test material (as cited in study report): , 99% pur
- Substance type: dark blue powder
- Physical state: solid
- Analytical purity: 99 %
- Impurities (identity and concentrations): moisture and water soluble matter < 1 %
- Lot/batch No.: 21993
- Storage condition of test material: at ambient temperature or in a cool store
-Degree of chlorination: ca 13 chlorine subsitutents on the rings
-Other identifiers in report: CAS-number and structure

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 UK
- Age at study initiation: ca. 5 weeks
- Weight at study initiation: 101 - 121 g
- Housing: 5 animals of the same sex per cage
- Diet: RMI (E) SQC pelleted diet, ad libitum
- Water: public drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 40 - 70 %
- Air changes: at least 10 per hr
- Photoperiod: 12hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

maize oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared for administration as a series of graded concentrations in maize oil. All formulations were prepared freshly each day and administered within four hours of preparation.
Animals received the test material or vehicle control formulations by gavage at a volume dose of 5 ml/kg bw. All animals were dosed once each day approx. at the same time, seven days per week. The volume administered to each animal was claculated from the body weight measured immediately before each administration.
The formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of test substance were determined in formulations prepared for one occasion of dosing during weeks 1 and 4 of treatment.

Duration of treatment / exposure:

daily for 28 (animals in the reversibility phase group) or 30 (animals in the main study group) consecutive days.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 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 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

All animals were treated for at least 28 days. Treatment and the recording of serial observations continued for all main study animals throughout the main study necropsy period (therefore main study animals were treated for 30 days). The reversibility phase commenced on day 29 and lasted for 16 days. The recording of serial observations continued throughout the reversibility necropsy period.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
All animals were observed at least twice daily for evidence of reaction to treatment or ill-health. Individual observations of all animals were also recorded before and after dosing on day of treatment. Additionally, a more detailed weekly exaination was performed on each animal. During acclimatization and recovery period, the animals and their cages were observed at least once daily.

BODY WEIGHT: Yes
Each animal was weighed during the acclimatization period, on the day of treatment and at weekly intervals throughout the treatment and reversibility period and before necropsy.

FOOD CONSUMPTION AND EFFICIENCY :
The weight of food supplied to each cage, the remaining and any an estimate of any spilled was recorded for each week throughout the treatment and reversibility periods. From these records the mean weekly consumption was calculated.
The group mean food conversion efficiencieswere calculated for each week of the treatment and reversibility periods.

HAEMATOLOGY: Yes
On day 30 of treatment (before dosing) blood samples were collected from all main study animals after overnight starvation. All samples were examined for the following characteristics:
Packed cell volume (PCV), hemoglobin concentration (HB), erythrocyte count (RBC), mean cell haemoglobin concentration (MCHC), mean cell haemoglobin (MCH), mean cell volume (MCV), total and differential leukocyte count (WBC), platelet count (PLAT), blood film - Romanowski stain for abnormal morphology and unusual cell types, incl. normoblasts, prothrombin time (PT) and activated partial thromboplastin time (PTTK).

CLINICAL CHEMISTRY: Yes
On day 30 of treatment (before dosing) blood samples were collected from all main study animals after overnight starvation. All samples were examined for the following characteristics:
Alkaline phosphatase activity (ALP), alanine amino-transferase (ALT), aspartate amino-transferase activity (AST), gamma glutamyl transpeptidase activity (GGT), glucose concentration (GLUC), total bilirubin concentration (BILT), total cholesterol concentration (CHOL), total glyceride concentration (TRIG), creatinine concentration (CREA), urea concentration, total protein concentration (TP), electrophoretic protein fractions (ALB, A-1, A-2, BETA, GAMMA), albumin-globulin ratio (A/G), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphate (Phos).
On day 16 of the recovery period, blood samples were obtained after overnight starvation and the samples were examined for ALT (males and females) and urea concentration (males only).

URINALYSIS: Yes
On day 30 of treatment, overnight urine samples were collected from all main study animals. Animals were deprived from water from approx. 12:30 hand placed into an invividual metabolism cage (at approx. 17 h) without food or water. Urine was collected until approx. 9:00 h the following day. Samples were examined for appearance (APP), volume (VOL), pH, specific gravity (SG), protein (PROT), glucose (GLUC), ketones (KET), bilirubin (BIL) and blood. Sediment from centrifugation was examined for epithelial cells (EP), polymorphonuclear leucocytes (P), erythrocytes (RBC), crystals (CRY), spermatozoa and precursors (S) or other abnormalities (A).

Sacrifice and pathology:

All animals were killed by carbon dioxide inhalation.

GROSS- and HISTOPATHOLOGY: Yes
All animals were subjected to a detailled necropsy, including a review of the history of each animal and a detailled examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic, abdominal and pelvic cavities and their viscera. The requisite organs were weighed, external and cut surfaces of organs and tissues were examined as appropriate. Abnormalities and interactions were noted and the required tissue samples preserved in fixative.
The following organs were taken and the weights were recorded: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, testes.
Adrenals, heart, kidneys, liver, spleen and testes were preserved for histopathology.
The following organs were preserved, but not processed histologically: Thoratic aorta, brain, bronchi, caecum, colon, duodenum, epididymides, eyes and optic nerves, harderian glands, ileum, jejunum, lachrymal glands, lungs, lymph nodes - mandibular and mesenteric, mammary glands - caudal and cranial, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, submandibular salivary glands, sciatic nerves, seminal vesicles, sceletal muscle - thigh, skin, spinal cord, sternum, stomach, thymus, thyroid with parathyroids, tongue, trachea, urinary bladder, uterus with cervix, vagina.
Femoral bone marrow smears were taken from all animals.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No animals died during the study. Blue staining of the coat or tail was observed in one male and one female receiving 200 mg/kg bw/day and in all animals receiving 1000 mg/kg bw/day. Blue staining of the dorsal coat occured predominantly in week 2 for affected high dosage males and in week 3 and 4 for affected high dosage females. Since this sign was not evident at the end of the treatment period its recovery could not be established. Staining of the tail first became evident during week 3 and persisted throughout the reversibility period in most animals.
There were no other signs of a reaction to treatment. There were a few incidences of salivation in association with the dosing procedure but these were either in control or low dose animals and were not attributed to treatment.
The blue staining is related to the blue colour of the pigment. It stained the feces blue and from there, secondary staining of coat and tail occurred. It is not an adverse finding.

BODY WEIGHT AND WEIGHT GAIN
The bodyweight gains of control and treated animals were essentially the same during the treatment period (see table 1) and the revsersibility phase. The slightly lower gain during the treatment period in females receiving 200 mg/kg bw/day and during the reversibility period in femals previously treated at 1000 mg/kg bw/day were considered to represent normal biological variation.

FOOD CONSUMPTION AND FOOD EFFICIENCY
The food consumption of control and treated animals was similar throughout the treatment period and reversibility phase. The food conversion efficiency of control and treated animals was essentially the same during the treatment period and the reversibility phase.

HAEMATOLOGY
Haematological investigations on day 30 of treatment showed no inter-group differences between control and treated animals that could be attributed to treatment. Inter-group differences that attained statistical significance (p<0.05) lacked dosage relationship, were minor or were confined to one sex and therefore considered to be of no toxicological significance.

CLINICAL CHEMISTRY
For mean plasma alanine amino-transferase activities, these were slightly high for females receiving 1000 mg/kg bw/day and slightly low for males receiving 1000 mg/kg bw/day. Examination of individual values showed that these inter-group differences arose as a result of two slightly high control male values and one slightly high value for a female given 1000 mg/kg bw/day. Consequently, the inter-group differences in plasma alanine aminotransferase activity were not attributed to treatment.

Other inter-group differences that attained statistical significance were either minor, lacked dosage relationship or were confined to one sex, and were therefore not attributed to treatment.

URINALYSIS
The composition of urine on day 30 was unaffected by treatment.

ORGAN WEIGHTS
No adverse effects on organ weights were observed.

GROSS PATHOLOGY
Macroscopic examination after 30 days of treatment revealed abnormal contents of the gastro-intestinal tract in two males and one female that received 1000 mg/kg bw/day. The contents were dark or blue and green. This is caused by the colour of the pigment and merely indicates is presence in the gastrointestinal tract.
There were no other macroscopic findings after 30 days of treatment or after 16 days of reversibility that was attributed to treatment or previous treatment with the test material.

HISTOPATHOLOGY:
There were no findings considered to be related to treatment with the test material.
2 males from the 1000 mg/kg bw/day group as well as one male from the 200 mg/kg bw/day group had dilatation and degeneration of the seminiferous tubules in the testes. This degeneration was, however, unilateral and was accompanied in one case by chronic inflammation of the epididymis. It is thought that these dilatations and degenerations may be associated with a blockage of the ducts distal to the testes and are unrelated to treatment.
Other findings were of the types commonly seen in rats of this age and occured with the expected frequency.

Effect levels

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at the limit dose.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Body weights of males and females during the treatment period

	GROUP:	1 (Male)	2 (Male)	3 (Male)	4 (Male)	1 (Female)	2 (Female)	3 (Female)	4 (Female)
WEEK
0	N	10	5	5	10	10	5	5	10
	MEAN	142	149	147	144	134	129	132	130
	S.D.	4.8	3.7	7.3	6.2	6.1	6.3	2.8	8.9
1	N	10	5	5	10	10	5	5	10
	MEAN	198	210	208	203	166	161	160	159
	S.D.	6.7	6.1	13.6	8.9	5.9	9.6	6.5	12.7
2	N	10	5	5	10	10	5	5	10
	MEAN	249	269	261	255	189	184	181	178
	S.D.	11.5	9.7	21	13.2	5.3	9.5	10.6	13
3	N	10	5	5	10	10	5	5	10
	MEAN	293	314	301	298	209	203	200	201
	S.D.	13.4	13.3	31.7	16.2	10.2	14	10.7	15.4
4	N	10	5	5	10	10	5	5	10
	MEAN	334	354	333	332	226	221	212	216
	S.D.	15	19	39.4	21.8	13.1	15.4	12.9	17.2
BW Gain 0-4	N	10	5	5	10	10	5	5	10
	MEAN	192	206	187	188	93	91	81	86
	S.D.	11.6	17.9	35.3	20.6	13.5	15.3	12.5	10.6
As % of control			107	97	98		98	87	92

Table 2: Liver weights of males and females

Dose group	control	40	200	1000	0 (recovery)	1000 (recovery)
male	16.7	15.4	15.8	15.6	20.6	19.8
SD	2	0.8	2.7	1.6	1.6	2.2
female	9.3	9.2	9.6	10.1	11	9.2
SD	0.8	1	0.8	0.5	1.2	1.2

Applicant's summary and conclusion

Conclusions:

Treatment of rats by gavage for 28 days did not cause treatment-related effects at the limit dose.