Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, (1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl derivs.

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

2014-2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

14-day gavage study with clinical chemistry, haematology, some organ weights and examination of plasma and liver concentrations of copper

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[[2,2',2''-[29H,31H-phthalocyaninetriyltris(methylene)]tris[1H-isoindole-1,3(2H)-dionato]](2-)-N29,N30,N31,N32]copper

EC Number:

261-638-5

EC Name:

[[2,2',2''-[29H,31H-phthalocyaninetriyltris(methylene)]tris[1H-isoindole-1,3(2H)-dionato]](2-)-N29,N30,N31,N32]copper

Cas Number:

59160-79-1

Molecular formula:

C59 H31 Cu N11 O6

IUPAC Name:

[[2,2',2''-[(29H,31H-phthalocyanine-C,C,C-triyl-kN29,kN30,kN31,kN32)tris(methylene)]tris[1H-isoindole-1,3(2H)-dionato]](2-)]-copper

Test material form:

solid: nanoform

Details on test material:

BET: 17.5 m2/g
TEM (min. Feret): 46.2 nm (D50)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 42 +/- 1 days
- Weight at study initiation:
- Fasting period before study: none
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2014-06-09 To:2014-06-24

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: drinkmg water containing 0.5% carboxymethylcellulose with about 5 mg/100 ml Cremophor EL

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Daily
The substance is a homogenous suspension.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

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: Data on structurally related copper phthalocyanines
- Rationale for animal assignment (if not random): on the basis of the animal weight.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, 7 and 14.

Food consumption and water consumption were determined on study days 3, 7 and 14.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 14
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters listed below were examined:
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes
11 . Preparation of blood smears (on ly evaluated blood smears will be archived)
12. Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 14
- Animals fasted: Yes
- How many animals: all
- Parameters listed below were examined.
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum -glutamyl transferase
5. Sodium
6. Potassium
7. Chloride
8. lnorg. phosphate
9. Calcium
10. Urea
11 . Creatinine
12. Glucose
13. Total bilirubin
14. Totalprotein
15. Albumin
16. Globulins
17. Triglycerides
18. Cholesterol

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

OTHER: The determination of copper concentrations in EDTA plasma samples after were performed at the test facility.
The total concentrations of the test substance in plasma were calculated indirectly from the determined copper concentrations (inductively-coupled-plasma mass-spectrometry; experimental error + /- 0,2 mg/kg Cu).
This part of the study was carried out in compliance with the Principles of Good Laboratory Practice.
On study day 15 EDTA blood samples (about 200 μL) were collected from fasted animals by puncturing the retro-bulbar venous plexus under isoflurane anesthesia. After plasma preparation, the samples were frozen at about -80°C prior to analysis.

Half of the liver tissue (divided in two parts) of all animals were deep frozen for each animal and stored at -80°C for the determination of the test substance by the analytical chemistry.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

Organ Weights:
Adrenal glands, Kidneys, Liver, Spleen

HISTOPATHOLOGY: Yes (all gross lesion, Adrenal glands, Kidneys, Liver, Spleen)

Statistics:

Food consumption, water consumption, body weight, body weight change: , Dunnett test (two-sided)
Clinical pathology parameters, organ weights and body weights of anesthetized animals: KRUSKAL-WALLIS and WILCOXON

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:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

No animal died prematurely in the present study.
No test substance-related, adverse findings were observed. During the administration period, discolored feces were observed in all animals of test group 1 and 2 (300 and 1000 mg/kg bw/d). These findings were clearly related to the color of the test substance and assessed as being non-adverse.
The discoloration of intestinal content (stomach, jejunum, colon) in treated animals was related to the color of test substance. No discoloration of organs was observed.

Effect levels

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No indication of systemic uptake from plasma and liver copper levels. No adverse effects on haematology, clinical chemistry, body weights, organ weights.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Red blood cell and coagulation parameters (males)

		control	300 mg/kg bw	1000 mg/kd bw
RBC	Mean	7.68 k	7.60	7.73
[tera/L]	S.d.	0.18	0.20	0.47
	N	5	5	5
	Median	7.69	7.50	7.75
HGB	Mean	8.6 k	8.5	8.7
[mmol/L]	S.d.	0.2	0.3	0.4
	N	5	5	5
	Median	8.6	8.7	8.8
HCT	Mean	0.420 k	0.416	0.424
[L/L]	S.d.	0.008	0.009	0.016
	N	5	5	5
	Median	0.418	0.418	0.423
MCV	Mean	54.7 k	54.7	55.0
[fL]	S.d.	1.1	1.5	2.1
	N	5	5	5
	Median	54.6	53.7	54.7
MCH	Mean	1.12 k	1.12	1.13
[fmol]	S.d.	0.02	0.04	0.03
	N	5	5	5
	Median	1.13	1.11	1.13
MCHC	Mean	20.47 k	20.52	20.55
[mmol/L]	S.d.	0.11	0.32	0.34
	N	5	5	5
	Median	20.46	20.56	20.68
RET	Mean	2.8 k	2.6	2.5
[%]	S.d.	0.5	0.5	0.6
	N	5	5	5
	Median	2.8	2.7	2.5
PLT	Mean	818 k	877	708
[giga/L]	S.d.	110	89	99
	N	5	5	5
	Median	774	911	703
HQT	Mean	36.7 k	38.8	38.5
[sec]	S.d.	0.9	1.9	1.4
	N	5	5	5
	Median	36.6	37.4	38.3

Red blood cell and coagulation parameters (females)

		control	300 mg/kg bw	1000 mg/kd bw
RBC	Mean	7.83 k	7.61	7.73
[tera/L]	S.d.	0.30	0.19	0.13
	N	5	5	5
	Median	7.67	7.59	7.81
HGB	Mean	8.5 k	8.4	8.6
[mmol/L]	S.d.	0.2	0.2	0.2
	N	5	5	5
	Median	8.5	8.4	8.6
HCT	Mean	0.405 k	0.398	0.406
[L/L]	S.d.	0.011	0.009	0.012
	N	5	5	5
	Median	0.398	0.401	0.412
MCV	Mean	51.8 k	52.2	52.5
[fL]	S.d.	2.1	0.9	1.1
	N	5	5	5
	Median	52.2	52.1	52.9
MCH	Mean	1.09 k	1.10	1.11
[fmol]	S.d.	0.04	0.02	0.02
	N	5	5	5
	Median	1.10	1.09	1.12
MCHC	Mean	21.09 k	21.08	21.16
[mmol/L]	S.d.	0.24	0.11	0.27
	N	5	5	5
	Median	20.99	21.08	21.23
RET	Mean	1.7 k	1.8	1.6
[%]	S.d.	0.2	0.4	0.5
	N	5	5	5
	Median	1.8	1.6	1.7
PLT	Mean	828 k	839	756
[giga/L]	S.d.	70	101	83
	N	5	5	5
	Median	846	801	770
HQT	Mean	35.1 k	35.1	35.9
[sec]	S.d.	2.1	1.5	1.4
	N	5	5	5
	Median	36.4	35.2	35.3

Total white and differential blood cell count (males)

		control	300 mg/kg bw	1000 mg/kd bw
WBC	Mean	5.57 k	5.62	5.36
[giga/L]	S.d.	0.96	1.75	1.98
	N	5	5	5
	Median	5.63	5.21	5.26
NEUTA	Mean	0.61 k	0.70	0.93
[giga/L]	S.d.	0.16	0.32	0.23
	N	5	5	5
	Median	0.64	0.65	0.93
LYMPHA	Mean	4.73 k	4.72	4.16
[giga/L]	S.d.	0.84	1.53	1.72
	N	5	5	5
	Median	4.72	4.30	4.29
MONOA	Mean	0.12 k	0.10	0.10
[giga/L]	S.d.	0.03	0.03	0.06
	N	5	5	5
	Median	0.11	0.09	0.08
EOSA	Mean	0.06 k	0.06	0.14
[giga/L]	S.d.	0.02	0.03	0.09
	N	5	5	5
	Median	0.06	0.05	0.09
BASOA	Mean	0.01 k	0.01	0.01
[giga/L]	S.d.	0.01	0.00	0.01
	N	5	5	5
	Median	0.01	0.01	0.01
LUCA	Mean	0.03 k	0.03	0.03
[giga/L]	S.d.	0.02	0.01	0.01
	N	5	5	5
	Median	0.03	0.03	0.02
NEUT	Mean	11.0 v	12.6	18.3 **
[%]	S.d.	2.2	3.9	4.4
	N	5	5	5
	Median	10.9	13.6	15.6
LYMPH	Mean	84.9 v	83.8	76.4 *
[%]	S.d.	2.3	4.3	5.8
	N	5	5	5
	Median	86.2	82.6	79.3
MONO	Mean	2.1 k	1.7	1.8
[%]	S.d.	0.6	0.3	0.8
	N	5	5	5
	Median	2.1	1.7	1.6
EOS	Mean	1.2 k	1.1	2.7
[% ]	S.d.	0.4	0.3	1.9
	N	5	5	5
	Median	1.0	1.1	1.7
BASO	Mean	0.3 k	0.2	0.3
[%]	S.d.	0.1	0.1	0.1
	N	5	5	5
	Median	0.2	0.2	0.2
LUC	Mean	0.6 k	0.6	0.5
[%]	S.d.	0.2	0.1	0.2
	N	5	5	5
	Median	0.6	0.6	0.6

Total white and differential blood cell count (females)

		control	300 mg/kg bw	1000 mg/kd bw
WBC	Mean	4.53 k	4.29	4.49
[giga/L]	S.d.	0.72	0.91	1.74
	N	5	5	5
	Median	4.51	4.45	3.59
NEUTA	Mean	0.38 k	0.47	0.55
[giga/L]	S.d.	0.03	0.15	0.15
	N	5	5	5
	Median	0.39	0.38	0.50
LYMPHA	Mean	3.98 k	3.67	3.78
[giga/L]	S.d.	0.68	1.00	1.53
	N	5	5	5
	Median	3.96	3.94	3.04
MONOA	Mean	0.08 k	0.06	0.07
[giga/L]	S.d.	0.03	0.02	0.05
	N	5	5	5
	Median	0.08	0.05	0.06
EOSA	Mean	0.07 k	0.05	0.06
[giga/L]	S.d.	0.02	0.01	0.05
	N	5	5	5
	Median	0.07	0.05	0.05
BASOA	Mean	0.01 k	0.01	0.01
[giga/L]	S.d.	0.00	0.01	0.01
	N	5	5	5
	Median	0.01	0.01	0.01
LUCA	Mean	0.02 k	0.03	0.02
[giga/L]	S.d.	0.00	0.01	0.01
	N	5	5	5
	Median	0.02	0.03	0.01
NEUT	Mean	8.5 k	11.7	13.1
[%]	S.d.	1.4	5.6	3.3
	N	5	5	5
	Median	8.2	8.5	11.8
LYMPH	Mean	87.7 k	84.7	83.6
[%]	S.d.	1.1	5.8	3.2
	N	5	5	5
	Median	87.8	87.5	84.0
MONO	Mean	1.6 k	1.5	1.5
[%]	S.d.	0.5	0.3	0.5
	N	5	5	5
	Median	1.6	1.4	1.6
EOS	Mean	1.4 k	1.1	1.2
[%]	S.d.	0.1	0.1	0.4
	N	5	5	5
	Median	1.4	1.2	1.1
BASO	Mean	0.3 k	0.3	0.2
[%]	S.d.	0.1	0.1	0.1
	N	5	5	5
	Median	0.2	0.3	0.3
LUC	Mean	0.5 v	0.6	0.4
[%]	S.d.	0.2	0.1	0.1
	N	5	5	5
	Median	0.5	0.6	0.4

Enzymes (males)

		control	300 mg/kg bw	1000 mg/kd bw
ALT	Mean	0.85 k	0.87	0.87
[µkat/L]	S.d.	0.20	0.07	0.14
	N	5	5	5
	Median	0.84	0.82	0.86
AST	Mean	1.66 k	1.67	1.70
[µkat/L]	S.d.	0.16	0.36	0.29
	N	5	5	5
	Median	1.68	1.56	1.71
ALP	Mean	2.50 k	2.92	2.65
[µkat/L]	S.d.	0.11	0.55	0.55
	N	5	5	5
	Median	2.52	2.76	2.38
GGT_C	Mean	0	0	0
[nkat/L]	S.d.	0	0	0
	N	5	5	5
	Median	0	0	0

Enzymes (females)

		control	300 mg/kg bw	1000 mg/kd bw
ALT	Mean	0.60 k	0.71	0.67
[µkat/L]	S.d.	0.10	0.13	0.18
	N	5	5	5
	Median	0.56	0.64	0.67
AST	Mean	1.52 k	1.62	1.69
[µkat/L]	S.d.	0.06	0.18	0.23
	N	5	5	5
	Median	1.51	1.56	1.58
ALP	Mean	1.54 k	2.18	2.09
[µkat/L]	S.d.	0.43	0.89	0.66
	N	5	5	5
	Median	1.73	1.75	2.28
GGT_C	Mean	0	0	0
[nkat/L]	S.d.	0	0	0
	N	5	5	5
	Median	0	0	0

Applicant's summary and conclusion

Conclusions:

There is no indication of systemic uptake after ingestion based on the absence of findings and the absence of elevated copper concentrations in plasma after 14 days of exposure.