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

Endpoint summary

Description of key information

For the UVCB substance with a higher average substitution grade, a 14-day study in rats with 300 and 1000 mg/kg bw of the substance showed absence of toxicity. No indication of systemic uptake as determined via serum copper concentrations was observed. For support, data on the smaller copper phthalocyanine core (CAS 147-15-8) is used. It shows lack of effects in rats and mice upon subchronic feed exposure. 

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:

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

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

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

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

Dose / conc.:

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

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.

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

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.

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.

Critical effects observed:

no

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

For a similar substance, a 14-day study in rats with 300 and 1000 mg/kg bw of the substance showed absence of toxicity as determined by clincical chemistry, haematology, organ weight determinations, clinical observations and body weight measurements. Intestinal passage was indicated by a blue discoloration of feces; there was no discoloration of internal organs or of urine. For the full subacute toxicity study, read-across is done to the core structure.

Read-across to the blue pigment copper phthalocyanine (CAS 147-14-8) is justified because the target substance phthalimidomethyl copper phthalocyanine is as inert as its core structure. All available toxicological data (acute toxicity, irritation, sensitization, 14-day oral study, genotoxicity) show absence of adverse effects. The measured solubility in water is as low as for copper phthalocyanine. The solubility in octanol is higher, but with 1 mg/L still very low. The phthalimido group is unreactive and does not introduce a new hazard. For details on phthalimide it is referred to the dissiminated ECHA dossier and to the data matrix in the read-across justification. Phthalimide is not classified based on experimental data submitted to ECHA for a 10-100 tpa registration including an OECD 422 study (accessed November 7th, 2015). The same data was also used in the OECD HPV programme (SIAM 20, 2005).

The same pattern of an inert core and a non-reactive derivative is observed for the violet pigment quinacridone and its phthalimido quinacridone derivative (CAS 332142-67-3). The latter is of interest because the molecular weight of both the core and the derivative are lower than that of the target substance. This would make any hypothetical systemic uptake more likely. The phthalimido quinacridone derivative (CAS 332142-67-3) is nontoxic in all studies (subacute oral toxicity at the limit dose, genotoxicity, acute toxicity, irritation and sensitization; see data matrix). If of interest, data on the quinacridone core (Pigment Violet 19, CAS 1047-16-1) it is referred to the dissiminated REACH dossier. No adverse effects are were reported in any study.

 

Organic pigments are poorly soluble in water and organic solvents. For analytical purposes of the target substance, concentrated sulphuric acid needs to be used as other solvents are not suitable. In the absence of solubility, transport processes are not possible and no systemic uptake takes place.

 

For both copper phthalocyanine and its phthalimido derivative, copper concentrations in tissues were determined after 90-day and 14-day oral dosing of doses of 1000 mg/kg bw or higher. For neither substance, there was an increase in systemic copper concentrations. This is a strong indicator that there is no systemic uptake. In case of another organic copper pigment (Pigment Yellow 129) which disintegrates in stomach acid, 14-day oral dosing of 300 and 1000 mg/kg bw caused a clear and dose-dependent increase in liver copper concentrations. Liver is the known organ for copper storage. The fact that even 3-months feed application of up to 4500 mg/kg bw of rats and mice did not result in increased systemic copper concentrations shows that this core structure is inert. Indeed, this result was used as argument by the US authorities not to perform a carcinogenicity study under the US National Toxicology Program.

 

In conclusion, it is predicted that the NOEL for a 28-day oral toxicity study (OECD 407) will be at the limit dose.

A full read-across justification document with data matrix is attached in the toxicokinetic section of IUCLID.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study on test substance

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no significant toxic effects at doses of less than 300 mg/kg bw upon subacute oral exposure in rats. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Directive EC 202/1182.