Hydrogen [29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with dodecylamine (1:1)

Endpoint summary

Description of key information

Subacute gavage dosing of rats caused histopathology findings in liver, mesenteric lymph nodes, kidneys (males only) and adrenal glands (males only) at 1000 mg/kg bw. The NOEL is 300 mg/kg bw. The study was performed accoring to OECD guideline 422 and under GLP.

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:

experimental study

Adequacy of study:

key study

Study period:

2014-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline 422 and GLP compliant study with well-characterized testing material

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France or Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 Dec 2014 To: 30 Jan 2015

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing
and were homogenized to a visually acceptable level. No adjustmentwas made for specific gravity/density of the test substance. Adjustment
was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.



VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance can be homogeneously suspendend in propylene glycol. It is poorly soluble in water.
- Concentration in vehicle: adjusted to volume
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes will be calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were taken at the test facility on a single occasion during the treatment period (formulations were prepared and sampled on 08 December 2014). The samples were dispatched on dry ice to the laboratory where they were analysed to assess accuracy of preparation, homogeneity and stability in vehicle over 5 hours at room temperature under normal laboratory light conditions. Formulations were analysed using LC-DAD.

Duration of treatment / exposure:

The males and females were exposed for at least 14 days prior to mating, during mating, and up to the day prior to scheduled necropsy (at least 28 days of dosing). Females were not dosed during littering.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on a 14-day dose range finding study in which dose levels of 300 and 1000 mg/kg bw/day were tested and no dose limiting effects were seen.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.



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: Yes


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked:
White blood cells (WBC)
Differential leukocyte count:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Red blood cells
Reticulocytes
Red blood cell distribution width (RDW)
Haemoglobin
Haematocrit
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Platelets
Prothrombin Time (PT)
Activated Partial Thromboplastin Time (APTT)



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked:
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline Phosphatase (ALP)
Total protein
Albumin
Total Bilirubin
Bile acids
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of treatment
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex and static righting reflex) / grip strength (fore- and hind-limb grip strength) / locomotor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

Organ weights for five animals :
Adrenal glands
Spleen
Brain
Testes (all animals)
Epididymides (all animals)
Thymus
Heart
Uterus (including cervix)
Kidneys
Prostate
Liver
Seminal vesicles including coagulating glands
Ovaries
Thyroid including parathyroid

HISTOPATHOLOGY: Yes
Adrenal glands
(Pancreas)
(Aorta)
Peyer's patches [jejunum, ileum] if detectable
Brain - cerebellum, mid-brain, cortex
Pituitary gland
Caecum
Preputial gland
Cervix
Prostate gland
Clitoral gland
Rectum
Colon
(Salivary glands - mandibular, sublingual)
Coagulation gland
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides
Skeletal muscle
Eyes (with optic nerve (if detectable) and Harderian gland)
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Female (and male) mammary gland area
Spleen
Femur including joint
Sternum with bone marrow
Heart
Stomach (forestomach and glandular stomach)
Ovaries
Ileum
Testes
Jejunum
Thymus
Kidneys
Thyroid including parathyroid if detectable
(Lacrimal gland, exorbital)
(Tongue)
(Larynx)
Trachea
Liver
Urinary bladder
Lung, infused with formalin
Uterus
Lymph nodes - mandibular, mesenteric
Vagina
(Nasopharynx)
All gross lesions
(Esophagus)

Other examinations:

staging of spermatogenesis

Statistics:

-If the variables can be assumed to follow a normal distribution, the Dunnett-test ( many-to-one t-test) based on a pooled variance estimate is applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) is applied if the data cannot be assumed to follow a normal distribution.
- The Fisher Exact-test is applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test is applied to motor activity data to determine intergroup differences. In case intergroup differences are seen, the Wilcoxon test is applied to compare the treated groups to the control group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation after dosing

Mortality:

mortality observed, treatment-related

Description (incidence):

salivation after dosing

Body weight and weight changes:

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:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver and mesenteric lymph node of males and females treated at 1000 mg/kg bw/day and in the urinary bladder and adrenal glands of males treated at 1000 mg/kg bw/day

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Blue discoloration of the faeces occurred at 100, 300 and 1000 mg/kg bw/day in all animals. Blue discoloration of the tail was noted at 300 mg/kg bw/day (in all males and a few females) and at 1000 mg/kg bw/day (most males and females). Blue discoloration of the skin/fur on the back, head or shoulder was noted in only one or a few animals of each test group. This discoloration reflected the blue colour of the test substance and was considered not to be toxicologically relevant.

Salivation was noted after dosing in all animals at 1000 mg/kg bw/day, starting a few days after initiation of treatment. Salivation was also seen consistently at 300 mg/kg bw/day in most males and a few females, and at 100 mg/kg bw/day in 5 of 10 males. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).


GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations.

Bluish contents of the gastro-intestinal tract was noted in most animals given the test substance. Blue discoloration of skin (tail) was noted at 300 mg/kg bw/day (all males, one female) and 1000 mg/kg bw/day (all males, 9/10 females). This discoloration was related to treatment, but not adverse as it reflected the colour of the test substance.

Enlarged spleen was noted in two males at 100 mg/kg bw/day, two males at 300 mg/kg bw/day and three males at 1000 mg/kg bw/day. In the absence of treatment-related changes in the weight or microscopic appearance of the spleen, these macroscopic findings were considered not to be related to treatment.


HISTOPATHOLOGY: NON-NEOPLASTIC

Liver:
-Microgranulomas were present in 4/6 males (1 minimal, 2 slight, 1 moderate) and in 1/5 females (moderate) treated at 1000 mg/kg bw/day.
-Single cell necrosis (apoptosis) was present in 2/6 males (2 minimal) and in 1/5 females (slight) treated at 1000 mg/kg bw/day.

Mesenteric lymph node:
-Increased macrophage foci were present in 4/5 males (3 slight, 1 moderate) treated at 1000 mg/kg bw/day and at increased incidence and/or severity in 4/5 females (3 slight, 1 moderate) treated at 1000 mg/kg bw/day, compared to 4/5 control (3 minimal, 1 slight), 2/5 100 mg/kg bw/day (2 minimal) and 2/5 300 mg/kg bw/day (2 minimal) treated females.

Urinary bladder:
-Hyperplasia/hypertrophy of the urothelium was present in 2/5 males (1 minimal, 1 slight) treated at 1000 mg/kg bw/day.

Adrenal glands:
-A slightly increased incidence and/or severity of vacuolation of the zona fasciculata was present in 3/5 males (2 minimal, 1 slight) treated at 1000 mg/kg bw/day, compared to 1/5 control (minimal), and 1/5 300 mg/kg bw /day (minimal) treated males.


One male (male no. 15) from the low dose group (100 mg/kg bw/day) couple showed massive tubular atrophy in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring. This animal had no normal spermatogenic staging profile. This finding was considered incidental because it involved a single animal of the low dose group.

The microgranulomas represent a multifocal inflammatory response to a difficult to digest and eliminate insult by the test substance and this is not normally seen in the liver. This, together with the single cell necrosis in the liver, was considered to be an adverse finding. The increased macrophage foci in the mesenteric lymph node also likely represent the response to a difficult to digest and eliminate insult by the test substance and therefore this was also considered adverse. The hyperplasia/hypertrophy of the urothelium in the urinary bladder was considered to be an adverse finding. The minimal increase in normally occurring vacuolation of the zona fasciculata in the adrenal glands was considered to be nonadverse.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on liver, kidney, mesenteric lymph nodes and adrenal glands at 1000 mg/kg bw

Critical effects observed:

not specified

Stability and homogeneity: No test substance was detected in the control formulations. The concentrations analysed in the formulations of the test groups were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%). The long term storage samples were stable at ≤-70°C for at least 36 days at the high concentration level (200 g/L) and for at least 21 days at the low concentration level (1.00 g/L).

Table 1: Histopathology findings occurring in both male and female rats

	Males				Females
Dose level (mg/kg bw/day):	0	100	300	1000	0	100	300	1000
Liver^a	5	5	5	6	5	6	6	5
Microgranulomas
Minimal	-	-	-	1	-	-	-	-
Slight	-	-	-	2	-	-	-	-
Moderate	-	-	-	1	-	-	-	1
Single cell necrosis  (apoptosis)
Minimal	-	-	-	2	-	-	-	-
Slight	-	-	-	-	-	-	-	1
Mesenteric lymph node^a	5	5	5	5	5	5	5	5
Increased macrophage foci
Minimal	-	-	-	-	3	2	2	-
Slight	-	-	-	3	1	-	-	3
Moderate	-	-	-	1	-	-	-	1

Table 2: Histopathology findings occurring in males rats only

	Males
Dose level (mg/kg bw/day):	0	100	300	1000
Urinary bladder^a	5	5	5	5
Hyperplasia/hypertrophy  urothelium
Minimal	-	-	-	1
Slight	-	-	-	1
Adrenal glands^a	5	5	5	5
Vacuolation zona fasciculata
Minimal	1	-	1	2
Slight	-	-	-	1

^a = Number of tissues examined from each group.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Valid without restriction

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

The substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, propylene glycol, alone. Males were exposed for 2 weeks prior to mating, during mating, and up to termination for 29 days. The females were exposed for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 4 days of lactation (total exposure period 42-56 days). The study was performed according to OECD guideline 422 and under GLP. This summary only reports on effects on repeated dose toxicity.

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

Salivation occurred after dosing in all rats treated with 1000 mg/kg bw/day, in most males and a few females at 300 mg/kg bw/day, and in five out of ten males at 100 mg/kg bw/day.This was considered to be a physiological response rather than a sign of systemic toxicity.

 Other treatment-related clinical and/or macroscopic findings included blue discoloration of the faeces and bluish contents of the gastro-intestinal tract in most animals given the test substance, blue discoloration of the tail at 300 and 1000 mg/kg bw/day, and, less frequently, blue discoloration of the skin/fur. These findings were attributable to the blue colour of the test substance and were not toxicologically relevant. 

At histopathological examination, treatment-related changes were noted in the liver (microgranulomas, single cell necrosis) and mesenteric lymph node (increased macrophage foci) of males and females at 1000 mg/kg bw/day, and in the urinary bladder (hyperplasia/hypertrophy of the urothelium) and adrenal glands (vacuolation of the zona fasciculata) of males at 1000 mg/kg bw/day.The microgranulomas represent a multifocal inflammatory response to a difficult to digest and eliminate insult by the test substance and this is not normally seen in the liver. This, together with the single cell necrosis in the liver, was considered to be an adverse finding. The increased macrophage foci in the mesenteric lymph node also likely represent the response to a difficult to digest and eliminate insult by the test substance. Thehyperplasia/hypertrophy of the urothelium in the urinary bladderwas considered to be an adverse finding. The minimal increase in normally occurring vacuolation of the zona fasciculatain the adrenal glands was considered to be non-adverse.

 No treatment-related or toxicologically relevant changes were noted in any of the remaining parental parameters investigated in this s

tudy (i.e. functional observations, body weight, food consumption, clinical laboratory investigations and organ weights). The NOEL is 300 mg/kg bw.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; digestive: other; urogenital: kidneys

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. No serious irreversible effects were observed at dose levels of less than 150 mg/kg bw upon subacute exposure. As a result the substance is not considered to be classified for repeated dose toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

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. Effects were only observed at 1000 mg/kg bw. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive EC944/2013.