29H,31H-phthalocyanine

Endpoint summary

Description of key information

- Oral: NOAEL = 1000 mg/kg bw/day, subchronic

- Inhalation: NOAEC = 30 mg/m3, subacute

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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

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

Dose / conc.:

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

Dose / conc.:

200 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 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.

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.

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.

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.

Critical effects observed:

no

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

Conclusions:

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

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Guideline for 28-Day Repeated Dose Toxicity Test in Mammalian Species (Chemical Substances Control Law of Japan)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

including 14-day recovery

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Pigment Green No. 7
- Analytical purity: 99.1 %

Species:

rat

Strain:

other: Crj: CD(SD) IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 138 - 156 g(males), 110 - 120 g(females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/-3°C
- Humidity (%): 53 - 71%

Further details available upon translation.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Pigment Green 7 is generally insoluble, it can be suspended in corn oil.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

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

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 animals per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

TEST SUBJECTS
Age of animals at study initiation: 5 weeks
Number of animals: 5 males and 5 females per group

STUDY DESIGN
No satellite groups
Post-exposure period: 14 days (control and 1000 mg/kg bw groups)

Observations and examinations performed and frequency:

Observations (frequency): general status (daily), body weight (study initiation, once per week), food consumption (daily), hematological examination (autopsy), urinalysis (4 days prior to autopsy)

Sacrifice and pathology:

Terminal kill: males and females, days 29 and 43
Organs examined at necropsy: brain, liver, kidney, adrenal gland, testis, ovary, pituitary, eyes, lung, stomach, thyroid gland, heart, spleen, bladder, bone marrow
Gross necropsy and histopathology was performed

For blood chemistry, see attachment.

Details on results:

No effects of pigment green No. 7 were detected in terms of general condition, body weights, food consumption, hematological or blood chemistry parameters, urinanalysis, organ weights, or pathological examinations in males or females. No deaths occured in either sex.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

no

Tables can be viewed in the attachment.

No clinical observations were recorded in female and male rats. No mortalities occurred.

No treatment-related effects were detected on bodyweights and food consumption

No evidence of treatment-related effects were found in female and male rats by evaluation of hematology, coagulation, biochemistry, urine analysis.    

No treatment-related effects on organ weights both in female and male rats.

No treatement-related abnormalities were observed by necropsy and histopathological evaluations.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

other: Guideline for 28-Day Repeated Dose Toxicity Test in Mammalian Species (Chemical Substances Control Law of Japan)

Principles of method if other than guideline:

TEST SUBJECTS
Age of animals at study initiation: 5 weeks
Number of animals: 5 males and 5 females per group

VEHICLE: corn oil

OBSERVATION:
The animals were observed daily for body weight changes and food consumption. Hematological examination, as well as blood and urinanalysis, and pathological examination were also done.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 99.04 %

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: 134 - 156 g in males and 109 - 138 g in females
- Housing: in pairs
- Diet: laboratory animal solid feed (MF: Oriental Yeast Co., Ltd.)
- Water (ad libitum): tap water (filtered + ultraviolet radiation)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): approx. 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

The volume of the test substance suspension was 10 ml/kg body weight.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

140 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:

6 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No obvious toxicity was observed, except for mild weight gain suppression in female rats in the 1000 mg/kg group, in a preliminary 7-day repeated dose oral test of 0, 100, 300 and 1000 mg/kg of the test substance. Therefore, the high dosage was set to be 1000 mg/kg, with intermediate and low dosages of 140 and 20 mg/kg, respectively, representing a ratio of approximately 7.

- Post-exposure recovery period in satellite groups: yes for high dose and control group (14 days)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of the study, then once per week

FOOD CONSUMPTION: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at planned necropsy
- Anaesthetic used for blood collection: Yes (intra-abdominal thiopental anesthesia)
- Animals fasted: Not specified
- How many animals: all animals
- Parameters examined: erythrocyte count, leucocyte count, thrombocyte count, plus hemoglobin level, hematocrit, leucocyte differentiation, reticulocyte count, prothrombin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at planned necropsy
- Animals fasted: Not specified
- How many animals: all animals
- Parameters examined: total protein, albumin, A/G ratio (calculated from total protein and albumin results), glucose, triglyceride, total cholesterol, urea nitrogen, creatinine, calcium, inorganic phosphorus, GOT, GPT, y-GTP, ALP, sodium, potassium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: days prior to autopsy and at the end of the administration period
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: pH, occult blood, protein, glucose, ketone body, bilirubin, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

Terminal kill was on days 29 or 43.

GROSS PATHOLOGY: Yes (brain, liver, kidney, adrenal gland, testis, ovary, pituitary glands, eyes (including accessory glands), lung, stomach, thyroid gland (including parathyroid gland), heart, spleen, bladder, bone marrow (femur))

HISTOPATHOLOGY: Yes (heart, liver, kidney, adrenal gland and spleen from male and female rats in control and 1000 mg/kg groups)

Other examinations:

Weights of brain, liver, kidney, adrenal gland, testis and ovary were measured.

Statistics:

- Bartlett's test for homogeneity of variances: When variances were homogeneous, a one-way ANOVA was used, followed by a comparison of
means with Dunnett's or Scheff's test. When variances were not homogeneous, a Kruskal-Wallis test was employed, followed by a Dunnett-type or Scheff-type rank sum test.
- Urine data were analyzed with Armitage's x² test.
- The significance level was set at less than 5%.

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Thrombocyte count decreased in female rats of the 20 mg/kg group at completion of test substance administration.
- Prothrombin time was shortened in male rats in the 1000 mg/kg group after recovery.
As these changes were not dosage-dependent and the magnitude of the changes was small, they were considered as incidental changes within the range of physiological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Triglycerides increased in male rats of the 140 mg/kg group.
- Sodium decreased in the 1000 mg/kg group at the completion of test substance administration.
As these changes were not dosage-dependent and the magnitude of the changes was small, they were considered as incidental changes within the range of physiological variation.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- At the end of the recovery period ovary weight was increased in female rats in the 1000 mg/kg group.
As the ratio of ovary to body weight showed no difference, and no change in ovary weight was observed at completion of test substance administration, the change was considered as incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- At the completion of test substance administration grey-white spots were present in the spleen, and bleeding in die lung, nodules in die diaphragm side of the liver, serous fluid retention in ovarian capsule, and adhesions between the lung and the thoracic wall were observed.
As no uniform tendencies were indicated in these incidences, these changes were regarded as incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Hyaline deposits in the renal tubule epithelium were seen in male rats in the control group and in all rats in the 1000 mg/kg group.
As this was a mild change and no differences in deposit levels emerged between this group and the cotnrol group, this was regarded as incidential.

- Small myocardial degeneration lesions, extramedullary hematopoiesis and localized capsulitis of the spleen, diffuse adipose formation in the liver, halophilic changes in the renal tubule epithelium, renal cysts and hyaline casts in the renal tubule were found occasionally in each group.
As no tendencies were observed in the incidences, these changes were regarded as incidential.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse findings

Critical effects observed:

no

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

; no clinical chemistry, hematology, or urinalysis were conducted and no organ weights were taken (as recommended in OECD Guideline 408). In accition, copper levels were determined in liver and kidneys

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 97.8 %

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Harlan Industries
- Weight at study initiation: males: 70 - 105 g; females: 70 - 100 g
- Housing: polycarbonate cages: groups of 5 rats per cage
- Diet: weighed portions of Purina Lab Chow in meal form, mixed together with weighed portions of the test material (see details at "doses/concentrations")
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS:
- Temperature: 21 - 23 °C
- Humidity: 40 - 60 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) were selected for both males and females. The selected doses were prepared by mixing weighed portions of purina Lab Chow in meal form with weighed portions of the test material. For each dose level, one weekly lot of 4500 g was prepared.

The actual mixtures were composed of the following ingredients:
- Dose level 5.0 % (w/w): 225 g test material and water + 4275 g meal
- Dose level 2.5 % (w/w): 112.5 g test material and water + 4387.5 g meal
- Dose level 1.25 % (w/w): 56.25 g test material and water + 4443.75 g meal
- Dose level 0.6 % (w/w): 27 g test material and water + 44735 g meal
- Dose level 0.3 % (w/w): 13.5 g test material and water + 4486.5 g meal

Each diet was mixed in a Patterson-Kelly twin shelled V blender for 15 min.
The doses were mixed one or two days prior to the week of their use in the study, and stored at 23 °C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One analysis was performed to determine the accuracy of mixture concentrations; results were within +- 10 % of the desired dose concentration.

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily

Dose / conc.:

0.3 other: % in the diet

Dose / conc.:

0.6 other: % in the idiet

Dose / conc.:

1.25 other: % in the diet

Dose / conc.:

2.5 other: % in the diet

Dose / conc.:

5 other: % in the diet

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, plain diet

Details on study design:

- Five dose levels of 0.3, 0.6, 1.25, 2.5 and 5.0 % in feed were used in this study (approx. 300, 600, 1200, 2300 and 4600 mg/kg bw for males [based on 15.4 g/d average food consumption, 0.167 kg average bw] and approx. 300, 600, 1250, 2500 and 5000 mg/kg bw for females [based on 12.3 g/d average food consumption, 0.123 kg average bw], respectively).
- All dose levels were prepared on a weight per weight basis. There were five dose level groups and one group of controls per sex, with ten individuals of each sex in each dosage and control group. Each dosed group received dosed feed mixture on 91 consecutive days.

Positive control:

not required

Observations and examinations performed and frequency:

Animals were observed twice each day for clinical signs, with at least six hours between observations. All clinical signs were recorded daily. Additional studies included blood sampling for the animal disease screening program from ten control rats, five males and five females.

Sacrifice and pathology:

- Rats were necropsied on day 92 and 93.
- Gross examination were performed on all animals from all dosage groups.
- Microscopic examinations were performed on all tissues from all animals in the control group and the highest dose treatment group: Kidney, liver, lung, heart, testis, epididymis, prostate, stomach, thyroid, skin, cecum, pancreas and spleen.

Other examinations:

Copper analyses were completed in the liver and kidney tissues and the formalin preserving those tissues from male mice in the highest dose group (5 % w/w) and control groups. See details and results in endpoint "7.1.1. Basic toxicokinetics".

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were dose-related trends in body weights among both male and female rats, when body weights are expressed in percent differentials of weight gains compared with untreated controls. Male rats at the highest doses of 5.0 % and 2.5 % had differential weight gains of -11 % and -5 % respectively. Female rats at the top doses of 5.0 and 2.5 % had differential weight gains of -9.0 %. These changes in dosed group body weights are not severe and may not be a meaningful sign of toxicity.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were slight trends in patterns of diet consumption between dosed and control rats of both sexes, with dosage groups eating about 1 g more of the higher concentrations of the dosed feed. This may be an adjustment in diet intake to offset the relatively high portion of non-caloric dyestuff present in the diet. Daily average diet consumption for female rats ranged from 11.5 g (controls) to as much as 12.7 g (1.25 %); for male rats, the range was from 14.6 g (controls) to 16.6 g (2.5 %).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

The authors recommended an dosage level of 5 % and 2.5 % test substance to be used in the chronic study, due to lack of compound-related lesions.

Dose descriptor:

NOAEL

Effect level:

ca. 4 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse findings

Dose descriptor:

NOAEL

Effect level:

ca. 5 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse findings

Critical effects observed:

no

Reference 5

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer reviewed well documented study.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

; no clinical chemistry, hematology, or urinalysis were conducted and no organ weights were taken (as recommended in OECD Guideline 408).

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 97.8 %

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Harlan Industries
- Age at study initiation: 8.5 weeks
- Weight at study initiation: males: 20 - 24 g; females: 15 - 18 g
- Housing: polycarbonate cages: groups of 5 mice per cage
- Diet: weighed portions of Purina Lab Chow in meal form, mixed together with weighed portions of the test material (see details at "doses/concentrations")
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 23 °C
- Humidity: 40 - 60 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) were selected for both males and females. The selected doses were prepared by mixing weighed portions of purina Lab Chow in meal form with weighed portions of the test material. 12 % water was added to the test material as a dust control agent prior to mixing with the meal. For each dose level, one weekly lot of 4500 g (+ 12 % water compensation) was prepared.

The actual mixtures were composed of the following ingredients:
- Dose level 5.0 % (w/w): 225 g test material and water + 4275 g meal
- Dose level 2.5 % (w/w): 112.5 g test material and water + 4387.5 g meal
- Dose level 1.25 % (w/w): 56.25 g test material and water + 4443.75 g meal
- Dose level 0.6 % (w/w): 27 g test material and water + 44735 g meal
- Dose level 0.3 % (w/w): 13.5 g test material and water + 4486.5 g meal

Each diet was mixed in a Patterson-Kelly twin shelled V blender for 15 min.
The doses were mixed one or two days prior to the week of their use in the study, and stored at 23 °C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One analysis was perfomed to determine the accuracy of the mixture concentration. Results were within +- 10 % of the desired dose concentration.

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily

Dose / conc.:

0.3 other: % in the diet

Dose / conc.:

0.6 other: % in the diet

Dose / conc.:

1.25 other: % in the diet

Dose / conc.:

2.5 other: % in the diet

Dose / conc.:

5 other: % in the diet

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, plain diet

Details on study design:

- 10 animals were used per sex and dose group.
- Five dose levels of 0.0, 0.3, 0.6, 1.25, 2.5 and 5.0 % in feed were used in this study (approx. 0, 1000, 2000, 4000, 8000 or 16000 mg/kg bw/day for males, [based on 8.2 g/d average food consumption, 0.026 kg average bw] and approx. 0, 1200, 2500, 5000, 10000 and 20000 mg/kg bw/day for females [based on 7.7 g/d average food consumption, 0.019 kg average bw]).
- The selected doses were prepared by mixing together weighed portions of Purina Lab Chow in meal form with weighed portions of the chemical.
- Each dosed group received dosed feed mixture on 91 consecutive days.

Observations and examinations performed and frequency:

Animals were observed twice each day for clinical signs, with at least 6 hours between observations. All clinical signs were recorded daily. Additional studies included blood sampling for the animal disease screening program from 10 control mice, 5 males and 5 females.

Sacrifice and pathology:

Mice were necropsied on day 92 and 93.
- Gross examination was performed on all animals from all dosage groups.
- Microscopic examinations were performed on following organs from all animals in the control group and the highest dose treatment group: Kidney, liver, lung, heart, epididymis, stomach, thyroid, skin (only in control group: bone marrow, urinary bladder, testis).

Other examinations:

Copper analyses were completed in the liver and kidney tissues and the formalin preserving those tissues from male mice in the highest dose group (5 % w/w) and control groups. See details and results in endpoint "7.1.1. Basic toxicokinetics".

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

4 deaths occurred during the study: 3 males at 1.25 % and 1 control male. These deaths were not considered to be test substance-related. There were no early dead females during the course of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A -9 % to +8 % differential weight gain was seen in the dosed females. Animals in the highest dose levels had less weight gain than controls. However, given the normal variability in mouse body weight measurements, this differences did not necessarily reflect a manifestation of toxicity. Dosed male mouse groups showed a positive weight differential in all groups except the 0.6 % dosage group. In this group during week 12 a corresponding weight loss was observed, which was not fully recovered during the final week of the study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no trends in diet consumption among dosed animals compared with controls in either male or female mice. Average daily consumption among male mice dosed groups and controls ranged from 7.6-9.5 g; female mice dosed groups and controls ranged from 7.5 to 7.8 g. While male ranges were somewhat erratic, there was no indication of dose-related effects.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

A dosage level of 5 % and 2.5 % test substance was recommended to be used in the chronic study, due to lack of compound-related lesions.

Dose descriptor:

NOAEL

Effect level:

ca. 16 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse findings

Dose descriptor:

NOAEL

Effect level:

ca. 20 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse findings

Critical effects observed:

no

Reference 6

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study (Japanese guideline). The original report was written in Japanese, an abstract and the relevant results (detailled) were available in English.

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: technical grade

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

200 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:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Administration period: 28 days for males and females
Post-exposure period: Terminal sacrifice was on day 29 or 43 (14-day recovery for high dose and control group)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION: Yes
HAEMATOLOGY: Yes
CLINICAL CHEMISTRY: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

After the 28 days of administration, a slight, but significant decrease in red blood cell count (RBC) and decrease of hemoglobin (Hb) and packed cell volume (PCV) were detected in the 200 and 1000 mg/kg male groups. These slight changes were dose dependent but within the normal biological range. After the recovery period, a statistically significant increase of erythroblasts was detected in the 1000 mg/kg female group.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Increases of absolute organ weights of lung, spleen, adrenal and salivary gland and a tendency for increased relative organ weights of the spleen, evident in the 1000 mg/kg male group, were detected. These changes were not statistically or biologically significant.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse findings

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Reference 7

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

; no clinical chemistry, hematology, or urinalysis were conducted and no organ weights were taken. Extra investigation on copper content in liver and kidneys included.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: The chemical analysis, performed at Midwest Research Institute indicated that the purity was 104.7 % +- 1.1 % (elemental analysis)

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Industries
- Weight at study initiation: males: 72 - 94 g; females: 70 - 90 g
- Housing: polycarbonate cages: groups of 5 rats per cage
- Diet: weighed portions of Purina Lab Chow in meal form, mixed together with weighed portions of the test material (see details at "doses/concentrations")
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 23 °C
- Humidity: 40 - 60 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: feed

Vehicle:

other: 12 % water was added to the test material as a dust control agent

Details on oral exposure:

Dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) were selected for both males and females. The selected doses were prepared by mixing weighed portions of purina Lab Chow in meal form with weighed portions of the test material. 12 % water was added to the test material as a dust control agent prior to mixing with the meal. For each dose level, one weekly lot of 4500 g (+ 12 % water compensation) was prepared.

The actual mixtures were composed of the following ingredients:
- Dose level 5.0 % (w/w): 252 g test material and water + 4275 g meal
- Dose level 2.5 % (w/w): 126 g test material and water + 4387.5 g meal
- Dose level 1.25 % (w/w): 63 g test material and water + 4443.75 g meal
- Dose level 0.6 % (w/w): 30.24 g test material and water + 44735 g meal
- Dose level 0.3 % (w/w): 15.12 g test material and water + 4486.5 g meal

Each diet was mixed in a Patterson-Kelly twin shelled V blender for 15 min.
The doses were mixed one or two days prior to the week of their use in the study, and stored at 23 °C.
One analysis was perfomed to determine the accuracy of the mixture concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One analysis was performed to determine the accuracy of mixture concentrations; results were within +/- 10 % of the desired dose concentration.

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily

Dose / conc.:

0.3 other: % in the diet

Dose / conc.:

0.6 other: % in the diet

Dose / conc.:

1.25 other: % in the diet

Dose / conc.:

2.5 other: % in the diet

Dose / conc.:

5 other: % in the diet

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, plain diet

Details on study design:

- Five dose levels of 0.0, 0.3, 0.6, 1.25, 2.5 and 5.0 % in feed were used in this study (approx. 0, 250, 500, 1100, 2200 and 4500 mg/kg bw for both sexes [based on 16.4 g/d average food consumption, 0.182 kg average bw for males and on 11.55 g/d average food consumption, 0.130 kg average bw] for females).
- The selected doses were prepared by mixing together weighed portions of Purina Lab Chow in meal form with weighed portions of the chemical. 12 % water was added to the chemical as a dust control agent prior to mixing with the meal.
- Each dosed group received 91 consecutive days of dosed feed mixture.

Observations and examinations performed and frequency:

Animals were observed twice each day for clinical signs, with at least six hours between observations. All clinical signs were recorded daily. Additional studies included blood sampling for the animal disease screening program from 10 control rats, 5 males and 5 females.

Sacrifice and pathology:

- Rats were necropsied on day 92 and 93.
- Gross examination were performed on all animals from all dosage groups.
- Microscopic examinations were performed on all tissues from all animals in the control group and the highest dose treatment group: Kidney, liver, lung, heart, pancreas and pituitary.

Other examinations:

Copper analyses were completed in the liver and kidney tissues and the formalin preserving those tissues from male rats in the highest dose group (5 % w/w) and control groups. See details and results in endpoint "7.1.1. Basic toxicokinetics" in endpoint study record "Batelle 76-34-106002, rat".

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight data showed differential weight gains in dosed rats no greater than 10 % to the control group; except in the highest dose level females which had + 12 % differential gain. However, no trend was established through the groups in either males or females. The greatest range between any two female groups was 12 % to - 5 % between the two highest doses (5 % and 2 .5 %). The greatest range between any two male groups was 9 % to -9 % between the highest dose and the third highest dose (5 % and 1 .25 %).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopically the observed lesions were encountered in both the control and experimental high dose group with similar frequency and severity . The high incidence of pulmonary lesions in both groups is thought to be associated with Sendai virus infection. No histopathologic lesions considered to be compound-related were encountered in the tissues examined.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 4 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse findings

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No accumulation of copper in liver or kidneys

Critical effects observed:

no

Reference 8

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

; no clinical chemistry, hematology, or urinalysis were conducted and no organ weights were taken (as recommended in OECD Guideline 408). Extra investigations on copper contents in liver and kidneys were included.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: The chemical analysis, performed at Midwest Research Institute indicated that the purity was 104.7 % +- 1.1 % (elemental analysis)

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Industries
- Age at study initiation: 8.5 weeks
- Weight at study initiation: males: 16 - 23 g; females: 16 - 19 g
- Housing: polycarbonate cages: groups of 5 mice per cage
- Diet: weighed portions of Purina Lab Chow in meal form, mixed together with weighed portions of the test material (see details at "doses/concentrations")
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 23 °C
- Humidity: 40 - 60 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: feed

Vehicle:

other: 12 % water was added to the test material as a dust control agent

Details on oral exposure:

Dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) were selected for both males and females. The selected doses were prepared by mixing weighed portions of purina Lab Chow in meal form with weighed portions of the test material. 12 % water was added to the test material as a dust control agent prior to mixing with the meal. For each dose level, one weekly lot of 4500 g (+ 12 % water compensation) was prepared.

The actual mixtures were composed of the following ingredients:
- Dose level 5.0 % (w/w): 252 g test material and water + 4275 g meal
- Dose level 2.5 % (w/w): 126 g test material and water + 4387.5 g meal
- Dose level 1.25 % (w/w): 63 g test material and water + 4443.75 g meal
- Dose level 0.6 % (w/w): 30.24 g test material and water + 44735 g meal
- Dose level 0.3 % (w/w): 15.12 g test material and water + 4486.5 g meal

Each diet was mixed in a Patterson-Kelly twin shelled V blender for 15 min.
The doses were mixed one or two days prior to the week of their use in the study, and stored at 23 °C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One analysis was perfomed to determine the accuracy of the mixture concentration. Results were within +/- 10 % of the desired dose concentration.

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily

Dose / conc.:

0.3 other: % in the diet

Dose / conc.:

0.6 other: % in the diet

Dose / conc.:

1.25 other: % in the diet

Dose / conc.:

2.5 other: % in the diet

Dose / conc.:

5 other: % in the diet

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, plain diet

Details on study design:

- 10 animals were used per sex and dose group.
- Five dose levels of 0.0, 0.3, 0.6, 1.25, 2.5 and 5.0 % in feed were used in this study (approx. 0, 1000, 2000, 4000, 8000 and 16000 mg/kg bw for males [based on 7.3 g/d average food consumption, 0.023 kg average bw] and approx. 0, 1100, 2200, 4700, 9400 and 18700 mg/kg bw for females [based on 7.1 g/d average food consumption, 0.019 kg average bw], respectively).
- The selected doses were prepared by mixing together weighed portions of Purina Lab Chow in meal form with weighed portions of the chemical. 12% water was added to the chemical as a dust control agent prior to mixing with the meal.
- Each dosed group received on 91 consecutive days of dosed feed mixture.

Observations and examinations performed and frequency:

Animals were observed twice each day for clinical signs, with at least 6 hours between observations. All clinical signs (or negative observations) were recorded daily. Additionally, blood sampling was conducted from 10 control mice, 6 treated males and 4 treated females.

Sacrifice and pathology:

- Mice were necropsied on day 92 and 93.
- Gross examination were performed on all animals from all dosage groups.
- Microscopic examinations were performed on following organs from all animals in the control group and the highest dose treatment group: Kidneys, liver, lung, (only in control group: heart).

Other examinations:

Copper analyses were completed in the liver and kidney tissues and the formalin preserving those tissues from male mice in the highest dose group (5 % w/w) and control groups. See details and results in endpoint "7.1.1. Basic toxicokinetics" in endpoint study record "Batelle 76-34-106002, mouse".

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were seven early deaths during this study. Four male mice from four different dose or control groups died during weeks 3, 7, and 10, while 3 control female mice died during the week 3.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight data ranged from -7 to +11 % differential weight gain in the dosed females, without any dose-relation. Dosed male mouse groups showed a positive weight differential in all groups except the lowest dosage group of 0.3 %. No trends coinciding with dosage levels or food consumption are evident in these data.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no trends in diet consumption among dosed animals compared with controls in either male or female mice. Female control mice consumed 2 to 3 grams more feed than dosed animals. However, diet consumption data in this group were based on seven survivors during the last ten weeks of the study and have a higher value than expected. The dosed female mouse groups had average and expected levels of diet consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 16 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse findings

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 18 700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse findings

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

no guideline available

Principles of method if other than guideline:

5-day inhalation exposure with 21 days recovery group.
Ma-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, van Ravenzwaay B, Landsiedel R. 2009. Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance Inhal Toxicol 21, 102-118

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Blue powder
stored at room temperature

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation (means): Main group day 0: control group: 252 g; test groups: 252 - 254g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust-free bedding, supplied by SSNIFF, Soest, Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks (Typ NGM E-022), supplied by Abedd Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- Diet: Mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum.
- Water: Tap water, ad libitum

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

Mean relative humidities in the inhalation systems ranged between 40.4 and 52.9 %. Mean temperatures in the inhalation systems ranged between 21.3 and 22.4°C. They are within the range suggested by the respective testing guidelines.

Route of administration:

other: dust aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: see table 1

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. By means of dust generators the substance to be tested is generated into dust aerosols using compressed air in a mixing stage, mixed with conditioned air and passed into the inhalation systems via cyclonic separators. For each concentration, a solid particle generator (brush-generator) wias used for generating the dust. The concentration was adjusted by varying the piston feed and by varying the brush rotation. For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed via the cyclonic separator into the inhalation system.
- The following test substance flow, air flows and evaporation temperatures were scheduled: Test group; Substance flow (g/h); Supply air 1 conditioned (m³/h); Supply air 2 compressed (m³/h); Exhaust air (m³/h)
0; -; 6.0 ± 0.3; -; 5.7 ± 0.3
1; 0.10 – 0.25; 4.5 ± 0.3; 1.5 ± 0.3; 5.4 ± 0.3

EXPERIMENTAL PROCEDURE
- Head-nose exposure systems: The inhalation atmosphere was maintained inside aerodynamic exposure systems (test group 1 in INA 60, volume V ≈ 90 L, BASF SE, control group in INA 120, volume V ≈ 120 L) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.
- Exposures: The head-nose exposure technique was preferably selected for this aerosol/dust/ inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult. Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume. A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air. In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours from Monday to Friday to reach 5 exposures. The animals did not have access to water or feed during the exposure.
- Measurements of the exposure conditions: The following exposure parameters were recorded: Supply air (conditioned), Supply air 2 (compressed), Exhaust air, Chamber humidity, Chamber temperature, Real time concentration surveillance. No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

The air flows were constantly maintained in the desired range. An air change of about 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system. Mean relative humidities in the inhalation systems ranged between 39.6 and 53.5 %. Mean
temperatures in the inhalation systems ranged between 21.0 and 22.2°C.

VEHICLE
- Composition of vehicle: Conditioned supply air is activated charcoal filtered air conditioned to about 30% - 70% relative humidity and 20°C - 24°C. Compressed air is filtered air pressurized to about 6 bar.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CALCULATION OF NOMINAL CONCENTRATIONS: The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.
ANALYTICAL DETERMINATION OF CONCENTRATIONS: The concentration of the inhalation atmosphere in test group 1 was analyzed by gravimetry. This analytical method is judged to be valid because the test substance does not possess an appreciable vapor pressure. Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In the test group, the constancy of the dust atmosphere in the chamber was continuously monitored using scattered light photometers. The analyses were carried out at the Laboratory for Inhalation Toxicity of the Experimental Toxicology and Ecology of BASF SE.
- Sampling for gravimetric analyses: Equipment: Sampling equipment with probe (Millipore Corporation, Billerica, MA 01821, USA), Internal probe diameter: 7 mm, Filter: MN 85/90 BF (d = 4.7 cm), Vacuum pump (Millipore Corporation, Billerica, MA 01821, USA), Balance: Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany). Sampling: Sampling velocity: 1.25 m/s, Flow rate of sampling: 3 L/min, Sample volumes Test group 1: 90 L, Sampling site: immediately adjacent to the animals' noses at a separate spare port, Sampling frequency: as a rule, 3 samples per exposure and concentration group
- Gravimetrical method of analyses: A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust aerosol was drawn through the filter. The dust concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.
REAL TIME MONOTORING OF CONSTANCY OF CONCENTRATIONS: Scattered light photometer (VisGuard (Sigrist) in test group 1 was used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. The measurements were recorded using line recorders.
PARTICLE SIZE ANALYSIS:
- Definitions: EACD 50%(effective aerodynamic cutoff diameter 50%) defines the separation characteristic of each impactor stage. 50% of particles with the EACD given are deposited in the pertinent impactor stage; the remainder has reached one of the following stages. MMAD(mass median aerodynamic diameter) is the calculated aerodynamic diameter which divides the size distribution in half when measured by mass. Geometrical standard deviation (GSD) is the ratio of the estimated 84 percentile to the 50 percentile and indicates the slope of the cumulative particle size distribution curve.
- Equipment, sampling and method of determination: The particle size analysis was carried out with a cascade impactor. Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA), Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA), Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA), Sampling probe internal diameter 7 mm, Balance Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany), Mettler XP 205.(Mettler-Teldo AG, Greifensee, Switzerland)
- Sampling for particle size analyses: Preweighed metal collecting discs and a backup particle filter were placed into the cascade impactor and 2 samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals. A sample volume of 90 L was used in each test group. Method of analysis: Gravimetrical determination. The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the metal collecting disc and backup filter before and after sampling. The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter. Evaluation: The calculation of the particle size distribution was carried out in the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (DIN 66141: Darstellung von Korngrößenverteilungen and DIN 66161: Partikelgrößenanalyse, Beuth-Vertrieb GmbH, Berlin und Köln, Germany).
- Particle size distribution measurements with APS: Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used (TSI APS 3321). Frequency: on two days during the exposure period, with 3 repeats on each day.
- Particle size distribution measurements with Optical particle counter: For each test atmosphere measurements with an optical particle counter (WELAS 2000; (Palas® GmbH, Karlsruhe, Germany)) were performed to determine the size distribution of particles with diameters larger than 246 nm. The WELAS 2000 uses a white-light source to illuminate a measurement volume through which particles have to move singly. The measuring range of the sensor was 0.246 to 9.653 μm and the sampling flow rate 5 L/min.
- Particle size distribution measurements with scanning mobility particle sizer: To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 μm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.083 μm according to the software calculation. The duration of each measurement cycle was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily, for five consecutive days

Dose / conc.:

29.3 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
29.3 +/- 3.4 mg/m³
Basis:
analytical conc.

Dose / conc.:

9.7 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
9.7 +/- 1.7 mg/m3
Basis:
analytical conc.

Dose / conc.:

3 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
3.0 +/- 0.7 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

3/dose/group (main group or recovery group for microscopic examination)
5/dose/group (main group or recovery group for blood sampling and BAL)

Control animals:

yes, concurrent vehicle

Details on study design:

On study day 4 after exposure and on study day 25, 3 animals per group and time point were sacrificed underwent gross necropsy. Selected organs were weighed, a broad set of organs and tissues were preserved, respiratory tract was examined histologically.
On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters and acute phase proteins were examined in blood. After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Observations and examinations performed and frequency:

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

CLINICAL OBSERVATIONS: The clinical condition of the test animals was recorded once daily during the pre-exposure period and on post-exposure observation days on working days. On exposure days, clinical observation was performed at least 3 times daily, before, during and after exposure. During exposure only a group wise examination was possible.

BODY WEIGHT: The body weight of the animals was determined at the start of the pre-exposure (day -4), and then, as a rule, twice a week (Monday and Friday), as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weights from Monday to Friday. The main reason for this type of calculation is to show body weight change of the exposure week without the exposure-free weekend. It enables detection of minor decrease of body weight gain, which may be overlooked because the animals recover during the weekend. Group means were derived from the individual differences.

CLINICAL PATHOLOGY: In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The examinations for haematology and clinical chemistry were carried out in 5 animals per test group.

HAEMATOLOGY: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Prothrombin time (Hepato Quick’s test) (HQT) was measured. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

CLINICAL CHEMISTRY: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL).
ACUTE PHASE PROTEINS IN SERUM: Rat α2-macroglobulin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25A2M). Rat haptoglobin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25HPT). Both ELISA kits were measured with a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer.

BRONCHOALVEOLAR LAVAGE FLUID (BAL): The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline. The following examinations were carried out in 5 male animals per test group.
- Cytology in BAL: Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. Parameters: Total cell count (BALTCN), Macrophages (BALMPH), Polymorphonuclear neutrophils (BALPMN), Lymphocytes (BALLY), Eosinophils (BALEO), Monocytes (BALMO), Atypical cells (BALATY).
- Total Protein and enzymes in BAL: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the humoral parameters in the bronchoalveolar lavage fluid. Parameter: γ−Glutamyltransferase (GGT), Protein (MTP), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP), N-acetyl-β-Glucosaminidase (NAG)
- Antigens in BAL: The antigens were measured with MTP ELISAs at a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer. The following antigens were measured in BALF: Rat monocyte chemoattractant protein-1 (rat MCP-1) level measured with an Instant ELISA produced by Bender MedSystems, Vienna, Austria (cat. no BMS631INST), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8) measured with an ELISA produced by R&D Systems Inc., Minneapolis, US, (Quantikine rat CINC-1, cat. no. RCN100), Macrophage colony stimulating factor (M-CSF) measured with a Quantikine Mouse M-CSF ELISA produced by R&D Systems Inc., Minneapolis, USA (cat no. MMC00), Rodent osteopontin measured with an ELISA produced by R&D Systems, Inc., Minneapolis, US (Quantikine mouse osteopontin, cat. no. MOST00).

Sacrifice and pathology:

NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS: The following weights were determined in all animals sacrificed on schedule: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands.

HISTOPATHOLOGY: The following organs or tissues were fixed in 4% buffered formaldehyde or modified Davidson’s solution: All gross lesions, Adrenal glands, Brain with olfactory bulb, Bone marrow (femur), Epididymides, Eyes with optic nerve and eyelids, Heart, Kidneys, Larynx/Pharynx, Liver, Lungs, Lymph nodes (tracheobronchial and mediastinal lymph nodes), Nose (nasal cavity), Oesophagus, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Stomach (forestomach and glandular stomach), Spleen, Testes, Thyroid glands, Thymus, Trachea, Urinary bladder. From the liver, one additional slice of the Lobus dexter medialis and the Lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast. The testes were fixed in modified Davidson’s solution. Fixation was followed by histotechnical processing and examination by light microscopy. Tissues and organs to be examined histologically: All gross lesions (only affected animals), Nasal cavity (4 levels), Larynx (3 levels), Trachea, Lungs (5 lobes), Lymph nodes (tracheobronchial and mediastinal lymph nodes).

Statistics:

- Body weight, body weight change: A comparison of the dose group with the control group was performed using the student t-test (two-sided) for the hypothesis of equal means.
- Clinical Pathology parameters in Blood: Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (two-sided) for the equal medians.
- Clinical Pathology parameter in BALF, Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (one-sided) for the
equal medians.

Throughout the sections of the report, when intergroup differences are referred to as "significant " it implies that the differences have attained statistical significance (p ≤ 0.05) when compared with the control group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

One animal exposed to the low concentration died on study day 4 after exposure. As no other animals exposed to higher concentrations died, the death was considered not substance-related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Blue discoloration was observed in the lungs of all males of test groups 2 and 3 (10 and 30 mg/m³) and in the skin of the snout region of test groups 1, 2 and 3 (3, 10 and 30 mg/m³). In different regions of the gastrointestinal tract, the contents showed blue discoloration starting from test group 1 (3 mg/m³) in the glandular stomach and jejunum and from test groups 2 (10 and 30 mg/m³) in cecum and rectum.
All discolorations revealed the presence of the pigment and were regarded as treatment-related. In the lungs the blue discoloration correlated with the presence of pigment within macrophages.
One animal showed at necropsy a red discoloration in the skin of the snout.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see table 3

All of these findings were regarded as treatment-related and were considered to be adaptive and not adverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Acute phase proteins
Neither after the administration of the compound nor after the recovery period there were any alteration of the serum haptoglobin and alpha-2-macroglobulin levels.

Bronchoalveolar lavage fluid (BAL)
No treatment-related changes among bronchoalveolar cytology parameters were observed.

Total protein and enzymes in BAL
No treatment-related changes among BAL enzyme activities and total protein levels were observed.

Antigens in BAL
No treatment-related changes among BAL antigen levels were observed.

Gross necropsy
Blue discoloration was observed in the lungs of all males of test groups 2 and 3 (10 and 30 mg/m3) and in the skin of the snout region of test groups 1, 2 and 3 (3, 10 and 30 mg/m3). In different regions of the gastrointestinal tract, the contents showed blue discoloration starting from test group 1 (3 mg/m3) in the glandular stomach and jejunum and from test groups 2 (10 and 30 mg/m3) in cecum and rectum. All discolorations revealed the presence of the pigment and were regarded as treatment-related.
In the lungs the blue discoloration correlated with the presence of pigment within macrophages.
Animal No. 11 (deceased) showed at necropsy a red discoloration in the skin of the snout.
At the end of the recovery period, the blue discoloration of the lung was still visible.

Histopathology (see tables 2-4)
After exposure, in test group 3 and 2 (30 and 10 mg/m3), slight and minimal number of macrophages showing blue pigment-laden cytoplasms (histiocytosis with pigment particles) were seen in the alveoli with a tendency to accumulate in lumen of the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). Minimal epithelial hypertrophy and/or hyperplasia was noted in terminal bronchioles of 2 out of 3 males of test group 3 (30 mg/m3). This finding was characterized by the presence of high cuboidal epithelial cells, accompanied by a slight cytoplasmic basophilia. Single alveolar macrophages containing very few blue cytoplasmic pigment particles were seen in animals of test group 1 (3 mg/m3). A minimal number of pigmentladen macrophages was observed in the bronchus-associated lymphoid tissue (BALT) at all concentration levels (10, 30 and 300 mg/m3) without a dose-dependency.
At the end of the recover period all males of test group 3 (30 mg/m3) and 2 out of 3 males of test group 2 (10 mg/m3) showed a minimal number of pigment-laden alveolar macrophages with a tendency to accumulate in the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). In one male out of 3 of test group 2 (10 mg/m3) and all males of test group 1 (3 mg/m3) single macrophages containing very few cytoplasmic pigment particles were found in alveolar lumina. The bronchus-associated lymphoid tissue (BALT) of all males in test group 2 and 3 showed minimal number of pigment-laden macrophages. Single macrophages containing very few pigment particles were detected in the tracheobronchial and mediastinal lymph nodes of 2 out of 3 animals of test group 3 (30 mg/m3) and in the tracheobronchial lymph node of one out of 3 animals of test group 2 (10 mg/m3).

In the larynx of animals of test group 3 (30 mg/m3), minimal focal epithelial alteration was seen at the base of the epiglottis (level I). In general, minimal amount of pigment particles (admixed with mucus secretion or within macrophages) were observed in the lumen of some animals in the larynx and nasal cavity.

Single macrophages containing very few pigment particles were detected in the tracheobronchial and mediastinal lymph nodes of 2 out of 3 animals of test group 3 (30 mg/m3). All other findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Deceased animal: One animal showed in the lungs single alveolar macrophages containing very few cytoplasmic pigment particles and minimal number of pigment-laden macrophages in the BALT (bronchus-associated lymphoid tissue). These findings were regarded as treatment-related and adaptive and revealed the presence of the test-substance. In the larynx, minimal epithelial alteration (level I) and minimal focal squamous metaplasia (level II) were seen, which were assessed as treatment-related and adaptive (Kaufmann et al., 2009). No histopathological findings were detected in the other examined organs that can explain the death of this animal.

Dose descriptor:

NOAEC

Effect level:

30 mg/m³ air

Based on:

test mat.

Remarks:

mean measured concentration

Sex:

male

Basis for effect level:

other: No adverse effects observed at the highest tested dose

Critical effects observed:

no

Particle size distribution was measured by different devices. The data are presented in table 1 below and in the attached pdf file. Cascade impactor measurements showed test atmospheres with high fraction of respirable particles. It seems that MMAD decreased slightly with increasing atmospheric dust concentration. APS measurements in the atmospheres of test groups 1 and 2 showed comparable MMADs to those of cascade impactor. The data of test group 3 showed higher MMAD than in test group 1 and 2, whereas cascade impactor measurements showed a tendency other way round.

The count median diameters measured by WELAS and SMPS showed the fine nature of the dust in the test atmospheres. Particle count concentrations increased with increasing mass concentrations. Slightly higher count median diameters were measured by WELAS, which is in the nature of the different measuring principle of these two devices. In general, the measured values by WELAS and SMPS were consistent. Both showed a slightly lower count median diameter of dust in test group 3 than in the groups 1 and 2, as observed in MMADs measured by cascade impactor. Summarizing the particle size measured by different devices, the test atmospheres consisted of respirable particles. The MMAD of test group 3 was slightly lower than in test groups 1 and 2. With increasing test concentration, higher forces applied during dust generation pressing the test material against the rotating brush might have caused a higher shear force on agglomerates. Thus, smaller particles may be generated. Same tendency was showed by

WELAS and SMPS. APS was the only one device showing higher MMAD in test group 3 than in the others. This phenomena cannot be explained.

Table 1 Particle size distributions measured by different devices

Test groups	1	2	3
Target concentration (mg/m3)	3	10	30
Marple cascade impactor MMAD (µm) / GSD	1.1 µm / 3.6 1.1 µm / 2.8	0.8 µm / 2.6 0.7 µm / 2.7	0.5 / 4.9 0.6 / 2.8
APS 3321 MMAD (µm) / GSD	0.94 µm / 1.851 0.92 µm / 1.591 0.91 µm / 1.501 0.91 µm / 1.652 0.95 µm / 2.562 0.91 µm / 1.642	1.01 µm / 3.161 0.94 µm / 1.911 0.96 µm / 2.351 1.04 µm / 3.522 0.98 µm / 2.332 0.98 µm / 2.202	2.46 µm / 2.421 2.59 µm / 2.501 2.64 µm / 2.541 2.41 µm / 2.422 2.89 µm / 2.812 2.62 µm / 2.452
WELAS 2000 (µm): Count median diameter (Q0)	0.404	0.407	0.379
SMPS (µm): Count median diameter (Q0)	0.281	0.315	0.257
Count concentration of particles in WELAS 2000 (number particle/cm3)	7327	16555	171154
Count concentration of particles in SMPS (number particle/cm3)	18909	76637	454053

1 = measured on 25 Feb 2014

2 = measured on 26 Jun 2014

Table 2: Macroscopic findings

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Cecum
·   Discoloration of contents			2	3
Glandular stomach
·   Discoloration of contents		1	2	2
Jejunum
·   Discoloration of contents		1	1	3
Lungs
·   Discoloration			3	3
Rectum
·   Discoloration of contents			1	1
Skin
·   Discoloration		2	3	3

Table 3: Microscopic findings at the end of exposure

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Macrophages, pigment-laden			3	3
·   Grade 1			3
·   Grade 2				3
Pigment particles, in single macrophages		3
·   Present		3
Hypertrophy/hyperplasia, terminal bronchiole				2
·   Grade 1				2
BALT (bronchus-associated lymphoid tissue): macrophages, pigment-laden		3	2	1
·   Grade 1		3	2	1

Table 4: Microscopic findings at the end of recovery

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Macrophages, pigment-laden	0	0	2	3
·   Grade 1			2	3
Pigment particles, in single macrophages		3	1
·   Present		3	1
BALT (bronchus-associated lymphoid tissue): macrophages, pigment-laden	0	0	3	3
·   Grade 1			3	3

Conclusions:

Inhalation exposure of rats to up 30 mg/m³ test substance on 5 consecutive days did not lead to any treatment-related adverse effects within the respiratory tract. Some non-adverse adaptive changes fully regressed within a 3-week
recovery period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) was 30 mg/m³.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

30 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

no guideline available

Principles of method if other than guideline:

5-day inhalation exposure with 21 days recovery group.
Ma-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, van Ravenzwaay B, Landsiedel R. 2009. Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance Inhal Toxicol 21, 102-118

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Blue powder
stored at room temperature

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation (means): Main group day 0: control group: 252 g; test groups: 252 - 254g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust-free bedding, supplied by SSNIFF, Soest, Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks (Typ NGM E-022), supplied by Abedd Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- Diet: Mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum.
- Water: Tap water, ad libitum

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

Mean relative humidities in the inhalation systems ranged between 40.4 and 52.9 %. Mean temperatures in the inhalation systems ranged between 21.3 and 22.4°C. They are within the range suggested by the respective testing guidelines.

Route of administration:

other: dust aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: see table 1

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. By means of dust generators the substance to be tested is generated into dust aerosols using compressed air in a mixing stage, mixed with conditioned air and passed into the inhalation systems via cyclonic separators. For each concentration, a solid particle generator (brush-generator) wias used for generating the dust. The concentration was adjusted by varying the piston feed and by varying the brush rotation. For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed via the cyclonic separator into the inhalation system.
- The following test substance flow, air flows and evaporation temperatures were scheduled: Test group; Substance flow (g/h); Supply air 1 conditioned (m³/h); Supply air 2 compressed (m³/h); Exhaust air (m³/h)
0; -; 6.0 ± 0.3; -; 5.7 ± 0.3
1; 0.10 – 0.25; 4.5 ± 0.3; 1.5 ± 0.3; 5.4 ± 0.3

EXPERIMENTAL PROCEDURE
- Head-nose exposure systems: The inhalation atmosphere was maintained inside aerodynamic exposure systems (test group 1 in INA 60, volume V ≈ 90 L, BASF SE, control group in INA 120, volume V ≈ 120 L) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.
- Exposures: The head-nose exposure technique was preferably selected for this aerosol/dust/ inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult. Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume. A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air. In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours from Monday to Friday to reach 5 exposures. The animals did not have access to water or feed during the exposure.
- Measurements of the exposure conditions: The following exposure parameters were recorded: Supply air (conditioned), Supply air 2 (compressed), Exhaust air, Chamber humidity, Chamber temperature, Real time concentration surveillance. No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

The air flows were constantly maintained in the desired range. An air change of about 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system. Mean relative humidities in the inhalation systems ranged between 39.6 and 53.5 %. Mean
temperatures in the inhalation systems ranged between 21.0 and 22.2°C.

VEHICLE
- Composition of vehicle: Conditioned supply air is activated charcoal filtered air conditioned to about 30% - 70% relative humidity and 20°C - 24°C. Compressed air is filtered air pressurized to about 6 bar.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CALCULATION OF NOMINAL CONCENTRATIONS: The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.
ANALYTICAL DETERMINATION OF CONCENTRATIONS: The concentration of the inhalation atmosphere in test group 1 was analyzed by gravimetry. This analytical method is judged to be valid because the test substance does not possess an appreciable vapor pressure. Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In the test group, the constancy of the dust atmosphere in the chamber was continuously monitored using scattered light photometers. The analyses were carried out at the Laboratory for Inhalation Toxicity of the Experimental Toxicology and Ecology of BASF SE.
- Sampling for gravimetric analyses: Equipment: Sampling equipment with probe (Millipore Corporation, Billerica, MA 01821, USA), Internal probe diameter: 7 mm, Filter: MN 85/90 BF (d = 4.7 cm), Vacuum pump (Millipore Corporation, Billerica, MA 01821, USA), Balance: Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany). Sampling: Sampling velocity: 1.25 m/s, Flow rate of sampling: 3 L/min, Sample volumes Test group 1: 90 L, Sampling site: immediately adjacent to the animals' noses at a separate spare port, Sampling frequency: as a rule, 3 samples per exposure and concentration group
- Gravimetrical method of analyses: A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust aerosol was drawn through the filter. The dust concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.
REAL TIME MONOTORING OF CONSTANCY OF CONCENTRATIONS: Scattered light photometer (VisGuard (Sigrist) in test group 1 was used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. The measurements were recorded using line recorders.
PARTICLE SIZE ANALYSIS:
- Definitions: EACD 50%(effective aerodynamic cutoff diameter 50%) defines the separation characteristic of each impactor stage. 50% of particles with the EACD given are deposited in the pertinent impactor stage; the remainder has reached one of the following stages. MMAD(mass median aerodynamic diameter) is the calculated aerodynamic diameter which divides the size distribution in half when measured by mass. Geometrical standard deviation (GSD) is the ratio of the estimated 84 percentile to the 50 percentile and indicates the slope of the cumulative particle size distribution curve.
- Equipment, sampling and method of determination: The particle size analysis was carried out with a cascade impactor. Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA), Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA), Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA), Sampling probe internal diameter 7 mm, Balance Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany), Mettler XP 205.(Mettler-Teldo AG, Greifensee, Switzerland)
- Sampling for particle size analyses: Preweighed metal collecting discs and a backup particle filter were placed into the cascade impactor and 2 samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals. A sample volume of 90 L was used in each test group. Method of analysis: Gravimetrical determination. The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the metal collecting disc and backup filter before and after sampling. The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter. Evaluation: The calculation of the particle size distribution was carried out in the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (DIN 66141: Darstellung von Korngrößenverteilungen and DIN 66161: Partikelgrößenanalyse, Beuth-Vertrieb GmbH, Berlin und Köln, Germany).
- Particle size distribution measurements with APS: Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used (TSI APS 3321). Frequency: on two days during the exposure period, with 3 repeats on each day.
- Particle size distribution measurements with Optical particle counter: For each test atmosphere measurements with an optical particle counter (WELAS 2000; (Palas® GmbH, Karlsruhe, Germany)) were performed to determine the size distribution of particles with diameters larger than 246 nm. The WELAS 2000 uses a white-light source to illuminate a measurement volume through which particles have to move singly. The measuring range of the sensor was 0.246 to 9.653 μm and the sampling flow rate 5 L/min.
- Particle size distribution measurements with scanning mobility particle sizer: To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 μm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.083 μm according to the software calculation. The duration of each measurement cycle was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily, for five consecutive days

Dose / conc.:

29.3 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
29.3 +/- 3.4 mg/m³
Basis:
analytical conc.

Dose / conc.:

9.7 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
9.7 +/- 1.7 mg/m3
Basis:
analytical conc.

Dose / conc.:

3 mg/m³ air (analytical)

Remarks:

Doses / Concentrations:
3.0 +/- 0.7 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

3/dose/group (main group or recovery group for microscopic examination)
5/dose/group (main group or recovery group for blood sampling and BAL)

Control animals:

yes, concurrent vehicle

Details on study design:

On study day 4 after exposure and on study day 25, 3 animals per group and time point were sacrificed underwent gross necropsy. Selected organs were weighed, a broad set of organs and tissues were preserved, respiratory tract was examined histologically.
On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters and acute phase proteins were examined in blood. After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Observations and examinations performed and frequency:

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

CLINICAL OBSERVATIONS: The clinical condition of the test animals was recorded once daily during the pre-exposure period and on post-exposure observation days on working days. On exposure days, clinical observation was performed at least 3 times daily, before, during and after exposure. During exposure only a group wise examination was possible.

BODY WEIGHT: The body weight of the animals was determined at the start of the pre-exposure (day -4), and then, as a rule, twice a week (Monday and Friday), as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weights from Monday to Friday. The main reason for this type of calculation is to show body weight change of the exposure week without the exposure-free weekend. It enables detection of minor decrease of body weight gain, which may be overlooked because the animals recover during the weekend. Group means were derived from the individual differences.

CLINICAL PATHOLOGY: In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The examinations for haematology and clinical chemistry were carried out in 5 animals per test group.

HAEMATOLOGY: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Prothrombin time (Hepato Quick’s test) (HQT) was measured. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

CLINICAL CHEMISTRY: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL).
ACUTE PHASE PROTEINS IN SERUM: Rat α2-macroglobulin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25A2M). Rat haptoglobin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25HPT). Both ELISA kits were measured with a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer.

BRONCHOALVEOLAR LAVAGE FLUID (BAL): The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline. The following examinations were carried out in 5 male animals per test group.
- Cytology in BAL: Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. Parameters: Total cell count (BALTCN), Macrophages (BALMPH), Polymorphonuclear neutrophils (BALPMN), Lymphocytes (BALLY), Eosinophils (BALEO), Monocytes (BALMO), Atypical cells (BALATY).
- Total Protein and enzymes in BAL: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the humoral parameters in the bronchoalveolar lavage fluid. Parameter: γ−Glutamyltransferase (GGT), Protein (MTP), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP), N-acetyl-β-Glucosaminidase (NAG)
- Antigens in BAL: The antigens were measured with MTP ELISAs at a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer. The following antigens were measured in BALF: Rat monocyte chemoattractant protein-1 (rat MCP-1) level measured with an Instant ELISA produced by Bender MedSystems, Vienna, Austria (cat. no BMS631INST), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8) measured with an ELISA produced by R&D Systems Inc., Minneapolis, US, (Quantikine rat CINC-1, cat. no. RCN100), Macrophage colony stimulating factor (M-CSF) measured with a Quantikine Mouse M-CSF ELISA produced by R&D Systems Inc., Minneapolis, USA (cat no. MMC00), Rodent osteopontin measured with an ELISA produced by R&D Systems, Inc., Minneapolis, US (Quantikine mouse osteopontin, cat. no. MOST00).

Sacrifice and pathology:

NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS: The following weights were determined in all animals sacrificed on schedule: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands.

HISTOPATHOLOGY: The following organs or tissues were fixed in 4% buffered formaldehyde or modified Davidson’s solution: All gross lesions, Adrenal glands, Brain with olfactory bulb, Bone marrow (femur), Epididymides, Eyes with optic nerve and eyelids, Heart, Kidneys, Larynx/Pharynx, Liver, Lungs, Lymph nodes (tracheobronchial and mediastinal lymph nodes), Nose (nasal cavity), Oesophagus, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Stomach (forestomach and glandular stomach), Spleen, Testes, Thyroid glands, Thymus, Trachea, Urinary bladder. From the liver, one additional slice of the Lobus dexter medialis and the Lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast. The testes were fixed in modified Davidson’s solution. Fixation was followed by histotechnical processing and examination by light microscopy. Tissues and organs to be examined histologically: All gross lesions (only affected animals), Nasal cavity (4 levels), Larynx (3 levels), Trachea, Lungs (5 lobes), Lymph nodes (tracheobronchial and mediastinal lymph nodes).

Statistics:

- Body weight, body weight change: A comparison of the dose group with the control group was performed using the student t-test (two-sided) for the hypothesis of equal means.
- Clinical Pathology parameters in Blood: Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (two-sided) for the equal medians.
- Clinical Pathology parameter in BALF, Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (one-sided) for the
equal medians.

Throughout the sections of the report, when intergroup differences are referred to as "significant " it implies that the differences have attained statistical significance (p ≤ 0.05) when compared with the control group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

One animal exposed to the low concentration died on study day 4 after exposure. As no other animals exposed to higher concentrations died, the death was considered not substance-related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Blue discoloration was observed in the lungs of all males of test groups 2 and 3 (10 and 30 mg/m³) and in the skin of the snout region of test groups 1, 2 and 3 (3, 10 and 30 mg/m³). In different regions of the gastrointestinal tract, the contents showed blue discoloration starting from test group 1 (3 mg/m³) in the glandular stomach and jejunum and from test groups 2 (10 and 30 mg/m³) in cecum and rectum.
All discolorations revealed the presence of the pigment and were regarded as treatment-related. In the lungs the blue discoloration correlated with the presence of pigment within macrophages.
One animal showed at necropsy a red discoloration in the skin of the snout.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see table 3

All of these findings were regarded as treatment-related and were considered to be adaptive and not adverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Acute phase proteins
Neither after the administration of the compound nor after the recovery period there were any alteration of the serum haptoglobin and alpha-2-macroglobulin levels.

Bronchoalveolar lavage fluid (BAL)
No treatment-related changes among bronchoalveolar cytology parameters were observed.

Total protein and enzymes in BAL
No treatment-related changes among BAL enzyme activities and total protein levels were observed.

Antigens in BAL
No treatment-related changes among BAL antigen levels were observed.

Gross necropsy
Blue discoloration was observed in the lungs of all males of test groups 2 and 3 (10 and 30 mg/m3) and in the skin of the snout region of test groups 1, 2 and 3 (3, 10 and 30 mg/m3). In different regions of the gastrointestinal tract, the contents showed blue discoloration starting from test group 1 (3 mg/m3) in the glandular stomach and jejunum and from test groups 2 (10 and 30 mg/m3) in cecum and rectum. All discolorations revealed the presence of the pigment and were regarded as treatment-related.
In the lungs the blue discoloration correlated with the presence of pigment within macrophages.
Animal No. 11 (deceased) showed at necropsy a red discoloration in the skin of the snout.
At the end of the recovery period, the blue discoloration of the lung was still visible.

Histopathology (see tables 2-4)
After exposure, in test group 3 and 2 (30 and 10 mg/m3), slight and minimal number of macrophages showing blue pigment-laden cytoplasms (histiocytosis with pigment particles) were seen in the alveoli with a tendency to accumulate in lumen of the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). Minimal epithelial hypertrophy and/or hyperplasia was noted in terminal bronchioles of 2 out of 3 males of test group 3 (30 mg/m3). This finding was characterized by the presence of high cuboidal epithelial cells, accompanied by a slight cytoplasmic basophilia. Single alveolar macrophages containing very few blue cytoplasmic pigment particles were seen in animals of test group 1 (3 mg/m3). A minimal number of pigmentladen macrophages was observed in the bronchus-associated lymphoid tissue (BALT) at all concentration levels (10, 30 and 300 mg/m3) without a dose-dependency.
At the end of the recover period all males of test group 3 (30 mg/m3) and 2 out of 3 males of test group 2 (10 mg/m3) showed a minimal number of pigment-laden alveolar macrophages with a tendency to accumulate in the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). In one male out of 3 of test group 2 (10 mg/m3) and all males of test group 1 (3 mg/m3) single macrophages containing very few cytoplasmic pigment particles were found in alveolar lumina. The bronchus-associated lymphoid tissue (BALT) of all males in test group 2 and 3 showed minimal number of pigment-laden macrophages. Single macrophages containing very few pigment particles were detected in the tracheobronchial and mediastinal lymph nodes of 2 out of 3 animals of test group 3 (30 mg/m3) and in the tracheobronchial lymph node of one out of 3 animals of test group 2 (10 mg/m3).

In the larynx of animals of test group 3 (30 mg/m3), minimal focal epithelial alteration was seen at the base of the epiglottis (level I). In general, minimal amount of pigment particles (admixed with mucus secretion or within macrophages) were observed in the lumen of some animals in the larynx and nasal cavity.

Single macrophages containing very few pigment particles were detected in the tracheobronchial and mediastinal lymph nodes of 2 out of 3 animals of test group 3 (30 mg/m3). All other findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Deceased animal: One animal showed in the lungs single alveolar macrophages containing very few cytoplasmic pigment particles and minimal number of pigment-laden macrophages in the BALT (bronchus-associated lymphoid tissue). These findings were regarded as treatment-related and adaptive and revealed the presence of the test-substance. In the larynx, minimal epithelial alteration (level I) and minimal focal squamous metaplasia (level II) were seen, which were assessed as treatment-related and adaptive (Kaufmann et al., 2009). No histopathological findings were detected in the other examined organs that can explain the death of this animal.

Dose descriptor:

NOAEC

Effect level:

30 mg/m³ air

Based on:

test mat.

Remarks:

mean measured concentration

Sex:

male

Basis for effect level:

other: No adverse effects observed at the highest tested dose

Critical effects observed:

no

Particle size distribution was measured by different devices. The data are presented in table 1 below and in the attached pdf file. Cascade impactor measurements showed test atmospheres with high fraction of respirable particles. It seems that MMAD decreased slightly with increasing atmospheric dust concentration. APS measurements in the atmospheres of test groups 1 and 2 showed comparable MMADs to those of cascade impactor. The data of test group 3 showed higher MMAD than in test group 1 and 2, whereas cascade impactor measurements showed a tendency other way round.

The count median diameters measured by WELAS and SMPS showed the fine nature of the dust in the test atmospheres. Particle count concentrations increased with increasing mass concentrations. Slightly higher count median diameters were measured by WELAS, which is in the nature of the different measuring principle of these two devices. In general, the measured values by WELAS and SMPS were consistent. Both showed a slightly lower count median diameter of dust in test group 3 than in the groups 1 and 2, as observed in MMADs measured by cascade impactor. Summarizing the particle size measured by different devices, the test atmospheres consisted of respirable particles. The MMAD of test group 3 was slightly lower than in test groups 1 and 2. With increasing test concentration, higher forces applied during dust generation pressing the test material against the rotating brush might have caused a higher shear force on agglomerates. Thus, smaller particles may be generated. Same tendency was showed by

WELAS and SMPS. APS was the only one device showing higher MMAD in test group 3 than in the others. This phenomena cannot be explained.

Table 1 Particle size distributions measured by different devices

Test groups	1	2	3
Target concentration (mg/m3)	3	10	30
Marple cascade impactor MMAD (µm) / GSD	1.1 µm / 3.6 1.1 µm / 2.8	0.8 µm / 2.6 0.7 µm / 2.7	0.5 / 4.9 0.6 / 2.8
APS 3321 MMAD (µm) / GSD	0.94 µm / 1.851 0.92 µm / 1.591 0.91 µm / 1.501 0.91 µm / 1.652 0.95 µm / 2.562 0.91 µm / 1.642	1.01 µm / 3.161 0.94 µm / 1.911 0.96 µm / 2.351 1.04 µm / 3.522 0.98 µm / 2.332 0.98 µm / 2.202	2.46 µm / 2.421 2.59 µm / 2.501 2.64 µm / 2.541 2.41 µm / 2.422 2.89 µm / 2.812 2.62 µm / 2.452
WELAS 2000 (µm): Count median diameter (Q0)	0.404	0.407	0.379
SMPS (µm): Count median diameter (Q0)	0.281	0.315	0.257
Count concentration of particles in WELAS 2000 (number particle/cm3)	7327	16555	171154
Count concentration of particles in SMPS (number particle/cm3)	18909	76637	454053

1 = measured on 25 Feb 2014

2 = measured on 26 Jun 2014

Table 2: Macroscopic findings

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Cecum
·   Discoloration of contents			2	3
Glandular stomach
·   Discoloration of contents		1	2	2
Jejunum
·   Discoloration of contents		1	1	3
Lungs
·   Discoloration			3	3
Rectum
·   Discoloration of contents			1	1
Skin
·   Discoloration		2	3	3

Table 3: Microscopic findings at the end of exposure

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Macrophages, pigment-laden			3	3
·   Grade 1			3
·   Grade 2				3
Pigment particles, in single macrophages		3
·   Present		3
Hypertrophy/hyperplasia, terminal bronchiole				2
·   Grade 1				2
BALT (bronchus-associated lymphoid tissue): macrophages, pigment-laden		3	2	1
·   Grade 1		3	2	1

Table 4: Microscopic findings at the end of recovery

Test group Concentration (mg/m3)	0 (0)	1 (3)	2 (10)	3 (30)
Animals examined	3	3	3	3
Macrophages, pigment-laden	0	0	2	3
·   Grade 1			2	3
Pigment particles, in single macrophages		3	1
·   Present		3	1
BALT (bronchus-associated lymphoid tissue): macrophages, pigment-laden	0	0	3	3
·   Grade 1			3	3

Conclusions:

Inhalation exposure of rats to up 30 mg/m³ test substance on 5 consecutive days did not lead to any treatment-related adverse effects within the respiratory tract. Some non-adverse adaptive changes fully regressed within a 3-week
recovery period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) was 30 mg/m³.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

30 mg/m³

Study duration:

subacute

Species:

rat

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

All substances in this category share a similar structure with a molecular weight > 500 g/mol. Based on the high molecular weight and insolubility, the substances in this category will not be able to penetrate biological membranes. Thus, no systemic exposure is expected. This is confirmed by the repeated dose toxicity studies for three substances in this category: CAS 147-14-8, CAS 1328-53-6, CAS 68987-63-3. The repeated dose toxicity data can be used for read-across to all substances in this category. All studies consistently show absence of adverse effects at the limit dose.

 

CAS No. 147-14-8:

Oral

A GLP-compliant 28 day subacute study was conducted, according to the Japanese guideline for 28 Day Repeated Dose Toxicity Test of Chemicals with Wistar rats, which received 0, 40, 200 and 1000 mg/kg/day copper phthalocyanine by gavage (JETOC 1995, K2). The test material is described to be of technical grade. Food consumption and body weight were determined. The animal’s state of health was checked by cage side observations as well as by detailed clinical observations. Clinicochemical and hematological examinations were carried out at the end of the administration period and after a recovery period of 14 days. All animals were subjected to gross-pathological assessment, followed by histopathological examination. No changes in general condition, body weight gain or food consumption were detected in any of the groups. After the 28 days of administration, a slight, but significant decrease in red blood cell count (RBC) and decrease of hemoglobin (Hb) and packed cell volume (PCV) were detected in the 200 and 1000 mg/kg male groups. These slight changes were dose dependent, but within the normal variation range. After the recovery period, a significant increase of erythroblasts was detected in the 1000 mg/kg female group. Also after the recovery period, slight increases of absolute organ weights of lung, spleen, adrenal and salivary gland and a tendency for increased relative organ weights of the spleen were evident in the 1000 mg/kg male group in comparison to the control group. These differences were of statistical, but not of biological relevance. No histopathological changes due to administration of phthalocyanine blue were detected. Therefore, a NOAEL of 1000 mg/kg bw per day was determined for male and female rats under the test conditions chosen.

 

In two 90 day subchronic feeding studies conducted by Batelle (1979, K2) with F344 rats and B6C3F1 mice, male and female animals received dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) copper phthalocyanine, administered in the food (approx. 0, 250, 500, 1100, 2200 and 4500 mg/kg bw for rats of both sexes, approx. 0, 1000, 2000, 4000, 8000 and 16000 mg/kg bw for male mice, and approx. 0, 1100, 2200, 4700, 9400 and 18700 mg/kg bw for female mice, calculated on average food consumption for 91 consecutive days. Animals were observed twice each day for clinical signs, with at least 6 hours between observations. All clinical signs were recorded daily. Gross examinations were performed on all animals from all dosage groups. Microscopic examinations were performed from kidney, liver, lung, heart, pancreas and pituitary from all animals in the control group and the highest dose treatment group. No clinical chemistry, hematology, or urinalysis were conducted and no organ weights were taken. No substance related mortality was reported for rats. There were seven early deaths for mice. Four male mice from four different dose or control groups and 3 control female mice died during the study. During the course of the 91-day study, neither treatment related signs of toxicity, nor abnormal clinical signs were observed in both rats and mice. Diet consumption among both male and female rats did not vary widely, nor showed any dose related trends. In mice, there were also no trends in diet consumption among dosed animals compared with controls in either male or female mice. No substance related changes were reported on macroscopic and histopathological examination of both rats and mice. This study was originally performed to serve as a range-finder for a chronic feeding study. It included determination of copper levels in kidneys and livers to find out if there was indication of uptake after ingestions. Since there was no significant increase in copper concentration, Pigment Blue 16 was considered to be inert and not absorbed, and the chronic toxicity study was not performed.

 

CAS No. 1328-53-6:

Oral

A GLP-compliant 28 day subacute study was conducted, according to the Japanese guideline for 28 Day Repeated Dose Toxicity Test of Chemicals with Wistar rats, which received 0, 100, 300 and 1000 mg/kg/day polychloro copper phthalocyanine by gavage using corn oil as vehicle (Mitsubishi 2000, K1). Food consumption and body weight were determined. The animal’s state of health was checked by cage side observations as well as by detailed clinical observations. Clinicochemical and hematological examinations as well as urinalysis were carried out at the end of the administration period and after a recovery period of 14 days. No deaths occurred in either sex. No effects of polychloro copper phthalocyanine were detected in terms of general condition, body weights, food consumption, hematological or blood chemistry parameters, urinalysis, organ weights, or pathological examination in males or females.

 

In two 90 day subchronic studies conducted by Batelle (1979, K2) with F344 rats and B6C3F1 mice, male and female animals received dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) copper phthalocyanine, administered in the food (approx. 300, 600, 1200, 2300 and 4600 mg/kg bw for male rats, approx. 300, 600, 1250, 2500 and 5000 mg/kg bw for female rats as well as approx. 0, 1000, 2000, 4000, 8000 or 16000 mg/kg bw/day for male mice and approx. 0, 1200, 2500, 5000, 10000 and 20000 mg/kg bw/day for female mice, calculated on average food consumption, for 91 consecutive days. Animals were observed twice each day for clinical signs, with at least 6 hours between observations. All clinical signs were recorded daily. Gross examinations were performed on all animals from all dosage groups. Microscopic examinations were performed from kidney, liver, lung, heart, pancreas and pituitary from all animals in the control group and the highest dose treatment group. No clinical chemistry, hematology, or urinalysis were conducted and no organ weights were taken. No substance related mortality was reported in rats, in mice 4 deaths occurred during the study which were not considered to be test substance related. No treatment related abnormal clinical signs were observed in either rats or mice. In mice, there were no trends in diet consumption among dosed animals compared with controls in either male or female mice. In rats there were slight trends in patterns of diet consumption between dosed and control rats of both sexes, with dosage groups eating about 1 g more of the higher concentrations of the dosed feed. In rats there were dose-related trends in body weights among both male and female rats, when body weights are expressed in percent differentials of weight gains compared with untreated controls. Male rats at the highest doses of 5.0 % and 2.5 % had differential weight gains of -11 % and -5 % respectively. Female rats at the top doses of 5.0 and 2.5 % had differential weight gains of -9.0 %. In mice, a -9 % to +8 % differential weight gain was seen in the dosed females. Animals in the highest dose levels had less weight gain than controls. Dosed male mouse groups showed a positive weight differential in all groups except the 0.6 % dosage group. In this group during week 12 with a corresponding weight loss was observed, which was not fully recovered during the final week of the study. However, given the normal variability in rodent body weight measurements, this differences did not necessarily reflect manifestation of toxicity. No substance related changes were reported on macroscopic and microscopic examination of the animals.

 

In another study (Mitsubishi, K2), four dose levels of 0, 20, 140 and 1000 mg/kg bw/day of the test material were administered to male and female rats by gavage. The NOAEL for the repeated dose toxicity was considered to be 1000 mg/kg/day for both sexes under the experimental conditions chosen.

 

CAS No. 68987-63-3:

Oral

A GLP-compliant 28 day subacute study was conducted following OECD guideline 407 (Huntington Life Sciences 1997, K1). Oral administration at dosages of 40, 200 and 1000 mg/kg/day in corn oil was generally well tolerated. No target organ was identified. The blue staining seen in the intermediate and high dosage groups, and the abnormal contents of the gastro-intestinal tract in high dosage animals were considered to be due to the test material, since it was a blue colour, and was therefore of no toxicological significance. The NOAEL was determined to be 1000 mg/kg bw.

 

CAS 12239-87-1:

Inhalation

Repeated dose inhalation toxicity was not performed as the substances are inert poorly soluble particles when manufactured as powder. They are non volatile and not inhalable if imbedded in a matrix such as a coating, a plastic article or a suspension.

A 5-day inhalation study with three-week recovery was performed with Pigment Blue 15 (BASF 2016, K1). This study examined both systemic and local effects. The NOAEC was determined to be the highest tested dose of 30 mg/m³.

 

Repeated dose dermal toxicity

No studies on repeated dose dermal toxicity are available. The molecular weights of the substances in the catagory are > 500 g/mol which in combination with poor solubility is an indication of low skin permeability. Considering that no hazard was identified at the limit dose for the oral route, there is no concern for a hazard via the dermal route. Testing for the dermal route is not needed.

Justification for classification or non-classification

Based on the available information classification upon repeated exposure is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.