2,9-bis(2-phenylethyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

Endpoint summary

Description of key information

Oral: NOAEL (28d) >= 1501 mg/kg bw/d, rat, according to OECD 407, GLP

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

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)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Physical state: black powder
- Analytical purity: 99 %
- Storage condition of test material: room temperature
- Lot/batch No.: 96-2363

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Manston, Kent
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: males: 130 - 156 g; females: 117 - 145 g
- Housing: 5 per cage
- Diet (e.g. ad libitum): SQC Rat and Mouse Diet No . 1 Expanded, Special Diet Services Limited, Witham, Essex, UK
- Water (e.g. ad libitum): free access to mains water from polycarbonate bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24 °C
- Humidity (%): 28 - 60 %
- Air changes (per hr): least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixture was prepared for use throughout the study, 2 - 3 days prior to the start of the study and were used throughout the 28-day dosing period.
- Mixing appropriate amounts with (Type of food): The test material was incorporated into the diet at concentrations of 750, 3750 and 15000 ppm as follows: A known amount of test material was mixed with a small amount of basal laboratory diet. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for 30 minutes at a constant speed, setting 1, in a Hobart QE 200 mixer. The diet was stored in labelled black plastic bags and contained in bins with lids when not in use.
- Storage temperature of food: not reported.

VEHICLE
- Concentration in vehicle: incorporated into the diet at concentrations of 50, 250 and 1000 ppm

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

expected concentration: 50 mg/kg;
analytical results:
44/42 mg/kg starting value;
37/36 mg/kg value after 10 days;
43/39 mg/kg value after 32 days;
The test substance is stable in Kliba 343 Mehl for the expected concentration (ca . 50 mg/kg Kliba Mehl) over a period of 32 days within the limits of the errors of the analytical method.

Stability under test conditions: The stability of the test material when mixed with rodent diet was determined by the sponsor. The results showed that the test material is stable under these conditions for at least 32 days. Dietary admixtures were therefore prepared 2 to 3 days prior to the start of the study and were used throughout the 28-day dosing period.

Duration of treatment / exposure:

28 days

Frequency of treatment:

continuously

Dose / conc.:

750 ppm

Remarks:

Mean: 79.25 mg/kg bw/day (actual ingested; males); 78.0 mg/kg bw/day (actual ingested; females)

Dose / conc.:

3 750 ppm

Remarks:

Mean: 404.25 mg/kg bw/day (actual ingested; males); 380.0 mg/kg bw/day (actual ingested; females)

Dose / conc.:

15 000 ppm

Remarks:

Mean: 1572.75 mg/kg bw/day (actual ingested; males); 1501.25 mg/kg bw/day (actual ingested; females)

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Post-exposure recovery period in satellite groups: Animals from satellite groups 5 (additional control group) and 6 (additional high dose group) were maintained for a further 14 days treatment free period following termination of treatment.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on the day before the start of treatment (day 0) and on days seven, fourteen, twenty-one and twenty-eight and in the case of satellite group animals on days thirty-five and forty-two. Bodyweights were also recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological and blood chemical investigations were performed on all animals from groups 1 to 4 at the end of the treatment period (day 28). Parameters showing possible treatment-related changes in these animals were examined in satellite group animals at the end of the treatment-free period (day 42).
- Anaesthetic used for blood collection: Yes (identity)
Blood samples were obtained by orbital sinus puncture under Halothane BP anaesthesia.
- Animals fasted: No
- How many animals:
- Parameters examined: measured on blood collected into tubes containing lithium heparin anti-coagulant:
Haematocrit (Hct), Haemoglobin (Hb), Erythrocyte count (RBC), Total leucocyte count (WBC ), Erythrocyte indices - mean corpuscular haemoglobin (MCH), - mean corpuscular volume (MCV), - mean corpuscular haemoglobin concentration (MCHC). Samples were withdrawn into tubes containing potassium EDTA anticoagulant for platelet counts (PLT) and differential leucocyte counts. Clotting time (CT) was assessed by 'Hepato Quick' time using samples collected into sodium citrate solution (0 .11 mol/L).

CLINICAL CHEMISTRY: Yes, see for the schedule, HAEMATOLOGY
- Parameters examined: measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Blood urea, Total protein, Albumin, Albumin/globulin ratio (by calculation), Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Creatinine
Alkaline phosphatase (AP), Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Glucose, Gamma glutamyl transpeptidase (yGT), Triglycerides, Total Cholesterol, Total Bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: during the final week of dosing (week 4)
- Metabolism cages used for collection of urine: Yes
Urine samples were collected over a period of approximately 16 hours, by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection, but without access to food.
- Animals fasted: Yes
- Parameters examined: Volume, Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Reducing substances, Blood.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organ weights

HISTOPATHOLOGY: Yes
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights: The following organs from animals that were killed at the end of the study, dissected free from fat, were weighed before fixation:
Adrenals, Brain, Gonads, Heart, Kidneys, Liver, Pituitary, Spleen.
Histopathology: Samples of the following tissues were removed from all animals and preserved in 10% buffered formalin.
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & Bone Marrow (femur), Pituitary, Brain Prostate, Caecum, Rectum, Colon, Salivary glands, Duodenum Sciatic nerve, Eyes, Seminal vesicles, Gross lesions Skin (hind limb), Heart, Spleen, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, Liver, Thyroid/parathyroid, Lungs, Trachea, Lymph nodes (cervial and mesenteric), Urinary bladder, Muscle (skeletal), Uterus, Oesophagus.
The following preserved tissues from all high dose and control group animals (groups 1 and 4) were prepared as paraffin blocks, sectioned at nominal thickness of 5 um and stained with haematoxylin and eosin for subsequent microscopic examination: Adrenals, Spleen, Heart, Kidneys, Liver.

Statistics:

Relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal Wallis non-parametric analysis of variance and Mann Whitney U-Test.
Probability values were calculated as: p < 0 .001 ***; P < 0. 01 **; P < 0 .05 *; p >= 0.05 (not significant)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinically observable signs of toxicity were noted in test or control animals throughout the study.
Extensive black/green staining of the external body surface was seen, from day 2 until the end of the treatment period, in animals (males and females) treated with 15000 ppm. Similar, but less extensive, staining was also observed in animals treated with 3750 ppm over the same period. This staining persisted in the satellite treatment groups but diminished with time, with only the animals tails being discoloured by the end of the treatment-free period. Black faeces was common to all treatment groups from day 4 onwards and was also apparent in the satellite treatment animals until day 31, after which the faeces of the satellite high dose groups appeared normal. No external staining was noted in animals treated with 750 ppm.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study .

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight gain in all test animals was comparable with that seen in controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in food consumption during the study.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in weekly food efficiency during the study.

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

no effects observed

Description (incidence and severity):

No overt intergroup differences were detected.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean values and standard deviations for test and control group animals are given in the tables below (statistically significant differences are indicated).
An anaemia was demonstrated in females treated with 15000 ppm, but not in males from the same dose group, identified by statistically significant reductions in haemoglobin levels, erythrocyte counts and haemotrocrit. Some animals showed values outside the normally expected range for rats of this strain and age but the intergroup difference was exacerbated by some rather high values in the control group. Calculation of mean corpuscular volume showed a slight increase in high dose females compared with controls but all values appeared normal. There is, therefore, no convincing evidence to suggest reticulocytosis and since there were no other changes that could be considered indicative of haemolysis it is possible that the anaemia was megaloblastic in nature. A slight but statistically significant reduction in platelet counts could have been associated with this, but all individual values remained within the normal range. The anaemia was very slight and apparently completely reversible since no such changes were seen in satellite high dose females after fourteen days without treatment. The effect was confined to the high dose group and an apparent increase in mean corpuscular haemoglobin, for females from all treatment groups, was considered to be entirely fortuitous since true hyperchromic cells do not exist.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant reduction in blood glucose was noted in males treated at 750 (111 +/- 10 mg/dL) and 15000 (116 +/- 13 mg/dL) ppm compared with control (133 +/- 16) although all values were within the normal range for animals of this strain and age. In addition, a true dose-related response was not elicited and the difference between test and control groups was considered not to be treatment-related. High dose males also showed a significant reduction in the calculated albumin/globulin ratio but as there were no significant changes in either serum albumin level or total protein and the values were within the normal range, the change is not thought to represent a toxicological effect. Female animals treated at 3750 (353 +/- 134 IU/L) and 15000 (353 +/- 86 IU/L) ppm showed reduced alkaline phosphatase levels compared to controls (500 +/- 77 IU/L), but none of the values could be considered abnormal and, a reduction in this enzyme could not be considered toxicologically significant.
Other minor statistically significant differences were confined to low and intermediate treatment groups only and, as such, were not dose-related .

Urinalysis findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes were detected in any of the urine parameters measured.

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):

No treatment-related differences in organ weight were detected between test and control animals.
A slight reduction in both liver and kidney weights, relative to body weight, was seen in male animals treated at 15000 ppm with only the decrease in relative liver weight being maintained in the satelite group males. However, none of the values could be considered abnormal for rats of this strain and age. In the absence of histopatholigcal abnormalities and any associated relevant changes in either blood chemical or haematological parameters, these reductions are unlikely to be toxicologically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Animals treated with 3750 and 15000 ppm had either slight or extensive black/green staining of the external body surface, respectively, at necropsy . Black/green staining of the non-glandular epithelium of the stomach was observed in animals from all the test groups . In addition, the contents of the gastro intestinal tract of all animals fed laboratory diet containing the test material were coloured black/green.
Although satellite treatment group animals still had black/green staining on their tails, there was no residual staining of the stomach epithelium at the end of the fourteen-day treatment-free period.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

>= 1 501.25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Effect level:

>= 1 572.75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

 

GROUP MEAN HAEMATOLOGICAL VALUES AND STANDARD DEVIATIONS (S.D.) - FEMALES
TEST MATERIAL
GROUP NUMBER	DIETARY CONCENTRATION (ppm)		Hb	RBC	Hct	MCH	MCV	MCHC	WBC	DIFFERENTIAL (10^9/L)					CT	PLT
			(g/dL)	(10^12/L)	(%)	(pg)	(fL)	(g/dL)	(10^9/L)	Neut	Lymph	Mono	Eos	Bas	(secs)	(10^9/L)
1	0 (Control)	MEAN	15.9	7.49	44.3	21	59	36	10.1	0.72	9.31	0.00	0.07	0	26	999
		S.D.	0.8	0.62	4.5	1	2	3	1.73	0.33	1.87	0	0.11	0	1	81
2	750	MEAN	15.4	6.86	41.8	23**	61	37	10.9	1.16	9.51	0.11	0.12	0	28	1001
		S.D.	0.6	0.38	2.7	1	3	2	1.83	0.79	2.14	0.19	0.08	0	2	74
3	3750	MEAN	15.8	7.02	41.8	23**	60	38	10.73	1.24	9.41	0.02	0.06	0	26	1003
		S.D.	0.6	0.63	3	1	1	2	1.44	0.78	0.89	0.05	0.08	0	1	103
4	15000	MEAN	14 .6*	6.35**	39 .3*	23**	62*	37	9.79	0.93	8.76	0.01	0.08	0	27	859*
		S.D.	1.5	0.63	4	1	1	1	1.97	0.52	1.75	0.03	0.15	0	2	104
5	0 (Control)	MEAN	15.5	7.6	44.7	20	59	-	-	-	-	-	-	-	-	877
	Satelite Group	S.D.	0.4	0.28	2.1	0	2	-	-	-	-	-	-	-	-	55
6	15000	MEAN	15.3	7.43	43.4	21	58	-	-	-	-	-	-	-	-	812
	Satelite Group	S.D.	0.5	0.07	0.8	1	1	-	-	-	-	-	-	-	-	117
		* = significantly different from corresponding control group value p = 0.05
		** = significantly different from corresponding control group value p = 0 .01
		- = not applicable

Conclusions:

In conclusion, the NOAEL of this study is above the highest tested dose of 15000 ppm (1573 mg/kg bw/day for males and 1501 mg/kg bw/day for females).

Executive summary:

In a 28-day oral repeated dose study (according to OECD guideline 407 and under GLP), Sprague-Dawley rats (5/sex/dose) were administered the test substance at 750, 3750 and 15000 ppm in diet. These concentrations in the diet are equal to 79, 404 and 1573 mg/kg bw/day for males and 78, 380 and 1501 mg/kg bw/day for females. The following parameters were evaluated: clinical signs, body weight, food efficiency, water consumption and compound intake, haematology, blood chemistry, urinalysis, organ weights and histopathology. No mortality and no clinically observable signs of toxicity were noted in test or control animals throughout the study. Bodyweight gain, food consumption and food efficiency in all test animals was comparable with that seen in controls. A reductions in haemoglobin levels, erythrocyte counts, haemotrocrit and platelet count in females of the highest dose group were considered to be not treatment induced. These effects were fully reversible during the 14 day post exposure observation period of the satellite groups.

Other non-treatment related effects observed were reduction in blood glucose was noted in males treated at 750 and 15000, a reduction in the calculated albumin/globulin ratio in high dose males and a reduced alkaline phosphatase in female animals treated at 3750 and 15000 ppm.

Urine parameters remained unchanged, and all morphological changes observed during necropsy were considered to be without toxicological significance.

In conclusion, the NOAEL of this study is above the highest tested dose of 15000 ppm (1573 mg/kg bw/day for males and 1501 mg/kg bw/day for females).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 501 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral toxicity

In a 28-day oral repeated dose study (according to OECD guideline 407 and under GLP), Sprague-Dawley rats (5/sex/dose) were administered the test substance at 750, 3750 and 15000 ppm in diet (SafePharm Laboratories, 1990). These concentrations in the diet are equal to 79, 404 and 1573 mg/kg bw/day for males and 78, 380 and 1501 mg/kg bw/day for females. The following parameters were evaluated: clinical signs, body weight, food efficiency, water consumption and compound intake, haematology, blood chemistry, urinalysis, organ weights and histopathology. No mortality and no clinically observable signs of toxicity were noted in test or control animals throughout the study. Bodyweight gain, food consumption and food efficiency in all test animals was comparable with that seen in controls. A reductions in haemoglobin levels, erythrocyte counts, haemotrocrit and platelet count in females of the highest dose group were considered to be not treatment induced. These effects were fully reversible during the 14 day post exposure observation period of the satellite groups.

Other non-treatment related effects observed were reduction in blood glucose was noted in males treated at 750 and 15000, a reduction in the calculated albumin/globulin ratio in high dose males and a reduced alkaline phosphatase in female animals treated at 3750 and 15000 ppm.

Urine parameters remained unchanged, and all morphological changes observed during necropsy were considered to be without toxicological significance.

In conclusion, the NOAEL of this study is above the highest tested dose of 15000 ppm (1573 mg/kg bw/day for males and 1501 mg/kg bw/day for females).

 

Further toxicological data of category members:

The test article belongs to the "perylene based organic pigments" category (see attached document for details on category members and for read across justification). According to the category approach, missing toxicity endpoints can be addressed with data available for other category members. Regarding repeated dose toxicity, reliable data are available for other members of the "Perylene based pigments category". All of these data are taken into account for the evaluation and assessment of the repeated dose toxicity of the test article.

Additional data is available for the oral and the inhalation route of exposure. After the oral exposure in further 28-day and/or 90-day repeated dose toxicity studies no adverse effects could be observed at the highest dose tested. In additional studies for the inhalation route of exposure local effects in the lung could be observed. After 5 and/or 90-day exposure inflammation processes were induced in the lung that were not fully reversible in two of the studies. Systemic effects after inhalation exposure could not be observed, even at the highest dose tested.

 

 

Statement on the Human Health Hazard Assessment of the Members of the Perylene Pigment Category including the Justification for Classification or Non-Classification

In the following statement the human health hazard of the Perylene pigments is assessed in the broader context of inhalation toxicity of poorly soluble particles. Therefore, a joint evaluation of particle specific properties (solubility, surface activity) and toxicological data of the perylene pigments was performed. Based on expert judgment, a classification or non-classification is derived for the individual members of the Perylene pigment category.

 

Particle specific investigations:

Solubility:

The available data consistently demonstrate that Perylene pigments are poorly soluble substances.  All category members have a low water solubility (solubility < 0.1 mg/L for all substances) and low solubility in n-octanol (< 10 mg/l for all substances). While this indicates poor solubility, it is not sufficient to conclude the assessment, as biosolubility may differ significantly from the solubility in water. Therefore, the OECD ‘Guidance document on inhalation toxicity studies’ suggests assessing the solubility of a solid material by measuring solubility in a simulated biofluid (OECD, 2018). The OECD guidance document further defines poor solubility if a material has a solubility of less than 0.1 g dissolved in 100 ml dissolvent within 24 hours. A test on biosolubility (static) and on dissolution kinetics (dynamic) in phagolysosomal simulant fluids was performed with all pigments of the category, except the intermediate product Pigment Red 224 (CAS 128-69-8), to determine the persistence after uptake in cells, e.g., alveolar macrophages. All substances tested were insoluble in phagolysosomal simulant fluid at pH 4.5 in the static and dynamic dissolution assay.

Surface Reactivity:

The surface reactivity of the pigment particles was investigated in chemico as well as in vitro using the FRAS (Ferric Reduction Ability of Serum) in combination with the EPR (Electron Paramagnetic Resonance) method and the in vitro macrophage assay. The assays were performed with all pigments of the category, except the intermediate Pigment Red 224 (CAS 128-69-8). None of the substances induced pro-inflammatory effects or cytotoxicity in rat alveolar macrophages according to the classification criteria of Wiemann et al. (2016.). The ability to induce biological oxidative damage in chemico was analyzed using the Ferric Reduction Ability of Serum (FRAS) assay and Electron Paramagnetic Resonance spectroscopy (EPR). The majority of the perylene pigments tested were classified as “passive” in both assays. Pigment Red 149 (CAS 4948-15-6) and 178 (CAS 3049-71-6), however, were classified as “passive” in the FRAS assay but “active” in EPR assay. Of note, the results of the red and violet Perylenes were consistently higher than those of the black Perylenes in the EPR assay. The black pigments appear to be significantly less surface active than the red and violet ones.

 

Toxicological in vivo data:

The available experimental data show, that the pigments of this category are not acutely toxic nor toxic after repeated oral exposure, not irritating to skin or eyes, do not cause skin sensitization, and are not genotoxic. In addition, no hazard concerning reproductive and developmental toxicity is concluded for members based on the currently available data. The only adverse effects observed were local effects after short-term and sub-chronic exposure via the inhalation route, whereas no systemic effects were observed after inhalation exposure.

Up to now, three studies on inhalation toxicity following repeated exposure are available, two 5-day short-term inhalation toxicity studies (STIS) according to OECD guideline 412 on Pigment Red 178 (CAS 3049-71-6) and 179 (CAS 5521-31-3) and a 90-day subchronic toxicity study according to OECD guideline 413 with Pigment Red 179.

Both pigments caused inflammation in the lung tissue at concentrations of 20mg/m³ and above in the STIS, with the effects being reversible within 3 weeks for Pigment Red 179. At the top dose of 60 mg/m³, Pigment Red 178 and 179 caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte counts in bronchioalveolar lavage fluid. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed. Most of these findings were also observed at 20 mg/m³ with reduced severity. For both pigments a NOAEC of 5 mg/m³ for local effects and a systemic NOAEC of above 60 mg/m³ was determined. The lung and the tracheobronchial lymph nodes were identified as target organs.

Due to the irreversibility of the effects of Pigment Red 178, the 90-day repeated dose inhalation study was performed with Pigment Red 178 following a worst-case approach. In this study, inflammation was observed at 20 and 5 mg/m³ as shown by increased inflammatory factors and protein levels in the lavage fluid, migration of inflammatory cells, cell debris, which correlated with the significantly increased lung weight and several histological changes in lungs. In line with the STIS results, there was not any indication for systemic effect. The effects were not reversible within 60 days. A NOAEC of 1 mg/m³ for local effects and a systemic NOAEC of above 60 mg/m³ was determined. The lung was identified as target organ. The observed effects were regarded as adverse and are presumably relevant for humans.

Overall, the perylene-based pigments are characterized by very low systemic toxicity even after repeated exposures to high doses. The absence of systemic effects observed after exposure via the oral or inhalation route and the insolubility observed in the solubility studies, indicate that the perylenes have a very low bioavailability. Therefore, the induction of local inflammation processes in the lung reported in the short-term inhalation studies as well as in the 90-day inhalation study are most likely induced by the surface activity of the particles. The results of the activity assays are evidence for the generation of reactive oxygen species at the particle surface for some of the Perylene pigments.

 

Conclusion:

As described above, the local effects after inhalation exposure are based on particle specific abiotic surface activity. Since the subgroups of the perylene pigments were demonstrated to differ in this property, a different evaluation of the pigments based on their measured abiotic surface activity is justified in this case.

 

Justification for Classification of Pigment Red 178 (CAS 3049-71-6), Pigment Red 179 (CAS 5521-31-3), Pigment Red 149 (CAS 4948-15-6), Pigment Violet 29 (CAS 81‑33-4) and intermediate product Pigment Red 224 (CAS 128-69-8):

As mentioned above, repeated inhalation exposure to Pigment Red 178 was shown to induce local inflammation processes in the lung at concentrations of 5 mg/m³ and above, but no systemic toxicity was observed in this subchronic inhalation study or any other of the toxicological in vivo studies available. The local effects to the lung were regarded as adverse and are presumably relevant for humans. Therefore, classification for Specific target organ toxicity (STOT RE) is justified under Regulation (EC) No. 1272/2008. However, due to the nature of the toxic effect observed, the guidance values mentioned in paragraphs 3.9.2.9.6 (Cat. 1) and 3.9.2.9.7 (Cat. 2), which take into account the duration of exposure and the dose/concentration which produced the effect(s), are not suitable for subclassification into category 1 or 2 in this case. Based on the explanations in chapter 3.9.2.9.8 these guidance values “are not intended as strict demarcation values”. Rather, in chapter 2 article 9 as well as in Annex I chapter 1.1.1, it is expressively permitted to “carry out an evaluation by applying a weight of evidence determination using expert judgement […] where the criteria cannot be applied directly to available identified information” and that “Expert judgement may also be required in interpreting data for hazard classification”. As outlined in chapter 3.9.1.3, adverse health effects in experimental animals relevant for STOT RE are defined as “toxicologically significant changes which have affected the function or morphology of a tissue/organ, or have produced serious changes to the biochemistry or haematology of the organism and these changes are relevant for human health”. One important reason for the deviation from the guidance values is that the local effects observed with the perylene pigments are not comparable to the effects that should be considered to support classification for STOT RE exposure according to chapter 3.9.2.7.3 such as e.g. morbidity or death, significant changes in biochemistry, significant organ damage noted as necropsy, microscopic changes indicative of necrosis or fibrosis. None of the listed effects nor any other effects indicative of a severe impairment of organ function or morphology were observed in the subchronic inhalation study on Pigment Red 178. As already mentioned above, in contrast to substances typically classified for STOT RE 2, it has a low systemic toxicity resulting from its insolubility and associated low bioavailability and the local effects observed in the respiratory tract can be most likely attributed to the surface activity of the pigment particles. Therefore, the effects are regarded as less severe compared to a STOT RE 1 classification. In order to still consider the adverse local effects to the lung, a classification for STOT RE 2 (H373, <lung>) is justified.

There are no data available on subchronic inhalation toxicity of Pigment Red 179 (CAS 5521-31-3), Pigment Red 149 (CAS 4948-15-6) and Pigment Violet 29 (CAS 81 33-4). In order to fill this data gap, a read-across from Pigment Red 178 (CAS 3049-71-6) was performed. The read-across is supported by the comparable high values measured in the EPR assay for the red and violet pigments as well as by the results from the STIS for Pigment 179. Therefore, Pigment Red 179, Pigment Red 149 and Pigment Violet 29 are classified for STOT RE 2 (H373, <lung>) too.

 

Justification for Non-classification of Pigment Black 31 (CAS 67075-37-0), Pigment Black 32 (CAS 83524-75-8), Perylen Black I (EC 479-300-2) and Perylen Black II (EC 475-310-6):

The induction of local inflammation processes in the lung reported in the short-term inhalation studies as well as in the 90-day inhalation study are most likely induced by the surface activity of the respective particles, as indicated in the results of the EPR. However, the values of the red and violet Perylenes were significantly higher than those of the black Perylenes in this assay. This means that although all category members are enormously similar in structural and physicochemical and toxicological parameters, there are differences between the substances in this property. The black pigments appear to be significantly less surface active than the red and violet ones and therefore are expected not to be less toxic to the lung.

Since, the results of the red and violet Perylenes were visibly higher than those of the black Perylenes it is justified to treat and classify the perylenes differently based on their activity measured in the EPR assay. This approach will be supported by two future short term inhalation studies investigating Pigment Black 32 and another of the much less active black pigments of this category. It is expected that no inflammatory, local effects will occur in these studies, further supporting the arguments for non-classification.

As described above, due to the local, partly non-reversible effects of Pigment Red 178, the red and violet pyrelene pigments must be classified as STOT RE2 under Regulation (EC) No. 1272/2008. However, as these effects are triggered by the surface activity of the pigments, a classification for the black pigments (Pigment Black 31/32 and Perylene Black I/II) is not justified. Therefore, no read-across from Pigment red 178 is performed.

 

References

OECD, 2018. Guidance document on inhalation toxicity studies. Series on testing and assessment No.39 (Paris).

Wiemann, Martin, et al. "An in vitro alveolar macrophage assay for predicting the short-term inhalation toxicity of nanomaterials." Journal of Nanobiotechnology 14.1 (2016): 1-27.

Regulation. "1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directive 67/548/EEC and 1999/45/EC and amending Regulation (EC) No 1907/2006." Official J Eur Union 353 (2008): 1.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008.

No data on repeated inhalation toxicity is available for the test substance, but data are available for the category member CAS 3049-71-6 (Pigment Red 178). In this study, repeated inhalation exposure to Pigment Red 178 was shown to induce local inflammation processes in the lung at concentrations of 5 mg/m³ and above, but no systemic toxicity was observed in this subchronic inhalation study or any other of the toxicological in vivo studies available for all category members.

The induction of local inflammation in the 90-day inhalation study, however, is most likely induced by the surface activity of the respective particles, as indicated in the results of the EPR (Please also refer to the Assessment Report in Chapter 13.2. or detailed explanations). However, the values of the red and violet Perylenes were significantly higher than those of the black Perylenes in this assay. This means that although all category members are enormously similar in structural and physicochemical and toxicological parameters, there are differences between the substances in this property. The black pigments appear to be significantly less surface active than the red and violet ones and therefore are expected not to be less toxic to the lung.

Since, the results of the red and violet Perylenes were visibly higher than those of the black Perylenes it is justified to treat and classify the perylenes differently based on their activity measured in the EPR assay. This approach will be supported by two future short term inhalation studies investigating Pigment Black 32 and another of the much less active black pigments of this category. It is expected that no inflammatory, local effects will occur in these studies, further supporting the arguments for non-classification.

As described above, due to the local, partly non-reversible effects of Pigment Red 178, the red and violet pyrelene pigments must be classified as STOT RE2 under Regulation (EC) No. 1272/2008. However, as these effects are triggered by the surface activity of the pigments, a classification for the black pigments (Pigment Black 31/32 and Perylene Black I/II) is not justified. Therefore, no read-across from Pigment red 178 to the test substance is performed.

As a result, the test substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.