7,17,28,38-tetraazatridecacyclo[24.16.2.2²,⁵.1⁸,¹².1²⁹,³³.0³,²².0⁴,¹⁹.0⁶,¹⁷.0²³,⁴³.0²⁷,³⁸.0⁴⁰,⁴⁴.0¹⁶,⁴⁶.0³⁷,⁴⁵]octatetraconta-1(42),2(48),3,5(47),6,8,10,12(46),13,15,19,21,23,25,27,29(45),30,32,34,36,40,43-docosaene-18,39-dione; 7,17,28,38-tetraazatridecacyclo[24.16.2.2²,⁵.1⁸,¹².1²⁹,³³.0³,²².0⁴,¹⁹.0⁶,¹⁷.0²³,⁴³.0²⁸,³⁹.0⁴⁰,⁴⁴.0¹⁶,⁴⁶.0³⁷,⁴⁵]octatetraconta-1(42),2(48),3,5(47),6,8,10,12(46),13,15,19,21,23,25,29,31,33,35,37(45),38,40,43-docosaene-18,27-dione

Endpoint summary

Description of key information

Oral: NOAEL (28d) >= 1000 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:

28 October 2005 - 19 December 2005

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:

; adopted on 27July 1995

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Physical state: Powder / black
- Storage condition of test material: Room temperature
- Analytical purity: 99.9%
- Lot/batch No.: Lot 2005-01
- Expiration date of the lot/batch: unlimited at room temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 41-43 days old
- Weight at study initiation: 100-112 g
- Housing: up to 5 of one sex to a cage, clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Code 1354 G, Techniplast Gazzada S.a.r.l., Buguggiate, Varese).
- Diet: commercially available laboratory rodent diet (4RF21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 to 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 28 October 2005 To: 2 December 2005 (main groups) or 19 December 2005 (recovery groups)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Following addition of the vehicle, the suspension was initially mixed manually with the aid of a spatula, then it was mixed by Silverson for approximately 2-3 minutes and finally stirred for approximately 5-6 minutes. The formulations were prepared daily.

administered dose volume: 10 ml/kg bw

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/ml.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the formulations prepared in weeks 1 and 4 were analysed to check the homogeneity and concentration. Results of the analyses were generally within the limits of acceptance (90-110%). In a single occasion, the measured concentration of group 2 was outside the limits of acceptance and was repeated on Day 2.

Duration of treatment / exposure:

4 consecutive weeks

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 (Five additional animals for each sex were included in the high and control groups for recovery assessment - two consecutive weeks)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from preliminary studies.
- Post-exposure recovery period in satellite groups: yes (2 weeks)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Mortality: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
Pre- and post-dose observations: All observations were recorded for individual animals. Examination of individual animals for signs of reaction to treatment was carried out daily prior to dosing, immediately after and approximately 1 and 2 hours after dosing.
Clinical signs and neurotoxicity assessment:
Once before commencement of treatment and once a week thereafter each animal was subjected to a detailed clinical examination, which included an evaluation of neurotoxicity. Animals were examined in an open arena for a period of a minimum of three minutes. Observed parameters: Removal (from cage), handling reactivity, lachrymation, palpebral closure, salivation, piloerection, rearing, spasms, myoclonia, mobility impairment, arousal (animal activity), vocalisation, stereotypies, unusual respiratory pattern, bizarre behaviour, urination, defecation, tremors, gait

In addition, from Day 6 of treatment, at each change of litter tray paper, an examination of the litter tray was performed and any signs were recorded. Once during week 4 of treatment and once during week 2 of recovery, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and assessment of grip strength were also performed.

Motor activity assessment (MA)
The motor activity (MA) of all animals was measured once during week 4 of treatment and once during week 2 of recovery by an automated activity recorder.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 4 of treatment, samples of blood were withdrawn from the retro-orbital sinus; prior to necropsy, blood samples were taken from the abdominal vena cava of the main phase rats from each group
- Anaesthetic used for blood collection: Yes (isofluorane anaesthesia)
- Animals fasted: Yes
- How many animals: all surviving male and female rats from each group
- Parameters checked : Haematocrit, haemoglobin, red blood cell count, reticulocyte count (not performed as no signs of anaemia were observed), mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells), abnormalities of the blood film, platelets, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During week 4 of treatment, samples of blood were withdrawn from the retro-orbital sinus; prior to necropsy, blood samples were taken from the abdominal vena cava of the main phase rats from each group
- Animals fasted: Yes
- How many animals: all surviving male and female rats from each group
- Parameters checked: Alkaline phosphatase,alanine aminotransferase, aspartate aminotransferase, gamma -glutamyltransferase, urea, creatinine, glucose, triglycerides, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G Ratio, sodium, potassium, calcium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: During week 4 of treatment, individual overnight (approximately 16 hours) urine samples were collected from all surviving male and female rats from each group
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: Appearance, volume, specific gravity, pH, protein, total reducing substances, glucose, ketones, bilirubin, urobilinogen, blood; t he sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: Epithelial cells, poly morphonuclear leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes (see: Detailed clinical observations)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Necropsy
The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Organ weights
From all animals the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues were fixed and preserved in 10% buffered formol saline (except testes and epididymides which were fixed in Bouin's solution and preserved in 70% ethyl alcohol). One slice from the liver lobes, the lobus dexter lateralis and the lobus sinister lateralis, was fixed in Carnoy's solution. Liver samples fixed in Carnoy's solution from the low, mid and recovery groups were processed onto the paraffin block.

Histopathological examination
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. Samples fixed in Carnoy' s solution were cut at 2-3 micrometre thickness. In the first instance the examination was restricted as follows:
a) Tissues from all animals in the control and high dose group killed after 4 weeks of treatment;
b) All abnormalities in all main phase groups.
Pertaining to the liver, only the sections fixed in Carnoy's solution were examined by the pathologist.

Statistics:

For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error variance. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No signs were observed at daily post-dose observations. Detailed clinical signs with neurotoxicity assessment did generally not show any signs which could be correlated to the treatment with the test item. Dark faeces (with a dose-related intensity) were seen in the litter tray of all animals treated with the test item for the duration of the treatment period. This sign was no longer evident by Day 6 of the recovery phase.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant differences in body weights were noted between treated animals and controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was not affected by treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes of toxicological significance were observed in haematological parameters. The slight increments, generally statistically significant, of red blood cells, haemoglobin and the decrement of mean corpuscular volume observed in females treated with 1000 mg/kg/day were considered to be incidental and without toxicological significance, as they were slight, not dose-related and fully within historical control values.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical chemistry parameters did not show any changes of toxicological importance. The slight, statistically significant differences observed in some parameters (aspartate aminotransferase, glucose, triglycerides, urea, albumin, sodium and calcium) were not dose related, not consistent between sexes and insufficient in magnitude to be of toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No significant changes were observed at analysis of urine parameters.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No significant differences between treated animals and controls were observed at evaluations of sensory reaction performed at the end of treatment. Motor activity measurements performed at the end of the treatment period did not show changes, which could be ascribed to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No changes of toxicological significance were observed in the weight of the organs.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No change of toxicological significance was reported at post mortem examination, in treated animals killed at the end of treatment or at completion of the recovery period, when compared with controls.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histopathological examination did not reveal any evident differences in the incidence of the findings observed in treated and control animals, which could be considered treatment related. The lesions described were considered evidence of spontaneous pathology, often seen in this species under our experimental conditions.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

Based on the results reported, the high-dose of 1000 mg/kg/day, when administered daily for 4 consecutive weeks, was considered the No Observed Adverse Effect Level (NOAEL).

Executive summary:

In a GLP-compliant oral subacute toxicity study following OECD test guideline 407, three groups, each of 5 male and 5 female Sprague Dawley rats received the test item by gavage at dosages of 100, 300 and 1000 mg/kg/day for 4 consecutive weeks. A fourth similarly constituted group received the vehicle alone (0.5% CMC) and acted as a control. Five additional animals for each sex were included in the high and control groups for recovery assessment.

No mortality occurred during the study and no clinical signs were observed at daily post-dose observations which could be correlated to the treatment with the test item. No significant differences between treated animals and controls were observed at evaluations of sensory reaction performed at the end of treatment. Motor activity measurements performed at the end of the treatment period did not show changes, which could be ascribed to treatment. No significant differences in body weights were noted between treated animals and controls. Food consumption was not affected by treatment. No changes of toxicological significance were observed in haematological and clinical parameters. No significant changes were observed at analysis of urine parameters. No changes of toxicological significance were observed in the weight of the organs. No change of toxicological significance was reported at Post mortem examination, in treated animals killed at the end of treatment or at completion of the recovery period, when compared with controls. The histopathological examination did not reveal any evident differences in the incidence of the findings observed in treated and control animals, which could be considered treatment related. The lesions described were considered evidence of spontaneous pathology, often seen in this species under our experimental conditions.

On the basis of these results, no signs of an adverse effect of the test item were seen at any of the dose levels investigated (100, 300 and 1000 mg/kg/day). Therefore, the high-dose of 1000 mg/kg/day, when administered daily for 4 consecutive weeks, was considered the No Observed Adverse Effect Level (NOAEL).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral toxicity

In a GLP-compliant oral subacute toxicity study following OECD test guideline 407, three groups, each of 5 male and 5 female Sprague Dawley rats received the test item by gavage at dosages of 100, 300 and 1000 mg/kg/day for 4 consecutive weeks (Research Toxicology Centre, 2006). A fourth similarly constituted group received the vehicle alone (0.5% CMC) and acted as a control. Five additional animals for each sex were included in the high and control groups for recovery assessment.

No mortality occurred during the study and no clinical signs were observed at daily post-dose observations which could be correlated to the treatment with the test item. No significant differences between treated animals and controls were observed at evaluations of sensory reaction performed at the end of treatment. Motor activity measurements performed at the end of the treatment period did not show changes, which could be ascribed to treatment. No significant differences in body weights were noted between treated animals and controls. Food consumption was not affected by treatment. No changes of toxicological significance were observed in haematological and clinical parameters. No significant changes were observed at analysis of urine parameters. No changes of toxicological significance were observed in the weight of the organs. No change of toxicological significance was reported at Post mortem examination, in treated animals killed at the end of treatment or at completion of the recovery period, when compared with controls. The histopathological examination did not reveal any evident differences in the incidence of the findings observed in treated and control animals, which could be considered treatment related. The lesions described were considered evidence of spontaneous pathology, often seen in this species under our experimental conditions.

On the basis of these results, no signs of an adverse effect of the test item were seen at any of the dose levels investigated (100, 300 and 1000 mg/kg/day). Therefore, the high-dose of 1000 mg/kg/day, when administered daily for 4 consecutive weeks, was considered the No Observed Adverse Effect Level (NOAEL).

 

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 could be 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.