Registration Dossier
Registration Dossier
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EC number: 226-971-2 | CAS number: 5580-58-5
Toxicological information
Endpoint summary
Administrative data
Description of key information
Oral:
subacute (28-days): NOAEL = 1000 mg/kg bw/day (OECD 407); read-across with CAS 5580-57-4
subacute (screening): NOAEL = 1000 mg/kg bw/day (OECD 422); read-across with CAS 68516-73-4 and 79953-85-8
Inhalative:
subacute (5 d): NOAEC (local) = 5 mg/m³ air; NOAEC (systemic) = 60 mg/m³ air; read-across with CAS 79953-85-8
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- READ ACROSS ANALOGUE APPROACH
The read-across hypothesis is fundamentally based on the same core structure of five ‘yellow disazo condensation pigments’ which optionally can serve as target or as source substances. None of the pigments are sufficiently soluble, either in water or in octanol for systemic uptake or metabolism. The molecular weight ranges from 716.6 g/mol (Pigment Yellow 155) to 1229.2 g/mol (Pigment Yellow 128). Therefore, the molecular weights of all ‘yellow disazo condensation pigments’ are well above the threshold of 500 g/mol, which is generally considered for low dermal and oral uptake [ECHA Guidance R. 7c, 2017]. Furthermore, for each of the substances, the critical body burden (CBB) is above the octanol solubility, which generally indicates a low uptake in biota and makes toxicity unlikely [ECHA Guidance R. 11, 2017].
An evidence of the low bioavailability for all five pigments is the absence of any systemic toxicity in the repeated-dose toxicity study with Pigment Yellow 128 (highest molecular weight), Pigment Yellow 93 (intermediate molecular weight) and Pigment Yellow 155 (lowest molecular weight). From theoretical considerations, uptake and metabolism should result in the release of aromatic amines; such compounds have a characteristic toxicity profile. As this was not observed, all five substances are not considered to be taken up by the body. This assumption is supported by results of several other toxicological and ecotoxicological studies with the pigments, in which no hazards were identified. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Key result
- 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
- Key result
- Critical effects observed:
- no
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From Nov 2021 to Oct 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- TEST MATERIAL
Batch no. 0004079400
CAS no. 79953-85-8
Purity 99.4 wt%
Expiry/Retest date 27 August 2022
Appearance Yellow powder
Storage conditions Room temperature - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar Hannover rat
- Details on species / strain selection:
- The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities. - Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females: nulliparous and non-pregnant
- Age at study initiation: 9 - 11 weeks old
- Weight at acquisition: 212-230 g for males and 182-193 g for females
- Housing arrival - mating: animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm, nesting material was provided inside suitable bedding bags and changed at least twice a week
- Housing during mating: animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor, each cage tray held absorbent material which was inspected and changed daily
- Housing after mating: the males were re-caged as they were before mating, females were transferred to individual solid bottomed cages for the gestation period, birth and lactation, nesting material was provided inside suitable bedding bags, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum, via water bottles
- Acclimation period: approx 2 weeks
DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 21 Dec 2021 (allocation) To: 17 Feb 2022 (last day of necropsy) - Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5% CMC in water, softened by reverse osmosis
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Dose volume: 10 mL/kg body weight, dose volumes for males and females were adjusted once per week for each animal according to the last recorded body weight, dose volumes for females during the gestation and lactation periods were calculated according to the last recorded body weight
- Dosing preparation: preparations were made daily, preparations were kept under magnetic stirring for at least 16 hours at room temperature and up to completion of dosing - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analytical method was validated in a separate study in the range from 10 to 100 mg/mL. In the same study, a 28 hour stability at room temperature and a 8-day stability at 2- 8°C were verified in the range from 10 to 100 mg/mL. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity).
- Duration of treatment / exposure:
- Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 39 days.
Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprising from 51 to 55 days. - Frequency of treatment:
- daily
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Group 1
Control - Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Group 2
Low-level dose - Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Group 3
Medium-level dose - Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- Group 4
High-level dose - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: dose levels of 100, 300 and 1000 mg/kg/day were selected based on information from subacute oral toxicity studies with structurally related pigments. 1000 mg/kg bw is the maximimum dose prescribed in the OECD testing guideline.
- Rationale for animal assignment: rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights., individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage
- Fasting period before blood sampling for clinical biochemistry: yes - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Mortality: checked twice daily
- Clinical signs: once before commencement of treatment and at least once daily during the study
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter
- Observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions (see table No. 1 for observed parameters and evaluation scale)
- Animals were examined in an open arena for a minimum of three minutes
BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed weekly from allocation to termination, females were weighed weekly from allocation to positive identification of mating and on days 0, 7, 14 and 20 post coitum, dams were also weighed on days 1, 4, 7, 13 post partum and just before to necropsy
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- the weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting from day 1 of dosing up to mating
- Individual food consumption for mated females were measured on gestation days 7, 14 and 20 post coitum starting from day 0 post coitum and on day 7 and 13 post partum starting from day 1 post partum
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (except for coagulation test in males)
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 2 were examined
- Coagulation parameters investigated: prothrombin time and activated partial thromboplastin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Animals fasted: Yes
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 3 were examined
SERUM HORMONES: Yes
- Time of blood sample collection: at termination, timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days
- Animals fasted: Yes
- How many animals: all parental animals, each litter (1 sample for males and 1 sample for females, when possible), blood samples from different pups was pooled to obtain the required volume
- Hormones analyzed: serum levels of total thyroxine (total T4) and thyroid stimulating hormone (TSH)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: once during study towards end of treatment, for males on day 39 and for females (with viable litters) on day 12 post partum
- Number of animals: 5 males and 5 females randomly selected from each dose group
- Studies conducted: grip strength and sensory reactivity to stimuli, motor activity assessment
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- Necropsy: the clinical history of adult animals 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
- All females were examined also for the following: number of visible implantation sites (pregnant animals), number of corpora lutea (pregnant animals)
- Pups: all pups found dead in the cage were examined for external and internal abnormalities, all culled pups sacrificed at day 4 post partum were subjected to an external examination, all live pups sacrificed on day 14 post partum were examined for external abnormalities, gonads were inspected from all pups in order to confirm the sex previously determined by external examination
ORGAN WEIGHTS:
- Parental animals: from all animals completing the scheduled test period, the organs indicated in table No. 4 were dissected free of fat and weighed, the ratios of organ weight to body weight was calculated for each animal
- Pups at day 14 post partum: thyroid was weighed from one male and one female pup selected for blood collection and preserved in 10% neutral buffered formalin, the thyroid weight was determined after fixation
HISTOPATHOLOGY: Yes
- Samples of all tissues (parental animals) required for histopathological examination are listed in table No. 4
- Samples of the tissue were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol)
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin, in addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS), the morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed
The examination was restricted as detailed below:
(i) Sexual organs (cervix, clitoral gland, ovaries, uterus and vagina) and thyroid glands from all parental females
(ii) Sexual organs (coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes) and thyroid glands from all parental males
(iii) Tissues specified in section 4.5.7 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term
(iv) Morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle) from all males in control and high dose groups
(v) All abnormalities in all groups - Other examinations:
- PARTURITION AND GESTATION LENGTH:
- Parturition check from day 20 - 25 post coitum, three rimes a day during working day or twice daily during weekends and Public Holidays
- Females which did not give birth after 25 days of post coitum period were sacrificed shortly after
- Gestation length calculated as the time between the day of successful mating (day 0 post coitum) and the day of birth
- Day of birth was defined as day 0 post partum
PUPS IDENTIFICATION, WEIGHT AND OBSERVATIONS:
- As soon as possible after parturition was considered complete (day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified
- Live pups were individually weighed on days 1, 4, 7 and 13 post partum
- Pups dying during the lactation period were weighed before the despatch to necropsy
- Observation was performed once daily for all litters from day 0 post partum until termination
- After culling, all pups were sacrificed with the dams on Day 14 post partum
ANOGENITAL DISTANCE (AGD):
- For each pup measured on day 1 post partum
- AGD was normalized to the cube root of body weight collected on day 1 post partum
NIPPLE COUNT CHECK:
- presence of nipples/areolae in male pups was checked on day 13 post partum
REPRODUCTIVE INDICES:
- Males: copulation index, fertility index
- Females: copulatory index, fertility index, pre-natal loss, post-natal loss at day 0, 4 and 13 post partum
- Pre coital interval
- Sex ratio of litter - Statistics:
- Standard deviations were calculated as appropriate.
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. - Clinical signs:
- no effects observed
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One control female was sacrificed for humane reason on Day 22 of gestation phase due to difficulties in parturition.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Body weight and body weight gain for male and female animals were comparable between treated and control groups through the study. The statistically significant decrease in body weight gain recorded in high dose males and mid-dose females were considered incidental.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food consumption was unaffected by treatment in both genders during the study.
- Food efficiency:
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- he statistically significant differences between contrTol and treated animals (neutrophils in males and platelets in females) were recorded only in animals dosed at 300 mg/kg/day, therefore they were considered to be incidental.
- Clinical biochemistry findings:
- no effects observed
- Endocrine findings:
- no effects observed
- Description (incidence and severity):
- The determination of T4 and TSH was performed on:
- Samples from all parental males from all groups
- Samples from pups on Day 14 post partum.
No treatment-related changes were recorded. Thyroid stimulating hormone was statistically significantly lower than controls in parental males (31%). This is due to 2 control animals, which showed values above the range of historical data, therefore the differences observed has no biological/toxicological significance.
The oestrous cyclicity of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated animals and control group was comparable.
No differences were recorded in pre-coital interval and copulation plugs between treated and control groups. Copulatory index and fertility index did not show any treatment-related differences. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Observation of treated animals at removal from the cage and in an open arena did not reveal significant changes when compared to controls.
No alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related microscopic observations at the end of the treatment period. There were no test item-related microscopic observations in the testis (stage were evaluation on PAS-stained slides). Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- effects observed, treatment-related
- Dose descriptor:
- NOAEL
- Remarks:
- systemic toxicity
- 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:
- The oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of systemic toxicity up to the highest tested dose level. Therefore, a NOAEL for general systemic toxicity was set to 1000 mg/kg bw/day.
- Executive summary:
Under the conditions of the OECD TG 422 and in compliance with GLP, a combined repeated dose toxicity study with the reproductive/developmental screening test was conducted in male and female Wistar rats. The test substance was administered daily as a solution to groups of 10 male and 10 female Wistar rats (P0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 1), 100 mg/kg bw/d (test group 2), 300 mg/kg bw/d (test group 3) and 1000 mg/kg bw/d (test group 4). 0.5% carboxymethylcellulose served as vehicle, control animals were dosed daily with the vehicle only. All doses were administered at a constant volume of 10 mL/kg body weight. For males, the duration of treatment covered a 2-week premating period and pairing with females until the day before necropsy (a total of 39 days). Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprised from 51 to 55 days.
No test-item related mortality occurred throughout the study and no treatment-related clinical signs were noted during the study. No signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reactivity to stimuli) were observed during the study in parental males and females. No differences in body weight and food consumption were observed in treated animals, compared to the control group. No treatment-related changes were observed in haematological (including coagulation and blood clotting time) or clinical chemistry parameters. Thyroid hormone evaluation in parental animals and pups sacrificed on day 14 post partum did not show treatment-related changes. No treatment-related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls. No treatment-related changes were observed at post mortem macroscopic observations and microscopic evaluation. No treatment-related changes were observed in reproductive and developmental parameters.
Copulatory and fertility indices did not show any treatment related differences among treated and control groups. Parturition, lactation, implantation, litter data and sex ratio did not show changes. No differences in the anogenital distance (normalised value) and no nipple were seen between control and treated groups both for male and female pups. Necropsy findings and thyroid weight in pups did not reveal any treatment-related effect.
Therefore, the no observed adverse effect level (NOAEL) for general systemic toxicity was determined to be 1000 mg/kg bw/day for male and female Wistar rats.
- Endpoint:
- short-term repeated dose toxicity: oral
- Remarks:
- combined repeated dose and reproduction / developmental screening
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012-1013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 wks
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Pigment Yellow 155 was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.
VEHICLE
- Concentration in vehicle: 1, 3, or 10 g/100mL, respectively - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- males treated 41 days (pre-mating, post-mating)
females treated 52 days (pre-mating, gestation, 4 days lactation) - Frequency of treatment:
- daily
- Remarks:
- Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
actual ingested - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, littering and lactation behavior of the dams, parturition behavior of the dams
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
BODY WEIGHT: Yes
- Time schedule for examinations: Before the start of the administration period. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
FOOD CONSUMPTION:
Food consumption was determined once a week for male and female parental animals, except during mating period, during gestation.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: no data
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 of each sex
- Parameters checked: leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: no data
- Animals fasted: Yes
- How many animals: 5 of each sex
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids
URINALYSIS: Yes
- Time schedule for collection of urine: males on day 35 and females on day 51
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional observational battery was performed in the first five parental males and females (with litter) per group at the end of the administration period. The motor activity assessment (MA) was carried out in the first five parental males and females (with litter) per group at the end of the administration period.
- Dose groups that were examined: all
- Battery of functions tested: functional observational battery / motor activity - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes (Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal gland, Epididymides, Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs, Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina)
HISTOPATHOLOGY: Yes (All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lung, Lymph nodes (mesenteric and axillary lymph nodes), Ovaries, Oviducts, Peyer’s patches, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina)
Organn weights: Epididymides, Testes, Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus - Statistics:
- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter where analysed by simultaneous com-parison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity where analysed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Blood parameters were analysed by non-parametric one-way analysis using KRUSKAL-WALLIS test for parameters with bidirectional changes. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. For parameters with unidirectional changes pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians was used.
Urinalysis parameters were analysed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Weight parameters were analysed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- yellow discolored feces, no mortality
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- yellow discolored feces, no mortality
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%)
- 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:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- CLINICAL SIGNS AND MORTALITY: No animal died prematurely in the present study. All male and female animals of test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) showed yellowish discolored feces towards the end of the study.
BODY WEIGHT AND WEIGHT GAIN: Mean body weights and mean body weight gain of the F0 males in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire study period. Except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%). Mean body weight and mean body weight gain of the F0 females in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.
FOOD CONSUMPTION: Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.
HAEMATOLOGY: No treatment-related, adverse changes among hematological parameters were observed.
In males of test group 1 (100 mg/kg bw/d) total white blood cell (WBC) counts were higher compared to controls, but this parameter was not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.
CLINICAL CHEMISTRY: No treatment-related changes among clinical chemistry parameters were observed.
In males of test groups 1 and 2 (100 and 300 mg/kg bw/d) cholesterol levels were decreased, but this parameter was not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.
URINALYSIS: No treatment-related changes among urinalysis parameters were observed.
NEUROBEHAVIOUR: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered as incidental. There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group. Regarding single intervals in males and females no significant deviations were detected.
ORGAN WEIGHTS: The weight decrease in absolute epididymis and liver weight in males of test group 2 (300 mg/kg bw/day) was regarded to be incidental due to a missing dose response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw). The decrease in thymus weights of females in test group 3 (1000 mg/kg bw/day) was also regarded to be incidental as no histopathologic correlate could explain the weight decrease (for details see table 1 and 2).
All other mean weight parameters did not show significant differences when compared to the control groups.
GROSS PATHOLOGY: Several animals of test group 3 (1000 mg/kg bw) revealed a yellow discoloration of the contents of the glandular stomach, small and large intestines. These findings are regarded to be treatment related (see table 3).
Each one male and female of test groups 1 and 2 (100 and 300 mg/kg bw/day) (animals No. 12, 21, 111, 125) revealed either some or all of the following findings: deposition or foci on the lung, deposition on the diaphragm, discoloration and enlargement of the mediastinal lymph nodes, deposition on the thymus, and deposition on the sternum. All these findings were regarded to be treatment related by gavage errors.
All other gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.
HISTOPATHOLOGY: The discoloration of the content in the digestive tract was regarded to be a consequence to the oral intake of the test substance which is of yellow color. Therefore, the gross findings in the remaining animals were not investigated in addition.
The discoloration and depositions described for animals No. 12, 21, 111, 125 in several organs of the thoracic cavity were multifocal granulomas with intrahistiocytically located yellow particles, interpreted as test substance. The granulomas were foreign body reactions interpreted as a consequence to a gavage error. The discoloration of the mediastinal lymph nodes was regarded to be the physiologic clearing route of the lung. Particles that were located intraalveolar were transported via macrophages to the regional lymph nodes and caused there the activation of the lymph nodes and the discoloration. Therefore these depositions and discolorations were caused by the test substance due to a gavage error and were not regarded to be an adverse finding.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: based on reduced food consumption and the decreased body weight change during the gestation
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- The pigment showed no adverse effects upon subacute oral dosings of rats.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2008 - 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP and OECD 407 compliant study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:SPF breeding, VELAZ s.r.o.., Kolec u Kladna, Czech Republic, RCH CZ 21760152
- Age at study initiation: 6 -7 weeks
- Weight at study initiation: ca 130g (females) and 170g (males)
- Fasting period before study: no information
- Housing: 2-3 rats of the same sex in one plastic cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 16.09.2008 To: 16.10.2008 - Route of administration:
- oral: gavage
- Vehicle:
- olive oil
- Details on oral exposure:
- The application form (test substance suspension in olive oil) was prepared daily just before administration.
- Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- The test substance is insoluble in many solvents. Low solubility of the test substance is declared by the sponsor and it was also checked up within the substance identity verification. In this study the substance was found also insoluble in olive oil. Therefore the test substance was administered as an olive oil suspension. A suitable analytical method was not found for homogeneity and stability testing. Since undissolved particles of the test substance are easily visible in the application form, homogeneity was checked by eye (suspension were mixed for 15 minutes by magnetic stirrer).
- Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- daily
- Remarks:
- Doses / Concentrations:
0, 160, 400 and 1000 mg/kg bw
Basis:
actual ingested - No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Range-finder study
- Rationale for selecting satellite groups: Reversibility of potential effects at the top dose.
- Post-exposure recovery period in satellite groups: 14 days - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Mortality, general health condition and general clinical symptoms were observed daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
At the first part of observation behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour. The second part was the observation during removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION:
- Food consumption: weekly
WATER CONSUMPTION : Yes
- Time schedule for examinations: twice per week
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28 and day 42 (for recovery animals)
- Anaesthetic used for blood collection: Yes (light ether narcosis)
- Animals fasted: 18h
- How many animals: all
- Parameters: total erythrocytes count, mean corpuscular volume, haematocrit, haemoglobin concentration, total leucocytes count, total platelets count, partial thromboplastin time, prothrombin time, fibrinogen, granulocytes, lymphocytes, monocytes
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28 and day 42 (for recovery animals)
- Animals fasted: 18h
- How many animals: all
- Parameters checked in table 1 were examined.
URINALYSIS: Yes
- Time schedule for collection of urine: day 28 and day 42 (for recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table 2 were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the last week of treatment and the last week of the recovery time.
- Dose groups that were examined: all
- Battery of functions tested: During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) pectoral legs, 2) pelvis legs, 3) all four legs. Grip power was expressed in Newtons. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, thymus, spleen, brain, pituitary gland and heart were recorded.
HISTOPATHOLOGY: Yes (see table 4) - Statistics:
- The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with satellite treated group.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of medians or averages. - Details on results:
- CLINICAL SIGNS AND MORTALITY: There were no unscheduled deaths during the test. No clinically observed signs of toxicity were detected. The animal' s health condition was very good during whole study and functional observation evidenced no effect ofthe test substance.
BODY WEIGHT AND WEIGHT GAIN: No adverse effect on body weight development was detected. The body weight of all treated and control groups were well-balanced without statistically significant difference during the whole application and recovery period. The intergroup differences were minimal and isolated, therefore they were regarded as fortuitous. No significant differences of average body weight increment were recorded in treated groups of males and females (in comparison with control).
FOOD/WATER CONSUMPTION AND COMPOUND INTAKE: No adverse effect on dietary intake or food conversion was detected. Food consumption of all groups was well-balanced. Sporadic slight intergroup reduction or increasing of food consumption and food conversion of males were considered to be a result of normal variation, which could not be related with administration of the test substance. Inspection of water consumption revealed no significant intergroup differences during the application period.
HAEMATOLOGY: The haemocoagulation examination detected statistically significant changes only in males. Shortening of protrombine time and prolongation of partial thromboplastin time were recorded at all dose levels but without dose-response relationship. These changes were not recorded in satellite treated males after 14-day recovery period - the effect of the test substance on this blood component was reversible. All changed parameters were within historical control range. These intergroup differences were considered to be adaptative response of a living organism to stress without toxicological significance.
The haematology parameters of red blood cells were without significant changes. Decreased value of haematocrite was measured in males of the lowest dose level only at the end of application period, but expected related changes of total erythrocyte count, mean corpuscular volume or haemoglobin volume were not detected. Changes of differential leucocyte count were recorded in both sexes but with statistical significance only in males at the middle dose level at the end of application period. Increased total leucocyte count was found out at the end of recovery period. These differences of haematological parameters were considered to be adaptative response to stress without toxicological significance.
CLINICAL CHEMISTRY: Statistical analysis of parameters revealed one significant difference in males (decreased calcium concentration against control at the lowest dose level). This variable factor was not important toxicologically because its fluctuation was within historical control range.
URINALYSIS: Urinalysis detected no treatment-related effects in males and females.
NEUROBEHAVIOUR: Functional observation evidenced no effect of the test substance. All inter and intra group differences in behavioural scores (upstanding, emiction, defecation) and sensory reactivity were considered to be a result of normal variation for rats of the strain and age used and were no toxicological importance.There were no treatment-related changes in functional performance parameters measured (grip strength).
ORGAN WEIGHTS: Biometry of organs detected intergroup difference in absolute and relative weight of thymus in males but statistically significant increase was found only in absolute weight in the middle dose level. Slight intergroup difference of uterus weight was recorded at treated females. These morphological changes are commonly observed in employed strain of females and are considered to be not of toxicological significance.
GROSS PATHOLOGY: Pathology examination revealed yellowish colour of chyme and coloured mucous membrane in digestive system at the end of application period with dose-related response. Other macroscopic findings were found out in stomach of treated males and females: congestion of mucous membrane, dilatation or focal changes on mucous membrane. These changes were not recorded at the end of recovery period - the effect of the test substance was reversible. Application of the test substance caused temporary colouring of the chyme but without marked effect on function of digestive tract.
HISTOPATHOLOGY: Metastatic mineralization in kidneys of females at the middle and highest dose levels were found out at the end of the application period. Due to absence of changed urinary parameters, this was considered to be no toxicological importance. Further sporadic pathologic changes were recorded: inflammation in prostate gland in males and hydrometra of uterus or cyst on ovaries in females. Frequencies of these microscopic findings in genital tract were similar in treated and control groups. This was considered to be of no toxicological importance. No neoplastic findings were recorded by histopathological examination.
OTHER FINDINGS: Yellowish colour of excrement in the treated animals was recorded from the 1st week of application to the 1st week of the recovery period. As the pigment is of yellow color, this intakes passage of the test item through the gastrointestinal tract. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: highest dose tested
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: highest dose tested
- Critical effects observed:
- not specified
Referenceopen allclose all
Table 1: Absolute weights: Male animals:
Male animals |
|||
Test groups (mg/kg bw/day) |
1 (100) |
2 (300) |
3 (1000) |
Epididymides |
101% |
94%* |
98% |
Liver |
93% |
88%* |
95% |
* p≤ 0.05
Table 2: Relative weights: female animals
Female animals |
|||
Test groups (mg/kg bw/day) |
1 (100) |
2 (300) |
3 (1000) |
Thymus |
90% |
105% |
82%** |
** p ≤ 0.01
Table 3: Gross lesions
Male animals |
Female animals |
|||||||
Test groups (mg/kg bw/day) |
0 (0) |
1 (100) |
2 (300) |
3 (1000) |
0 (0) |
1 (100) |
2 (300) |
3 (1000) |
Glandular stomach Discoloration of contents |
2 |
7 |
1 |
6 |
7 |
|||
Jejunum Discoloration of contents |
1 |
1 |
4 |
|||||
Ileum Discoloration of contents |
1 |
7 |
4 |
|||||
Cecum Discoloration of contents |
1 |
7 |
8 |
|||||
Colon Discoloration of contents |
1 |
5 |
3 |
|||||
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
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- READ ACROSS ANALOGUE APPROACH
The read-across hypothesis is fundamentally based on the same core structure of five ‘yellow disazo condensation pigments’ which optionally can serve as target or as source substances. None of the pigments are sufficiently soluble, either in water or in octanol for systemic uptake or metabolism. The molecular weight ranges from 716.6 g/mol (Pigment Yellow 155) to 1229.2 g/mol (Pigment Yellow 128). Therefore, the molecular weights of all ‘yellow disazo condensation pigments’ are well above the threshold of 500 g/mol, which is generally considered for low dermal and oral uptake [ECHA Guidance R. 7c, 2017]. Furthermore, for each of the substances, the critical body burden (CBB) is above the octanol solubility, which generally indicates a low uptake in biota and makes toxicity unlikely [ECHA Guidance R. 11, 2017].
An evidence of the low bioavailability for all five pigments is the absence of any systemic toxicity in the repeated-dose toxicity study with Pigment Yellow 128 (highest molecular weight), Pigment Yellow 93 (intermediate molecular weight) and Pigment Yellow 155 (lowest molecular weight). From theoretical considerations, uptake and metabolism should result in the release of aromatic amines; such compounds have a characteristic toxicity profile. As this was not observed, all five substances are not considered to be taken up by the body. This assumption is supported by results of several other toxicological and ecotoxicological studies with the pigments, in which no hazards were identified. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Key result
- Dose descriptor:
- NOAEC
- Remarks:
- local effects
- Effect level:
- 5 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: min. infiltration of neutrophils within bronchiolar epithelium, hyptrophy/hyperplasia of terminal bronchi (terminal and small), hyperplasia of type II pneumocytes, increased cellularity in the mediastinal lymph nodes. Findings in BALF
- Key result
- Dose descriptor:
- NOAEC
- Remarks:
- systemic effects
- Effect level:
- >= 60 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: only findings in BALF
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 mg/m³ air (analytical)
- System:
- respiratory system: upper respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Apr 22 - Oct 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- 5-day dust inhalation study in rats (with bronchoalveolar lavage, 3 weeks recovery period)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- yellow powder
- 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 (when supplied)
- Weight at study initiation (means):ca 265g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [P SU]) 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 Switze rland), ad libitum.
- Water: Tap water, ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%) 45 - 65
- Air changes (per hr): 15
Photoperiod (hrs dark / hrs light):: 12h/12h - Route of administration:
- inhalation: dust
- Type of inhalation exposure:
- nose/head only
- Vehicle:
- clean air
- Mass median aerodynamic diameter (MMAD):
- >= 0.66 - <= 0.77 µm
- Geometric standard deviation (GSD):
- 245
- Remarks on MMAD:
- All measurements of particle size resulted in MMADs between 0.66 and 0.77 µm with GSDs between 2.06 and 2.90. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 90.0 % and 97.6 %.
SMPS showed different geometric mean concentration than those measured by cascade impactor measurement. Major reason is that this geometric mean referred to count distribution, while cascade impactor measurement measured mass-based aerodynamic diameter.
The SMPS showed very high particle count concentrations in all concentrations. The geometric mean count diameters were between 266 nm and 295 nm. - Details on inhalation exposure:
- For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned air, and passed via the cyclonic separator and glass tube into the inhalation system
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 con
centration 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. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentrations of the inhalation atmospheres in test groups 1 - 3 were analyzed by gravimetry. This method was applicable because the test item possessed extremely low 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 these groups, the constancy of concentrations in each chamber was continuously monitored using scattered light photometers.
The particle size analysis was carried out with a cascade impactor with the following equipment:
• 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 6.9 mm
• Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)
Sampling for particle size analyses:Pre-weighed metal collecting discs and a backup particle filter were placed into the cascade impactor and two samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.
The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the filter/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.
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. During the exposure period, one measurement per concentration with 10 repeats each were performed.
Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures.
The air flows were constantly maintained in the desired range. An air change of about 65 to 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system. Daily mean relative humidities in the inhalation systems ranged between 33.7 and 49.6 %. Daily mean temperatures in the inhalation systems ranged between 20.6 and 22.1 °C. These values were within guideline recommendations. - Duration of treatment / exposure:
- 6h for 5 days
- Frequency of treatment:
- daily
- Dose / conc.:
- 4.98 mg/m³ air (analytical)
- Remarks:
- SD 0.27
- Dose / conc.:
- 20 mg/m³ air (analytical)
- Remarks:
- SD 1.0
- Dose / conc.:
- 60.4 mg/m³ air (analytical)
- Remarks:
- SD 2.1
- No. of animals per sex per dose:
- 10 (five for sacrifice after exposure and 5 for sacrifice after recovery)
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Dose selection rationale: Results of short-term inhalation studies with other inert organic pigments
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical bioche Particlesmistry: overnight
- Rationale for selecting satellite groups: Clearance of inert particles by lung macrophages is known to take time
- Post-exposure recovery period in satellite groups: 3 weeks- Positive control:
- not applicable
- Observations and examinations performed and frequency:
- A check for moribund or dead animals was carried out twice per day on working days. A check for moribund or dead animals was carried out once per day on weekends and holidays.
The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal.During exposure only a group wise examination was possible.
The animals were weighed prior to the pre-exposure period (study day -5), at the start of the exposure period (study day 0), at the end of the exposure period (study day 4), as well as on the study days 5, 12, 19 and 26.
Food consumption was determined once over the exposure period (study day 0 – study day 4), during the post-exposure period weekly and calculated as mean food consumption in grams per animal and day.The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible - Sacrifice and pathology:
- Clinical pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer).
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters of the animals were examined
Clinical chemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase,-Glutamyltransferase, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins,Triglycerides,Cholesterol
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.
Parameters and methods of cytological examination in BAL: Total cell count, Macrophages, Polymorphonuclear neutrophils, Lymphocytes, Eosinophils, Monocytes, Epithelial, Gamma−Glutamyltransferase, Protein, Lactate dehydrogenase, Alkaline phosphatase, N-acetyl-Beta-Glucosaminidase
Cytokines in BAL: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Rodent osteopontin
Necropsy
The animals were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes werelavaged, whereas the left lung lobe was ligated during lavage. Immediately after lung lavage,
the animals were necropsied and assessed by gross pathology.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus (fixed)
12. Thyroid glands (with parathyroid glands) (fixed)
All paired organs were weighed together (left and right).
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain with olfactory bulb
5. Epididymides
6. Esophagus
7. Eyes with optic nerve
8. Heart
9. Kidneys
10. Larynx/pharynx
11. Liver
12. Lungs
13. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
14. Nose (nasal cavity)
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cord)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder
Extend of histological processing and sub-sequent microscopical examinations in main group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal), nasal cavity (4 levels), trachea and in recovery group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal) - Other examinations:
- Lung lavage: The animals intended for lung lavage were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The right lung was lavaged in situ with physiological saline, whereas the left lung was ligated during this procedure.
- Statistics:
Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
BALF: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Organ weights: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.
Terminal body weight: Comparison of each group with the control group was performed using the Dunnett test (two-sided) for the hypothesis of equal means.- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- In males of test group 3 (60 mg/m3) absolute neutrophil counts were significantly higher compared to controls. The values were slightly above the historical control range (males, neutrophils 0.53-1.01 Giga/L). however, total white blood (WBC) counts as well as all other differential blood cell fractions were not changed. Therefore, this isolated alteration of absolute neutrophil counts in males of test group 3 was regarded as treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).
After the three-week recovery period, no changes of absolute neutrophil counts were observed. In males of test group 2 (20 mg/m3) absolute and relative, large unstained cell (LUC) counts were significantly higher compared to study controls, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related. - Clinical biochemistry findings:
- no effects observed
- Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The significant increase of absolute and relative lung weight in animals of test group 3 (60 mg/m³) and the relative lung weight in test group 2 (20 mg/m³) is regarded as treatment-related and correlates with histopathological findings.
(See tables below). Findings were reversible within the recovery period. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Two animals of test group 3 (60 mg/m³) showed a yellow discoloration of the mediastinal lymph nodes. These findings were regarded to be treatment-related.
After recovery, three animals of test group three showed a yellow discoloration, and so did one animal of the low dose group. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In the lungs animals of test group 3 (60 mg/m³) and 2 (20 mg/m³) revealed a minimal to moderate hyperplasia/hypertrophy of bronchioli (mainly small and terminal bronchi and the bronchio-alveolar transition were affected). Furthermore, an increase of alveolar histiocytes was observed which contained yellow particles within their cytoplasm in test group 2 and 3 animals (20 and 60 mg/m³) and a minimal infiltration of intra alveolar neutrophils in test group 3 animals (60 mg/m³). Within the BALT (Bronchio-alveolar lymphoid tissue) macrophages containing the above-mentioned particles were seen.
Test group 1 animals (5 mg/m³) showed no increase in histiocytes but the histiocytes that were present also revealed the above-mentioned yellow particles within their cytoplasm. These findings were regarded to be treatment-related.
The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes. In the mediastinal lymph nodes of test groups 2 and 3 (20 and 60 mg/m³) macrophages were observed that revealed the same yellow particles as described for the lungs. These findings were regarded to be treatment-related.
Recovery findings:
Comparable to the main groups yellow particles within minimally increased numbers of histiocytes were observed in treated recovery animals of test groups 2 and 3 (20 and 60 mg/m³). The histiocytes tended to accumulate in the area of the bronchio-alveolar transition. Macrophages with particles within the BALT were observed in animals of test group 2 and 3 (20 and 60 mg/m³) as well as particles within alveolar septae in test group 3, only. Alveolar histiocytes containing the particles in their cytoplasm were seen in animals of test group 1 (5 mg/m³) but were not increased in number.
A single animal (No. 39) of test group 3 (60 mg/m³) still showed hyperplasia/hypertrophy bronchiole, neutrophilic infiltrates and cellular debris intra alveolar in addition. It was regarded to be degenerating histiocytes as also free yellow particles were observed. All these findings were regarded to be treatment-related. The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes.
In the mediastinal lymph nodes of all treated test groups macrophages were observed that revealed comparable particles as described for the lungs. They were also found as agglomerated macrophages with particles. These findings were regarded to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Bronchoalveolar lavage fluid (BAL)
After the administration period in BAL of males of test group 3 (60 mg/m3) total cell counts as well as above all absolute and relative neutrophil cell and absolute eosinophil cell counts were increased (absolute eosinophil counts not statistically significantly). Additionally, a moderate increase of absolute and relative lymphocyte as well as absolute macrophage, monocyte and epithelial cell counts could be observed. In BAL of males of test group 2 (20 mg/m3) total cell counts were not changed, but absolute and relative neutrophil, lymphocyte counts as well as absolute monocyte cell counts were already increased. Relative macrophage counts in BAL of males in test groups 2 and 3 were decreased. These alterations were regarded as treatment related and adverse.
In males of test group 2 (20 mg/m3) absolute eosinophil counts were also increased, but not statistically significantly. However, the mean value was within the historical control range (males, absolute eosinophils 0.00-0.21 cn/µL BAL). Therefore, this change was regarded as incidental and not treatment related.
After the three-week recovery period all altered cell count values in BAL were back in the normal range. In males of test group 11 (5 mg/m3) relative lymphocyte counts were significantly increased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.
In BAL of males in test group 3 (60 mg/m³) N-acetyl--D-glucosaminidase (NAG) activities were also significantly increased, but the values were within the historical control range (males, NAG 10-79 nkat/L). The same was true for significantly increased total protein levels in BAL of males in test group 2 (20 mg/m3) as well as in GGT activities in BAL of males in test group 1 (5 mg/m3)( males, total protein 18-50 mg/L, GGT 25-64 nkat/L). Therefore, these changes were regarded as incidental and not treatment related.
After the three-week recovery period, all BAL parameter values were back in the normal range.
During the exposure period the animals all animals of the mid and high concentration (20 and 60 mg/m³) showed substance-contaminated fur. Moreover, substance-like discoloration of the fur was observed in all animals of the high concentration of the test substancewerewed substance-like discoloration of the fur on study day 6. - Key result
- Dose descriptor:
- NOAEC
- Remarks:
- local effects
- Effect level:
- 5 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: min. infiltration of neutrophils within bronchiolar epithelium, hyptrophy/hyperplasia of terminal bronchi (terminal and small), hyperplasia of type II pneumocytes, increased cellularity in the mediastinal lymph nodes. Findings in BALF
- Key result
- Dose descriptor:
- NOAEC
- Remarks:
- systemic effects
- Effect level:
- >= 60 mg/L air (analytical)
- Based on:
- test mat.
- Sex:
- male
- Remarks on result:
- other: only findings in BALF
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 mg/m³ air (analytical)
- System:
- respiratory system: upper respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Grade 1
- Grade 2
- Grade 1
- Grade 1
- Conclusions:
- Under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could was 5 mg/m3. The systemic NOAEC is above 60 mg/m³ (high concentration group).
- Executive summary:
Inhalation exposure of rats to 60 mg/m³ on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte and macrophage counts in bronchoalveolar lavage, while relative macrophage count was reduced in 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, as well as hyptrophy/hyperplasia of bronchioli. The absolute and relative lung weights were increased at the high concentration. Similar findings were also observed at 20 mg/m³ with reduced severity.
After the post-exposure period of 3 weeks, the effects resolved partly at the high concentration of 60 mg/m³ and was fully recovered at the mid concentration of 20 mg/m³.
Thus, under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could was 5 mg/m3. The systemic NOAEC is above 60 mg/m³ (high concentration group).
Referenceopen allclose all
Table 1: Test concentration and particle size distribution
Target concentration | Measurement | MMAD (µm) | GSD | Fraction < 3 µm |
5 mg/m³ | 1 | 0.77 | 2.90 | 90.0 % |
2 | 0.70 | 2.41 | 95.0 % | |
20 mg/m³ | 1 | 0.71 | 2.22 | 96.4 % |
2 | 0.66 | 2.67 | 93.9 % | |
60 mg/m³ | 1 | 0.72 | 2.06 | 97.6 % |
2 | 0.85 | 2.51 | 91.6 % |
Table 2: Particle count distribution measured by SMPS
Target concentration | Total count concentration (N/cm³) | Geometric mean diameter | Geometric standard deviation |
5 | 103723 | 266 | 1.75 |
20 | 340464 | 295 | 1.75 |
60 | 840216 | 288 | 1.75 |
Table 3 Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure).
Analyte | Study day 5 | Study day 30 | |||||
| Gr. 1 5 mg/m3 | Gr. 2 20 mg/m3 | Gr. 3 60 mg/m3 | Gr. 11 5 mg/m3 | Gr. 12 20 mg/m3 | Gr. 13 60 mg/m3 | |
Total Cells | 1.1 | 1.2 | 3.3** | 0.6 | 1.1 | 0.7 | |
Eosinophils | 3.1 | 10.4 | 24.4 | 0.1 | 0.3 | 0.4 | |
Lymphocytes | 2.0 | 2.8* | 7.9** | 1.5 | 1.0 | 1.0 | |
Macrophages | 1.1 | 1.1 | 2.3** | 0.6 | 1.1 | 0.7 | |
Neutrophils | 0.7 | 5.6** | 27.8** | 1.3 | 1.3 | 2.1 | |
Monocytes | 1.3 | 3.1* | 9.9** | 8.9 | 0.0 | 2.8 | |
Epithelial cells | 0.8 | 0.4 | 4.1* | 0.9 | 2.4 | 0.2 | |
One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01: + increase could not be calculated because of zero activity in controls
Table 4: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure)
Analyte | Study day 5 | Study day 30 | ||||
| Gr. 1 5 mg/m3 | Gr. 2 20 mg/m3 | Gr. 3 60 mg/m3 | Gr. 11 5 mg/m3 | Gr.2 2 20 mg/m3 | Gr. 13 60 mg/m3 |
Total Protein | 1.0 | 1.3* | 1.9** | 1.3 | 1.4 | 1.1 |
GGT | 1.2** | 2.4** | 3.9** | 1.0 | 1.3 | 1.0 |
LDH | 1.0 | 1.1 | 2.3** | 0.9 | 1.3 | 1.0 |
ALP | 1.2 | 1.9** | 2.8** | 1.1 | 1.0 | 1.2 |
NAG | 1.2 | 1.3 | 2.2* | 1.1 | 1.3 | 0.9 |
GGT =g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;
NAG = N-Acetyl-b--D-glucosaminidase, One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01
Table 5: Changes in antigen levels in BAL (x-fold of concurrent control means) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure)
Analyte | Study day 5 | Study day 30 | ||||
| Gr. 1 5 mg/m3 | Gr. 2 20 mg/m3 | Gr. 3 60 mg/m3 | Gr. 11 5 mg/m3 | Gr. 12 20 mg/m3 | Gr. 13 60 mg/m3 |
CINC-1/IL-8 | 1.5* | 4.1** | 4.2** | 1.1 | 1.4* | 1.3 |
Osteopontin | 1.1 | 1.1 | 4.3** | 1.3 | 1.4 | 2.5* |
.BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1
one-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01
Table 6a: Absolute and relative organ weights (main group)
Test group (mg/m³) | 01 (5) | 02 (20) | 03 (60) |
Lungs, absolute (and relative) | +8.8% (+ 3.3%) | +20.9% (+12.6%)* | +24.6%* (+21.8%)* |
*p <= 0.05; **p <= 0.01
Table 6b Relative changes of absolute organ weights (recovery group).
| Male animals | ||
Test group (mg/m³) | 11 (5) | 12 (20) | 13 (60) |
Brain | +8.8%** | +4.8%** | +3.5% |
Heart | +18.1%** | +5.8% | +15.0%* |
Spleen | +18.6% | +11.6% | +34.5%** |
NB All mean relative weight parameters did not show significant differences when compared to the control group
Table 7a: Histopathology ( Incidence and severity of histological findings in the lungs of main group animals)
Lungs | Male animals | |||
Test group (mg/m³) | 00 (0) | 01 (5) | 02 (20) | 03 (60) |
No. of animals | 5 | 5 | 5 | 5 |
Hyperplasia/hypertrophy, bronchioli, (m)f | 0 | 0 | 5 | 5 |
· Grade 1 |
|
| 3 | 1 |
· Grade 2 |
|
| 2 | 2 |
· Grade 3 |
|
|
| 2 |
Histiocytosis alveolar with particles, (m)f | 0 | 0 | 5 | 5 |
|
| 5 |
| |
|
|
| 5 | |
Particles within histiocytes* | 0 | 5 | 0 | 0 |
Balt: macrophages with particles (m)f | 0 | 0 | 4 | 4 |
· Grade 1 |
| 0 | 4 | 4 |
Infiltrate neutrophilic, (m)f | 0 | 0 | 0 | 4 |
· Grade 1 |
|
|
| 4 |
Table 7b Incidence and severity of histological findings in the lungs of recovery group animals
Lungs | Male animals | ||||||
Test group (mg/m³) | 01 (0) | 11 (5) | 12 (20) | 13 (60) | |||
No. of animals | 5 | 5 | 5 | 5 | |||
Hyperplasia/hypertrophy, bonchioli | 0 | 0 | 0 | 1 | |||
· Grade 1 |
|
|
| 1 | |||
Debris, cellular, (m)f | 0 | 0 | 0 | 1 | |||
· Grade 1 |
|
|
| 1 | |||
Histiocytosis alveolar with particles, (m)f | 0 | 0 | 5 | 5 | |||
|
| 5 | 5 | ||||
Macrophages with particles, interstitial (m)f | 0 | 0 | 0 | 2 | |||
|
|
| 2 | ||||
Particles within histiocytes* | 0 | 5 | 0 | 0 | |||
Balt:[WT1] macrophages with particles (m)f | 0 | 0 | 1 | 3 | |||
· Grade 1 |
|
| 1 | 2 | |||
· Grade 2 |
|
|
| 1 | |||
Infiltrate neutrophilic, (m)f | 0 | 0 | 0 | 1 | |||
· Grade 1 |
|
|
| 1 | |||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 60 mg/m³
- Study duration:
- subacute
- Species:
- rat
Repeated dose toxicity: inhalation - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- READ ACROSS ANALOGUE APPROACH
The read-across hypothesis is fundamentally based on the same core structure of five ‘yellow disazo condensation pigments’ which optionally can serve as target or as source substances. None of the pigments are sufficiently soluble, either in water or in octanol for systemic uptake or metabolism. The molecular weight ranges from 716.6 g/mol (Pigment Yellow 155) to 1229.2 g/mol (Pigment Yellow 128). Therefore, the molecular weights of all ‘yellow disazo condensation pigments’ are well above the threshold of 500 g/mol, which is generally considered for low dermal and oral uptake [ECHA Guidance R. 7c, 2017]. Furthermore, for each of the substances, the critical body burden (CBB) is above the octanol solubility, which generally indicates a low uptake in biota and makes toxicity unlikely [ECHA Guidance R. 11, 2017].
An evidence of the low bioavailability for all five pigments is the absence of any systemic toxicity in the repeated-dose toxicity study with Pigment Yellow 128 (highest molecular weight), Pigment Yellow 93 (intermediate molecular weight) and Pigment Yellow 155 (lowest molecular weight). From theoretical considerations, uptake and metabolism should result in the release of aromatic amines; such compounds have a characteristic toxicity profile. As this was not observed, all five substances are not considered to be taken up by the body. This assumption is supported by results of several other toxicological and ecotoxicological studies with the pigments, in which no hazards were identified. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Key result
- Dose descriptor:
- NOAEC
- Remarks:
- local effects
- Effect level:
- 5 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: min. infiltration of neutrophils within bronchiolar epithelium, hyptrophy/hyperplasia of terminal bronchi (terminal and small), hyperplasia of type II pneumocytes, increased cellularity in the mediastinal lymph nodes. Findings in BALF
- Key result
- Dose descriptor:
- NOAEC
- Remarks:
- systemic effects
- Effect level:
- >= 60 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: only findings in BALF
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 mg/m³ air (analytical)
- System:
- respiratory system: upper respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Apr 22 - Oct 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- 5-day dust inhalation study in rats (with bronchoalveolar lavage, 3 weeks recovery period)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- yellow powder
- 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 (when supplied)
- Weight at study initiation (means):ca 265g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [P SU]) 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 Switze rland), ad libitum.
- Water: Tap water, ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%) 45 - 65
- Air changes (per hr): 15
Photoperiod (hrs dark / hrs light):: 12h/12h - Route of administration:
- inhalation: dust
- Type of inhalation exposure:
- nose/head only
- Vehicle:
- clean air
- Mass median aerodynamic diameter (MMAD):
- >= 0.66 - <= 0.77 µm
- Geometric standard deviation (GSD):
- 245
- Remarks on MMAD:
- All measurements of particle size resulted in MMADs between 0.66 and 0.77 µm with GSDs between 2.06 and 2.90. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 90.0 % and 97.6 %.
SMPS showed different geometric mean concentration than those measured by cascade impactor measurement. Major reason is that this geometric mean referred to count distribution, while cascade impactor measurement measured mass-based aerodynamic diameter.
The SMPS showed very high particle count concentrations in all concentrations. The geometric mean count diameters were between 266 nm and 295 nm. - Details on inhalation exposure:
- For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned air, and passed via the cyclonic separator and glass tube into the inhalation system
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 con
centration 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. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentrations of the inhalation atmospheres in test groups 1 - 3 were analyzed by gravimetry. This method was applicable because the test item possessed extremely low 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 these groups, the constancy of concentrations in each chamber was continuously monitored using scattered light photometers.
The particle size analysis was carried out with a cascade impactor with the following equipment:
• 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 6.9 mm
• Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)
Sampling for particle size analyses:Pre-weighed metal collecting discs and a backup particle filter were placed into the cascade impactor and two samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.
The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the filter/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.
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. During the exposure period, one measurement per concentration with 10 repeats each were performed.
Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures.
The air flows were constantly maintained in the desired range. An air change of about 65 to 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system. Daily mean relative humidities in the inhalation systems ranged between 33.7 and 49.6 %. Daily mean temperatures in the inhalation systems ranged between 20.6 and 22.1 °C. These values were within guideline recommendations. - Duration of treatment / exposure:
- 6h for 5 days
- Frequency of treatment:
- daily
- Dose / conc.:
- 4.98 mg/m³ air (analytical)
- Remarks:
- SD 0.27
- Dose / conc.:
- 20 mg/m³ air (analytical)
- Remarks:
- SD 1.0
- Dose / conc.:
- 60.4 mg/m³ air (analytical)
- Remarks:
- SD 2.1
- No. of animals per sex per dose:
- 10 (five for sacrifice after exposure and 5 for sacrifice after recovery)
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Dose selection rationale: Results of short-term inhalation studies with other inert organic pigments
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical bioche Particlesmistry: overnight
- Rationale for selecting satellite groups: Clearance of inert particles by lung macrophages is known to take time
- Post-exposure recovery period in satellite groups: 3 weeks- Positive control:
- not applicable
- Observations and examinations performed and frequency:
- A check for moribund or dead animals was carried out twice per day on working days. A check for moribund or dead animals was carried out once per day on weekends and holidays.
The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal.During exposure only a group wise examination was possible.
The animals were weighed prior to the pre-exposure period (study day -5), at the start of the exposure period (study day 0), at the end of the exposure period (study day 4), as well as on the study days 5, 12, 19 and 26.
Food consumption was determined once over the exposure period (study day 0 – study day 4), during the post-exposure period weekly and calculated as mean food consumption in grams per animal and day.The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible - Sacrifice and pathology:
- Clinical pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer).
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters of the animals were examined
Clinical chemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase,-Glutamyltransferase, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins,Triglycerides,Cholesterol
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.
Parameters and methods of cytological examination in BAL: Total cell count, Macrophages, Polymorphonuclear neutrophils, Lymphocytes, Eosinophils, Monocytes, Epithelial, Gamma−Glutamyltransferase, Protein, Lactate dehydrogenase, Alkaline phosphatase, N-acetyl-Beta-Glucosaminidase
Cytokines in BAL: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Rodent osteopontin
Necropsy
The animals were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes werelavaged, whereas the left lung lobe was ligated during lavage. Immediately after lung lavage,
the animals were necropsied and assessed by gross pathology.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus (fixed)
12. Thyroid glands (with parathyroid glands) (fixed)
All paired organs were weighed together (left and right).
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain with olfactory bulb
5. Epididymides
6. Esophagus
7. Eyes with optic nerve
8. Heart
9. Kidneys
10. Larynx/pharynx
11. Liver
12. Lungs
13. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
14. Nose (nasal cavity)
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cord)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder
Extend of histological processing and sub-sequent microscopical examinations in main group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal), nasal cavity (4 levels), trachea and in recovery group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal) - Other examinations:
- Lung lavage: The animals intended for lung lavage were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The right lung was lavaged in situ with physiological saline, whereas the left lung was ligated during this procedure.
- Statistics:
Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
BALF: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Organ weights: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.
Terminal body weight: Comparison of each group with the control group was performed using the Dunnett test (two-sided) for the hypothesis of equal means.- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- In males of test group 3 (60 mg/m3) absolute neutrophil counts were significantly higher compared to controls. The values were slightly above the historical control range (males, neutrophils 0.53-1.01 Giga/L). however, total white blood (WBC) counts as well as all other differential blood cell fractions were not changed. Therefore, this isolated alteration of absolute neutrophil counts in males of test group 3 was regarded as treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).
After the three-week recovery period, no changes of absolute neutrophil counts were observed. In males of test group 2 (20 mg/m3) absolute and relative, large unstained cell (LUC) counts were significantly higher compared to study controls, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related. - Clinical biochemistry findings:
- no effects observed
- Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The significant increase of absolute and relative lung weight in animals of test group 3 (60 mg/m³) and the relative lung weight in test group 2 (20 mg/m³) is regarded as treatment-related and correlates with histopathological findings.
(See tables below). Findings were reversible within the recovery period. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Two animals of test group 3 (60 mg/m³) showed a yellow discoloration of the mediastinal lymph nodes. These findings were regarded to be treatment-related.
After recovery, three animals of test group three showed a yellow discoloration, and so did one animal of the low dose group. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In the lungs animals of test group 3 (60 mg/m³) and 2 (20 mg/m³) revealed a minimal to moderate hyperplasia/hypertrophy of bronchioli (mainly small and terminal bronchi and the bronchio-alveolar transition were affected). Furthermore, an increase of alveolar histiocytes was observed which contained yellow particles within their cytoplasm in test group 2 and 3 animals (20 and 60 mg/m³) and a minimal infiltration of intra alveolar neutrophils in test group 3 animals (60 mg/m³). Within the BALT (Bronchio-alveolar lymphoid tissue) macrophages containing the above-mentioned particles were seen.
Test group 1 animals (5 mg/m³) showed no increase in histiocytes but the histiocytes that were present also revealed the above-mentioned yellow particles within their cytoplasm. These findings were regarded to be treatment-related.
The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes. In the mediastinal lymph nodes of test groups 2 and 3 (20 and 60 mg/m³) macrophages were observed that revealed the same yellow particles as described for the lungs. These findings were regarded to be treatment-related.
Recovery findings:
Comparable to the main groups yellow particles within minimally increased numbers of histiocytes were observed in treated recovery animals of test groups 2 and 3 (20 and 60 mg/m³). The histiocytes tended to accumulate in the area of the bronchio-alveolar transition. Macrophages with particles within the BALT were observed in animals of test group 2 and 3 (20 and 60 mg/m³) as well as particles within alveolar septae in test group 3, only. Alveolar histiocytes containing the particles in their cytoplasm were seen in animals of test group 1 (5 mg/m³) but were not increased in number.
A single animal (No. 39) of test group 3 (60 mg/m³) still showed hyperplasia/hypertrophy bronchiole, neutrophilic infiltrates and cellular debris intra alveolar in addition. It was regarded to be degenerating histiocytes as also free yellow particles were observed. All these findings were regarded to be treatment-related. The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes.
In the mediastinal lymph nodes of all treated test groups macrophages were observed that revealed comparable particles as described for the lungs. They were also found as agglomerated macrophages with particles. These findings were regarded to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Bronchoalveolar lavage fluid (BAL)
After the administration period in BAL of males of test group 3 (60 mg/m3) total cell counts as well as above all absolute and relative neutrophil cell and absolute eosinophil cell counts were increased (absolute eosinophil counts not statistically significantly). Additionally, a moderate increase of absolute and relative lymphocyte as well as absolute macrophage, monocyte and epithelial cell counts could be observed. In BAL of males of test group 2 (20 mg/m3) total cell counts were not changed, but absolute and relative neutrophil, lymphocyte counts as well as absolute monocyte cell counts were already increased. Relative macrophage counts in BAL of males in test groups 2 and 3 were decreased. These alterations were regarded as treatment related and adverse.
In males of test group 2 (20 mg/m3) absolute eosinophil counts were also increased, but not statistically significantly. However, the mean value was within the historical control range (males, absolute eosinophils 0.00-0.21 cn/µL BAL). Therefore, this change was regarded as incidental and not treatment related.
After the three-week recovery period all altered cell count values in BAL were back in the normal range. In males of test group 11 (5 mg/m3) relative lymphocyte counts were significantly increased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.
In BAL of males in test group 3 (60 mg/m³) N-acetyl--D-glucosaminidase (NAG) activities were also significantly increased, but the values were within the historical control range (males, NAG 10-79 nkat/L). The same was true for significantly increased total protein levels in BAL of males in test group 2 (20 mg/m3) as well as in GGT activities in BAL of males in test group 1 (5 mg/m3)( males, total protein 18-50 mg/L, GGT 25-64 nkat/L). Therefore, these changes were regarded as incidental and not treatment related.
After the three-week recovery period, all BAL parameter values were back in the normal range.
During the exposure period the animals all animals of the mid and high concentration (20 and 60 mg/m³) showed substance-contaminated fur. Moreover, substance-like discoloration of the fur was observed in all animals of the high concentration of the test substancewerewed substance-like discoloration of the fur on study day 6. - Key result
- Dose descriptor:
- NOAEC
- Remarks:
- local effects
- Effect level:
- 5 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: min. infiltration of neutrophils within bronchiolar epithelium, hyptrophy/hyperplasia of terminal bronchi (terminal and small), hyperplasia of type II pneumocytes, increased cellularity in the mediastinal lymph nodes. Findings in BALF
- Key result
- Dose descriptor:
- NOAEC
- Remarks:
- systemic effects
- Effect level:
- >= 60 mg/L air (analytical)
- Based on:
- test mat.
- Sex:
- male
- Remarks on result:
- other: only findings in BALF
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 mg/m³ air (analytical)
- System:
- respiratory system: upper respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Grade 1
- Grade 2
- Grade 1
- Grade 1
- Conclusions:
- Under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could was 5 mg/m3. The systemic NOAEC is above 60 mg/m³ (high concentration group).
- Executive summary:
Inhalation exposure of rats to 60 mg/m³ on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte and macrophage counts in bronchoalveolar lavage, while relative macrophage count was reduced in 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, as well as hyptrophy/hyperplasia of bronchioli. The absolute and relative lung weights were increased at the high concentration. Similar findings were also observed at 20 mg/m³ with reduced severity.
After the post-exposure period of 3 weeks, the effects resolved partly at the high concentration of 60 mg/m³ and was fully recovered at the mid concentration of 20 mg/m³.
Thus, under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could was 5 mg/m3. The systemic NOAEC is above 60 mg/m³ (high concentration group).
Referenceopen allclose all
Table 1: Test concentration and particle size distribution
Target concentration | Measurement | MMAD (µm) | GSD | Fraction < 3 µm |
5 mg/m³ | 1 | 0.77 | 2.90 | 90.0 % |
2 | 0.70 | 2.41 | 95.0 % | |
20 mg/m³ | 1 | 0.71 | 2.22 | 96.4 % |
2 | 0.66 | 2.67 | 93.9 % | |
60 mg/m³ | 1 | 0.72 | 2.06 | 97.6 % |
2 | 0.85 | 2.51 | 91.6 % |
Table 2: Particle count distribution measured by SMPS
Target concentration | Total count concentration (N/cm³) | Geometric mean diameter | Geometric standard deviation |
5 | 103723 | 266 | 1.75 |
20 | 340464 | 295 | 1.75 |
60 | 840216 | 288 | 1.75 |
Table 3 Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure).
Analyte | Study day 5 | Study day 30 | |||||
| Gr. 1 5 mg/m3 | Gr. 2 20 mg/m3 | Gr. 3 60 mg/m3 | Gr. 11 5 mg/m3 | Gr. 12 20 mg/m3 | Gr. 13 60 mg/m3 | |
Total Cells | 1.1 | 1.2 | 3.3** | 0.6 | 1.1 | 0.7 | |
Eosinophils | 3.1 | 10.4 | 24.4 | 0.1 | 0.3 | 0.4 | |
Lymphocytes | 2.0 | 2.8* | 7.9** | 1.5 | 1.0 | 1.0 | |
Macrophages | 1.1 | 1.1 | 2.3** | 0.6 | 1.1 | 0.7 | |
Neutrophils | 0.7 | 5.6** | 27.8** | 1.3 | 1.3 | 2.1 | |
Monocytes | 1.3 | 3.1* | 9.9** | 8.9 | 0.0 | 2.8 | |
Epithelial cells | 0.8 | 0.4 | 4.1* | 0.9 | 2.4 | 0.2 | |
One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01: + increase could not be calculated because of zero activity in controls
Table 4: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure)
Analyte | Study day 5 | Study day 30 | ||||
| Gr. 1 5 mg/m3 | Gr. 2 20 mg/m3 | Gr. 3 60 mg/m3 | Gr. 11 5 mg/m3 | Gr.2 2 20 mg/m3 | Gr. 13 60 mg/m3 |
Total Protein | 1.0 | 1.3* | 1.9** | 1.3 | 1.4 | 1.1 |
GGT | 1.2** | 2.4** | 3.9** | 1.0 | 1.3 | 1.0 |
LDH | 1.0 | 1.1 | 2.3** | 0.9 | 1.3 | 1.0 |
ALP | 1.2 | 1.9** | 2.8** | 1.1 | 1.0 | 1.2 |
NAG | 1.2 | 1.3 | 2.2* | 1.1 | 1.3 | 0.9 |
GGT =g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;
NAG = N-Acetyl-b--D-glucosaminidase, One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01
Table 5: Changes in antigen levels in BAL (x-fold of concurrent control means) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure)
Analyte | Study day 5 | Study day 30 | ||||
| Gr. 1 5 mg/m3 | Gr. 2 20 mg/m3 | Gr. 3 60 mg/m3 | Gr. 11 5 mg/m3 | Gr. 12 20 mg/m3 | Gr. 13 60 mg/m3 |
CINC-1/IL-8 | 1.5* | 4.1** | 4.2** | 1.1 | 1.4* | 1.3 |
Osteopontin | 1.1 | 1.1 | 4.3** | 1.3 | 1.4 | 2.5* |
.BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1
one-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01
Table 6a: Absolute and relative organ weights (main group)
Test group (mg/m³) | 01 (5) | 02 (20) | 03 (60) |
Lungs, absolute (and relative) | +8.8% (+ 3.3%) | +20.9% (+12.6%)* | +24.6%* (+21.8%)* |
*p <= 0.05; **p <= 0.01
Table 6b Relative changes of absolute organ weights (recovery group).
| Male animals | ||
Test group (mg/m³) | 11 (5) | 12 (20) | 13 (60) |
Brain | +8.8%** | +4.8%** | +3.5% |
Heart | +18.1%** | +5.8% | +15.0%* |
Spleen | +18.6% | +11.6% | +34.5%** |
NB All mean relative weight parameters did not show significant differences when compared to the control group
Table 7a: Histopathology ( Incidence and severity of histological findings in the lungs of main group animals)
Lungs | Male animals | |||
Test group (mg/m³) | 00 (0) | 01 (5) | 02 (20) | 03 (60) |
No. of animals | 5 | 5 | 5 | 5 |
Hyperplasia/hypertrophy, bronchioli, (m)f | 0 | 0 | 5 | 5 |
· Grade 1 |
|
| 3 | 1 |
· Grade 2 |
|
| 2 | 2 |
· Grade 3 |
|
|
| 2 |
Histiocytosis alveolar with particles, (m)f | 0 | 0 | 5 | 5 |
|
| 5 |
| |
|
|
| 5 | |
Particles within histiocytes* | 0 | 5 | 0 | 0 |
Balt: macrophages with particles (m)f | 0 | 0 | 4 | 4 |
· Grade 1 |
| 0 | 4 | 4 |
Infiltrate neutrophilic, (m)f | 0 | 0 | 0 | 4 |
· Grade 1 |
|
|
| 4 |
Table 7b Incidence and severity of histological findings in the lungs of recovery group animals
Lungs | Male animals | ||||||
Test group (mg/m³) | 01 (0) | 11 (5) | 12 (20) | 13 (60) | |||
No. of animals | 5 | 5 | 5 | 5 | |||
Hyperplasia/hypertrophy, bonchioli | 0 | 0 | 0 | 1 | |||
· Grade 1 |
|
|
| 1 | |||
Debris, cellular, (m)f | 0 | 0 | 0 | 1 | |||
· Grade 1 |
|
|
| 1 | |||
Histiocytosis alveolar with particles, (m)f | 0 | 0 | 5 | 5 | |||
|
| 5 | 5 | ||||
Macrophages with particles, interstitial (m)f | 0 | 0 | 0 | 2 | |||
|
|
| 2 | ||||
Particles within histiocytes* | 0 | 5 | 0 | 0 | |||
Balt:[WT1] macrophages with particles (m)f | 0 | 0 | 1 | 3 | |||
· Grade 1 |
|
| 1 | 2 | |||
· Grade 2 |
|
|
| 1 | |||
Infiltrate neutrophilic, (m)f | 0 | 0 | 0 | 1 | |||
· Grade 1 |
|
|
| 1 | |||
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- LOAEC
- 5 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
Potential for repeated dose toxicity has been investigated for three 'yellow disazo condensation pigments'.
These include the one with the lowest molecular weight and the best chance of systemic uptake (Pigment Yellow 155) and one with aromatic amine elements that would be expected to cause adverse effects in case of metabolic degradation (Pigment Yellow 93). They are considered to be representative for all disazo yellow pigments. All other relevant parameters such as water and octanol solubility and aromaticity as well as the nature of linkages and hydrolysable groups are basically identical. A data matrix for both toxicology and physico-chemical endpoints is presented in the toxicokinetics section.
Repeated dose toxicity oral:
With C. I. Pigment Yellow 93 (CAS number 5580-57-4) a GLP subacute toxicity study according to OECD 407 (Synthesia, 2009) has been performed. The oral administration of Pigment Yellow 93 to rats (5/sex) by gavage for a period of twenty-eight consecutive days at dose levels 160, 400 and 1000 mg/kg/day produced no toxicologically significant changes in the parameters measured. No major functional changes in any organ systems or severe organ dysfunction were detected. Consistent changes in clinical biochemistry, haematology and urinalysis parameters, which indicate organ dysfunction, were not recorded at any dose level. Histopathological examination revealed no pathological changes, which could be related with administration of the test substance. Based on the results of laboratory investigations in clinical biochemistry, haematology and urinalysis and with respect to the results of histopathological examination the following conclusion about NOAEL can be suggested in this study:
The NOAEL (No Observed Adverse Effect Level) for males and females in this study is equal to 1000 mg/kg/day.
A GLP-compliant investigation of the toxicological effects resulting from repeated oral-gavage administration to rats was performed following OECD guideline 422 without deviations with Pigment Yellow 155 (BASF, 2013). Pigment Yellow 155 (CAS 68516-73-4) was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. Pigment Yellow 155 was administered to male rats for 41 days and to female rats for 52 days including time prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Treatment with the test item up and including 1000 mg/kg bw/day did not reveal any clinical signs or histological findings and did not affect reproduction. All dose-treated males and females had dose-related yellow discolored feces during the treatment period. This finding is considered to be a typical effect resulting from oral administration of a yellow dyestuff and not adverse.
Based on these results a general NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day. Therefore, it is concluded that all members of the 'yellow disazo condensation pigments' are not toxic when administered orally to rat for up 52 days and have not to be classified for repeated oral toxicity.
Under the conditions of the OECD TG 422 and in compliance with GLP, a combined repeated dose toxicity study with the reproductive/developmental screening test was conducted in male and female Wistar rats with Pigment Yellow 128 (CAS 79953-85-8). The test substance was administered daily as a solution to groups of 10 male and 10 female Wistar rats (P0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 1), 100 mg/kg bw/d (test group 2), 300 mg/kg bw/d (test group 3) and 1000 mg/kg bw/d (test group 4). For males, the duration of treatment covered a total of 39 days. Females were treated for 51 to 55 days. No test-item related mortality occurred throughout the study and no treatment-related clinical signs were noted during the study. No changes in body weight, food consumption, haematological or clinical chemistry parameters and no signs of neurotoxicity were observed during the study. Thyroid hormone evaluation did not show treatment-related changes. No treatment-related changes were observed in organ weights, post mortem macroscopic observations and microscopic evaluation. No treatment-related changes were observed in reproductive and developmental parameters.
Therefore, the no observed adverse effect level (NOAEL) for general systemic toxicity was determined to be 1000 mg/kg bw/day for male and female Wistar rats.
Repeated dose toxicity inhalative:
Inhalation exposure of rats to 60 mg/m³ on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte and macrophage counts in bronchoalveolar lavage, while relative macrophage count was reduced in 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, as well as hyptrophy/hyperplasia of bronchioli. The absolute and relative lung weights were increased at the high concentration. Similar findings were also observed at 20 mg/m³ with reduced severity. After the post-exposure period of 3 weeks, the effects resolved partly at the high concentration of 60 mg/m³ and was fully recovered at the mid concentration of 20 mg/m³. Thus, under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could was 5 mg/m³. The systemic NOAEC is above 60 mg/m³ (high concentration group).
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.
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