Calcium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-2-naphthoate

Endpoint summary

Description of key information

The NOAEL for chronic oral toxicity is established at 25 mg/kg bw based on repeated dose toxicity studies with analogue substances. Higher doses result in kidney damage. This is derived from subacute gavage studies with Pigment Red 57:1(Ca) (OECD 422, GLP, MHLW 1993).

No histopathology findings on kidneys were reported after a two-year skin painting study with mice with Pigment Red 57:1(Ca) (Carson 1984).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007 - 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Contains an additional recovery element. NOEL not identified for females.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 1996

Deviations:

yes

Remarks:

A 14 day recovery element was added.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Pigment Red 48:2
- CAS no. 7023-61-2
- Substance type: pigment
- Physical state: solid
- Analytical purity: 99%
- Impurities: No data
- Lot/batch No.: 061101
- Stability under test conditions: Stable, no change before and after the administration period. (confirmed by IR analysis)
- Storage condition of test material: Stored in a sealed container, at room temperature ( Measured temperature: 16.2-26.4 °C, Acceptance range: 10 -30 oC), in a dark place.

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan (Atsugi Breeding Center)
- Age at study initiation: 8 weeks
- Weight at study initiation: 326 - 376 g and 204 - 236 g
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4 - 23.6°C
- Humidity (%): 49 -65 %
- Air changes (per hr): 6 - 20
- Photoperiod (hrs dark / hrs light): 07:00 - 19:00 light phase

Route of administration:

oral: gavage

Vehicle:

other: 1% Tween 80 in water

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): substance is a poorly soluble pigment
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males : Total of 42 days ( 14 days before mating, 14 days for mating, and 14 days after mating)
Females : Total of 41-50 days (14 days before mating, during mating, pregnancy and up to lactation day 3)

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

- Control: 7 males and 12 females plus each five recovery group animals
- 40 mg/kg bw: 12 males and 12 females
- 200 mg/kg bw: 12 males and 12 females
- 1000 mg/kg bw: 7 males and 12 females plus each 5 recovery group animals

Control animals:

yes, concurrent vehicle

Details on study design:

Standard protocol for OECD 422 testing guideline

Positive control:

not applicable

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males and Females - on each administration day.

BODY WEIGHT: Yes
- Time schedule for examinations: Males - measured once a week on administration days 0, 7, 14, 21, 28, 35, and 42; Females - measured once a week on administration days 0, 7, 14, and 21, during pregnancy on days 0, 7, 14, and 20, during lactation on day 0 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined: Yes
- Time schedule for examinations : Males - measured once a week on administration days 0, 7, 14, 21, 28, 35, and 42; Females - measured once a week on administration days 0, 7, 14, and 21, during pregnancy on days 0, 7, 14, and 20, during lactation on day 0 and 4.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males - day after final administration day
- Anaesthetic used for blood collection: Yes
- Animals fasted: No data
- For parameter see table
- How many animals: five per group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 43 and day 57
- Animals fasted: No data
- How many animals: five per group
- Parameters checked in table

URINALYSIS: Yes
- Time schedule: day 40 and day 54
- How many animals: five males per group
- Parameters: pH, protein, glucose, ketones, bilirubin, occult blood, urobilironen, urine colour

NEUROBEHAVIOURAL EXAMINATION: Function tests and motor activity performed for 5 animals (week 6)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Organs investigated: heart, Lymph node, mandibular, Lymph node, mesenteric, Thymus, Spleen, Bone marrow, femur, Trachea, lung (and bronchus), Stomach, Small intestine - duodenum, Small intestine - jejunum, Small intestine - ileum, Large intestine - cecum, Large intestine - colon, Large intestine - rectum, Liver, Kidney, Urinary bladder, Testis, Epididymis, Seminal vesicle, Prostate, Coagulating gland, Pituitary, Thyroid, Parathyroid, Adrenal, Brain, Spinal cord, Sciatic nerve, Eyeball, Uterus, Ovary, Vagina

Histopathology evaluation was performed of the control and high dose group for all animals. If findings were observed, then also the slides of the lower dose groups and recovery animals were scored.

Other examinations:

Determination of organ weights (day 43 and day 57) Organ Weights: Thymus, Liver, Kidney, Testicles, Epididymis, Ovaries , Uterus, Brain, Heart

Statistics:

Multiple comparisons tests were performed with measured values : distribution uniformity was tested by Bartlett’s test , subsequently, one-way variance analysis was performed when the distribution was uniform, and Kruskal-Wallis’s test was performed when the homoscedastic was not recognized. Where significant differences were observed, Duneett’s test or Duneett’s type - multiple comparison tests were conducted.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Haematological findings:

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urine showed red coloration in some animals of the highest dose group on day 40, but not on day 54

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes attributed to the test substance

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased kidney weight was noted in males of the 1000 mg/kg group. These changes disappeared after a 2-week recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Abnormal contents (test substance-colored reddish contents) the digestive organs such as cecum and colon and reddish urine in males were also noted.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- In the histological examination of animals sacrificed after the dosing period, degeneration/necrosis of the proximal tubular epithelium in the kidney was noted in males of the 1000 mg/kg group and females of the 40 mg/kg group and higher. Moreover, degeneration/necrosis of the papillary ductal epithelium in females of the 1000 mg/kg group and mild basophilic tubule in males of the 1000 mg/kg group and females of the 200 and 1000 mg/kg groups were noted.

- No abnormal changes in the digestive system or other organs/tissues in the histological examination were noted.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance-colored stool (red) was observed in all animals of the 40 mg/kg group and higher.

Dose descriptor:

LOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Table 1: Histopathology findings in kidney - females (n = 5)

dose (mg/kg bw); R = recovery	0	40	200	1000	R0	R1000
Basophilic tubule grade 1	1	1	2	0	0	0
Basophilic tubule grade 2	0	0	1	1	0	0
Cell infiltration, inflammatory, focal, grade 1	1	0	0	0	0	0
Cell infiltration, inflammatory, pelvis, grade 1	0	0	0	0	1	0
Cyst	0	1	0	0	0	0
Degeneration/necrosis, papillary ductal epithelium, grade 1	0	0	0	1	0	0
Degeneration/necrosis, tubular epithelium, grade 1	0	1	1	0	0	0
Degeneration/necrosis, tubular epithelium, grade 2	0	0	2	2	0	0
Mineralization, medulla	0	0	0	0	2	1

Grade: 1 = Minimal; 2 = Mild; 3 = Moderate; 4 = Severe

Table 2: Histopathology findings in kidney - males (n = 5)

dose (mg/kg bw); R = recovery	0	40	200	1000	R0	R1000
Basophilic tubule grade 1	1	1	1	0	2	2
Basophilic tubule grade 2	0	0	0	4	0	0
Cell infiltration, inflammatory, focal, grade 1	0	0	0	1	0	0
Cyst	0	1	0	0	0	0
Degeneration/necrosis, tubular epithelium, grade 2	0	0	0	1	0	0
Dilatation, pelvis	0	1	0	0	0	0
Mineralization, medulla, grade 1	0	0	0	1	0	0
Mineralization, cortex, grade 1	0	1	1	1	0	0
Hyaline droplet, tubular epithelium, grade 1	0	0	0	0	0	1

Grade: 1 = Minimal; 2 = Mild; 3 = Moderate; 4 = Severe

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: dermal

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Justification for type of information:

Please see the category read-across justification in the category object.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOEL

Effect level:

>= 50 other: mg/kg bw twice weekly

Based on:

other: read-across

Sex:

male/female

Basis for effect level:

other: no adverse effects expected

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Oral exposure

Pigment Red 57:1(Ca) 5281-04-9

The study with Pigment Red 57:1(Ca) for repeated dose oral gavage toxicity was performed in Crj: CD(SD) rats with a product of 98% purity following OECD testing guideline 422 (MHLW, 1993) and the principles of GLP. Applied doses were 100, 300 and 1000 mg/kg/day using 5% Gum Arabic solution as vehicle. All animals survived to the end of the study. No clinical findings indicative of chemical toxicity were observed; red-stained faeces of dosed animals were due to the colour of the pigment. The mean body weight gain and food consumption in both sexes of dosed groups were comparable to those in the control groups throughout the study. No biologically significant changes in hematological parameters were noted in any dosed male groups. Males that received 300 mg/kg or more showed significantly decreased serum calcium and phosphorus levels. Further, significant decreases in serum potassium and total cholesterol levels, and significant increases in chloride and GOT levels were shown in the males that received 1000 mg/kg. Males that received 1000 mg/kg showed significant increases in relative kidney weights. Females that received 100 or 1000 mg/kg showed decreases in thymus weights in comparison with the controls. No other significant differences in organ weights were observed in either the males or the females. On the histopathology examination, predominant alterations suggesting effects in dosed rats were observed in the kidney. The lesions included an increased incidence of renal tubular epithelium regeneration in males receiving 300 mg/kg or more, and this and necrotic or foamy tubular epithelial cells in females that received 100 mg/kg or more. These lesions increased in severity and incidence in a dose-dependent manner.NOELsfor repeat dose toxicity are considered to be100 mg/kg bw/day for males and less than 100 mg/kg bw/day for females.

In another study rats were exposed 5 days a week to 1000 mg/kg bw/day for 30 days (22 doses in total). Half of the animals were not exposed during a recovery period, the other animals were sacrificed after the last exposure. No clinical signs except coloration of the feces was observed. No mortality, changes in food and water consumption, and effects on hematology, clinical biochemistry and urinalysis findings was observed. Slight inhibition of bodyweight gain which recovered during the recovery period was observed. Kidney weights were increased, although recovered after the recovery period. Morphological changes to the tubules of the kidney were observed, although no longer visible after the recovery period. TheNOAELwas determined to beless than 1000 mg/kg bw/day.

Pigment Red 57(Sr) 73612-29-0

Reversible kidney toxicity with a NOEL of 25 mg/kg bw and a LOAEL of 75 mg/kg bw was observed in the subacute oral toxicity study (OECD 407) with Pigment Red 57(Sr) using olive oil as vehicle (DIC 2006). Absolute and relative kidney weights were increased after four weeks, but not at the end of the treatment-free recovery period. No macroscopic findings were observed, but histopathology examinations showed that kidneys had a dose-dependent increase in atypical and typical basophilic tubules, degeneration and necrosis of tubular epithelium and dilation of distal and collecting tubules with a NOEL of 25 mg/kg bw. All males of the highest dose group of 200 mg/kg bw had orange coloured pigments in the cell debris. The pigment were still visible in two of five animals at the recovery sacrifice, all other histopathology findings were absent. Kidney findings occurred in females at a lower extent - slight degeneration and necrosis of tubular epithelium was observed only for the high dose group (200 mg/kg bw). Histopathology findings were not observed in animals of the recovery group. Body weight and food intake were not affected during the treatment period. During the recovery period, males of treatment group showed an increased food intake compared to control rats. Clinical signs consisted of salivation at the highest dose group during treatment.

Pigment Red 48:2(Ca) 7023-61-2

In the subacute oral toxicity study with Pigment Red 48:2(Ca) (OECD 422, MHLW 2009) using 1% Tween 80 in water as vehicle, the NOEL was determined to be 40 and less than 40 mg/kg bw for males and females, respectively. The test dosages were 40, 200, and 1000 mg/kg bw/day. In the histological examination of animals sacrificed after the dosing period, degeneration/necrosis of the proximal tubular epithelium in the kidney was noted in males of the 1000 mg/kg group and females of the 40 mg/kg group and higher. Moreover, degeneration/necrosis of the papillary ductal epithelium in females of the 1000 mg/kg group and mild basophilic tubule in males of the 1000 mg/kg group and females of the 200 and 1000 mg/kg groups were noted. Increased kidney weight was noted in males of the 1000 mg/kg group. These changes disappeared after a 2-week recovery period. Test substance-colored stool (red) was observed in all animals of the 40 mg/kg group and higher. Abnormal contents (test substance-colored reddish contents) the digestive organs such as cecum and colon and reddish urine in males were also noted. However, there were no abnormal changes in the digestive system or other organs/tissues in the histological examination. In other parameters, there were no changes attributed to the test substance on behavior test, body weight, food consumption, hematology, or blood chemistry.

Conclusion

Results for all analogue pigments are largely comparable. Kidney effects are clearly the most sensitive endpoint for the BONA metal laked pigments. Bolus dosing results in high peak exposures and high local concentrations and may overload the local detoxification capacities. The colourant is quickly taken up and eliminated via the kidney as indicated by dose-dependent urine coloration occurring within the first day of dosing. The regeneration of the renal epithelium indicates a cytotoxic effect, probably by the azo moiety or a metabolite.

In reproductive toxicity screening studies with Pigment Red 48:2(Ca) and 57:1(Ca) , pregnant females seemed somewhat more susceptible than males or non-pregnant females. However, the reported LOAELs of 40 and 100 mg/kg/day (lowest tested doses in each study, respectively) and the limited severity of the kidney effects at the LOAELs are considered compatible with the derived NOAEL of 25 mg/kg bw/day and no further adjustment is considered necessary. The cytotoxic effect of the Pigments Red lead to a constant regeneration of tubular epithelial cells as long as exposure continues. No pronounced increase in toxicity with dose was observed in any of the studies, i.e. higher doses did not induce qualitatively different kidney effects. Also, prolonged exposure up to 2 years did not increase kidney toxicity or induced qualitatively different effects. It appears that continuous application is better tolerated than bolus application. All kidney effects were completely reversible within tested recovery periods of 14 days. Effects at 40, 100, 200 or 300 mg/kg bolus dosing did not induce severe or serious damage, i.e., clear functional disturbance or morphological changes.

Taking all repeated-dose studies into account both the most relevant NOAELs are the one from a guideline 28-day study with Pigment Red 57(Sr) (25 mg/kg bw) and from the chronic feeding study with sodium salt of Pigment Red 57(Na) (26 mg/kg/bw). Twenty-five mg/kg bw is used as starting point for DNEL derivation because the value stems from the newest study with the most extensive analysis of parameters (including urinalysis) and is the lowest value.

Dermal exposure

Absence of histopathology findings in kidney upon 18 month skin painting study with mice was published (1984). The study was designed between the US FDA and an industry association to assess the safety of the use of Pigment Red 57:1(Ca) in lipstick and therefore, a limit dose of 50 mg/kg bw was chosen. Limited details are given in the literature. The main focus of the study was local effects, but a set of organs for each five males and females was investigated by histopathology. As kidney histopathology was the most sensitive endpoint, this investigation contributes to hazard assessment.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity according to Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008.