Barium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Endpoint summary

Description of key information

The NOEL 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 (OECD 422, GLP, MHLW 1993), Pigment Red 48:2 (OECD 422, GLP, MHLW 2009) and Pigment Red 57-Sr (OECD 407, GLP, DIC 2006).  In a chronic feeding study with the sodium salt of Pigment Red 57, exacerbation of spontaneous renal disease in aged rats was observed with a NOEL of 0.05 % (CTFA 1981a). The dietary concentration of 0.05% corresponds to an average dose of 26 mg/kg bw for males and 31 mg/kg bw for females. A NOEL of 25 mg/kg bw was identified in a two-year feeding study in rats with Pigment Red 53:1.  A NOEL of 30 mg/kg bw for rats and mice was identified for Barium as applied in Barium chloride dihydrate in drinking water for two years.
No histopathology findings on kidneys were reported after a two-year skin painting study with mice with Pigment Red 57:1 (Carson 1984).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1977 -1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Designed to meet US FDA requirements for long-term feeding studies, combines a fertility element, in-utero-exposure and long-term exposure. Histopathology of kidneys assessed for mid and low dose group as follow-up investigation and reported in amendment

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Charles River CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Wilmington, Massachusetts (USA)
- Age at study initiation: no information
- Weight at study initiation: 75 - 155 g (males) and 75 - 144g (females)
- Fasting period before study: not applicable
- Housing: individually, except for mating period and nursing period
- Diet (e.g. ad libitum): yes (Supplier Ralston Purina company)
- Water (e.g. ad libitum): yes
- Acclimation period: 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled at 71° Fahrenheit (recorded weekly starting week 31)
- Humidity (%): controlled at 53% (recorded weekly starting week 31)
- Air changes (per hr): controlled, no further data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: Sept 2, 1977 To: January 6, 1978 (F0-Generation) and December 1977 To:June 3, 1980 (F1-Generation)

Individual ear tags were used for identification. Rats were examined for physical abnormalities prior to entering the study. Rats were then randomly assigned to groups.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The main batch of feed for each group was sampled (three sub-samples representing three distinct areas of the batch) and assayed weekly during the first 13 weeks and every 4 weeks thereafter. Concentrations were determined to be 95 - 99% of target values.
Homogenicity was also confirmed.
Chemical analysis was performed at the sponsor's laboratories.

Duration of treatment / exposure:

F0-generation (not relevant for the endpoint chronic toxicity):
Rats were given the test item in the feed for 60 days prior to mating, during mating (maximum 7 days), during gestation and during lactation (21 days).F0-animals were sacrificed after weaning.

F1-animals (chronic toxicity study part)
exposure started in-utero. After weaning, rats received the control diet for two weeks and were then exposed via the diet until a survival rate of 20% was reached for the highest dose group; this was 95 weeks for males and 126 weeks for females.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 0.05, 0.3 and 2.0 %
Basis:
other: in the diet (measured)

Remarks:

Doses / Concentrations:
0, 26, 161 and 1117 mg/kg bw
Basis:
other: average calculated for males from food consumption and body weight

Remarks:

Doses / Concentrations:
0, 31, 189 and 1315 mg/kg bw
Basis:
other: average calculated for females from food consumption and body weight

No. of animals per sex per dose:

70 rats per sex per dose group for the F1-Generation (chronic toxicity study):
of these 10 per sex per dose group were sacrified at the age of 12 months (interim sacrifice)

Control animals:

yes, plain diet

Details on study design:

The F1 generation was used for the long-term part of the study. Parental animals were used for the fertility element and for in-utero exposure. F1 pups were counted and sexed on days 0,4,14 and 21 of lactation. Litters were housed with their dams until 21 days after birth. After that pups were housed together for two weeks and during that time, they received control diets and were selected for the long-term feeding part of the study.

Positive control:

none

Observations and examinations performed and frequency:

Clinical signs: Daily (pup stage), twice daily (adult stage)
Body weight and food consumption: weekly for the first 14 weeks, biweekly for the next 12 weeks and monthly thereafter. At the pup stage, weighing of the litter was done on days 0, 4 and 14
Hematology, biochemistry and urinalysis were performed for 10 animals per sex and dose group at 3, 6, 12, 18 and 21 months for both genders, for females, also after 24 months. Rats were fasted overnight prior to blood and urine collection.
At 20 months of study, blood was obtained from 2 unfasted rats/sex/group (one rat in apparent good health and one rat exhibiting signs of pulmonary distress). Blood was collected and sent to Microbiological Associates, Rockville, Maryland for viral serology analysis. Lung and trachea tissue from three male rats (two control and one high dose) were sent to Microbiological Associates in an attempt to isolate Sendai virus and mycoplasmas, respectively. Tissue samples from these rats were delivered to Kar Laboratory, Kalamazoo, Michigan for growth, isolation and characterization of the bacterial populations. At 20 months of study, additional blood sampling involving five rats/sex/groups was conducted in an attempt to identify the etiological agent responsible for the numbers of deaths which were occurring.


Hematology parameters: hemoglobin, hematocrit, total and differential leucocyte counts, total erythrocyte counts and total reticulocyte counts; for the 20,21 and 24 month investigation also mean corpulscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration

Biochemistry parameters: glucose, blood urea nitrogen, serum SGOT, SGPT, ALP, creatinine and total protein

Urinalysis parameters: pH, specific gravity, qualitative tests for protein, glucose, bilirubin, ketones and occult blood; description of color and appearance, microscopic analysis of sediments. Volumes were inadvertently recorded at 3, 6, 12 and 24 months.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (both interim and final sacrifice)
HISTOPATHOLOGY: Yes (both interim and final sacrifice: any gross lesion plus for control and highest dose group: abdominal aorta, adrenals, bone and bone marrow(femur), blood smear, brain (3 sections), esophagus, eye, ovaries, testes with epididymides, heart, colon, cecum, duodenum, ileum, kidneys, liver, lung and mainstream bronchi, lymph nodes (mediastinal and mesenteric), mammary gland, mandibular salivary gland, sciatic nerve, pancreas, pituitary, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, thymus, trachea, thyroid/parathyroid, urinary bladder, uterus/prostate
Kidney tissues from the low and mid dose groups (0.05% and 0.3%) were sectioned and examined microscopically (reported in study amendment).

Organ weights: brain, kidney, liver, spleen, testes, thyroid, heart, uterus, ovaries, adrenals

Other examinations:

ophthalmoscopic examinations (after pupillary dilation with 1% tropicamide, using binocular indirect ophthalmoscope):
-Schedule: month 3, 6 ,12 ,18 and 21 for both genders and month 24 for females

Statistics:

For fertility indices, gestation, pup survival indices: Chi-square test criterion with Yates correction on 2x2 contingency tables and/or Fisher's exact probability test. For mean number of liveborn pups per litter and litter mean pup weight: analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test as described by Steel and Torrie (1960) using Dunnett's multiple comparison tables (1964).
The same tests were used for the long-term study as appropriate. Life time table curves were computed using both Kaplan-Meier and standard methods. Homogenicity of curves was tested using Cox's test for life table data and the Gehan-Breslow generalized Kruskal-Wallis test.
The data on time to neoplastic lesion was analyzed by methods described by Thomas, Breslow and Gart (1977).

Details on results:

No compound-related effects were seen in behavior, survival, food consumption, ophthalmoscopic examinations, hematological, biochemical or urinalysis parameters.

Throughout the study, significant body weight depressions were seen for males at the 2.0% dosage level. When compared to control means, these decreases were considered moderate by 52 weeks and marked by 78 weeks of study. When analyzed statistically, significance was seen (when compared to combined control means) at all intervals analyzed between study weeks 10-91. Statistical significant decreases were also seen (when compared to combined control means) for females at the 2.0% dosage level at study weeks 2-4, 8, 9, 11-14 and 65. However, these decreases were not considered biologically significant.
The changes in body weight were reflected in changes in relative organ weights.

For animals that died between the interim and the terminal sacrifice, males of the highest dose group had a higher incidence of chronic nephritis, renal tubular epithelial hyperplasia, myocardial fibrosis, reticular hyperplasia of the spleen, atrophy/degeneration of the testicular tubules and pigment in the spleen (Table 3 and 4 for kidney histopathology). These lesions are common spontaneous or aging lesions in rats. There appeared to be an exacerbation of this spontaneous renal disease in aged rats in the mid and high-dose group male rats and in the high-dose group female rats (Table 2).
At the interim sacrifice, no test-item related histophathology findings were noted (Table 5).

After one year, males at the 2 % dose level had a slightly higher incidence of deaths (or sacrificed in extremis). The 20% survival rate for (2.0% dosage level) was reached at week 95 for males and at week 126 for females. (see table 1). Based on virology examinations, it was concluded that these deaths were of microbiological origin and not related to treatment with the test item. According to the veterinarian at The Charles River Breeding Laboratories, North Wilmington, Massachusetts, the PVM and KEV titers were higher than expected for rats at this age. in the two rats examined at 12 months.
No Sendal virus was isolated from the lung samples. Trachea samples from these ssme rats for mycoplasma isolation revealed that all three
samples were positive for M. Pulmonis. The most predominant organism was a species of Pseudomonas.

A dose-dependent orange coloration of urine and feces was caused by the colored test item and indicates elimination without destruction of the chromophore. Coloration was also observed for hair and skin.
No adverse effects were noted during the pup stage (day 0-23).

Dose descriptor:

NOEL

Effect level:

0.3 other: % in the diet (analytically verified)

Sex:

female

Basis for effect level:

other: increased incidence of chronic nephritis in aged rats

Dose descriptor:

NOEL

Effect level:

0.05 other: % in the diet (analytically verified)

Sex:

male

Basis for effect level:

other: Increased incidence of chronic nephritis in aged rats; reduced body weight observed at 2.0 %

Critical effects observed:

not specified

Table 1

dose	Number of surviving rats at terminal sacrifice*
% diet	Male (week 95)	Female (week 126)
0	29	15
0	17	16
0.05	18	16
0.3	24	15
2	9	11
*Initial number: 60 rats after interim sacrifice

Table 2

Incidence of non-neoplastic microscopic observations in kidney (females)
Terminal Sacrifice (TS) and Death and unscheduled sacrifices (DOS) between interim and terminal sacrifice
	Control 1		Control 2		0.05%		0.30%		2%
Lesion	TS	DOS	TS	DOS	TS	DOS	TS	DOS	TS	DOS
	43	15	43	16	39	17	43	15	48	11
abscess	1	1	3	0	4	0	4	0	2	0
aurolysis	0	0	0	0	0	0	1	0	2	0
cystic dilatation	0	3	0	1	1	0	2	0	2	1
chronic nephritis	10	7	16	10	22	12	26	15	31	6
cyst	0	0	0	1	1	1	0	0	1	0
degeneration	1	0	1	0	8	0	2	0	0	0
extramedullary hematopoiesis	0	0	0	0	0	0	0	0	1	0
granuloma	0	0	0	0	0	0	0	0	1	0
hematocyst	0	0	1	0	0	0	0	0	0	0
hydronephrosis	2	1	1	0	2	1	1	0	1	0
hyperplasia of pelvic epithelium	0	0	2	0	0	0	0	0	0	0
interstitial lymphocytic infiltrates	0	0	0	0	0	0	1	0	0	0
mineralization	9	3	7	2	2	2	6	1	1	0
necrosis	0	0	0	0	0	1	0	0	0	0
nephrosis	0	0	0	0	1	0	1	0	0	0
papillitis	0	0	0	0	0	0	2	0	0	0
pigmentation	0	0	0	0	0	0	1	0	0	0
pyelonephritis	0	0	1	0	1	0	2	0	2	1
pyelitis	1	1	1	0	4	3	4	1	3	0
within normal limits	22	4	20	3	5	2	5	3	0	0

Table 3

Incidence of non-neoplastic microscopic observations in kidney (males)
Death and unscheduled sacrifices (DOS) at 12 month to termination
	Control 1	Control 2	0.05%	0.30%	2%
Number examined	30	42	40	35	49
abscess	0	0	0	0	1
cystic dilatation, tubules	2	5	2	0	5
chronic nephritis	18	28	36	34	48
cortical cyst	0	0	2	3	3
hydronephrosis	1	0	0	0	0
hyperplasia of pelvic epithelium	0	1	0	1	2
interstitial lymphocytic/mononuclear/inflammatory cell infiltrates	3	6	0	1	0
tubular mineralization	0	0	0	1	2
nephrosis	1	0	0	0	0
necrotic papillitis	0	0	0	1	0
yellow-brown pigment, tubular epithelium	1	1	0	2	0
pyelonephritis/purulent exudate, pelvis	1	1	0	0	0
microcalculi pelvis	1	3	0	0	0
epithelial hyperplasia, tubules	0	0	0	1	8
arterial mineralization	0	0	0	0	1
hyaline droplet degeneration, tubules	0	0	0	0	1
cellular debris, lumina	0	0	0	0	1

Table 4

Incidence of non-neoplastic microscopic observations in kidney
Terminal sacrifice
Males
	Control 1	Control 2	0.05%	0.30%	2%
number of kidneys examined	29	17	18	25	9
cystic dilatation, tubules	1	0	0	0	0
chronic nephritis	22	16	15	22	9
cyst	0	0	2	1	0
hyperplasia of pelvic epithelium	1	0	0	0	0
interstitial lymphocytic/mononuclear/inflammatory cell infiltrates	2	0	0	0	0
pyelonephritis/pyelitis	0	0	2	2	0
microcalculi pelvis	1	0	0	0	0
interstitial fibrosis	1	0	0	0	0
ectatic capillaries	0	0	0	1	0

Table 5

Incidence of non-neoplastic microscopic observations in kidney
12-Months interim sacrifice and unscheduled sacrifice between 0-12 months
	Control 1		Control 2		0.05%		0.30%		2%
Lesion	M	F	M	F	M	F	M	F	M	F
number examined	11	12	11	11	11	10	10	10	12	11
chronic nephritis	6	4	10	3	7	3	7	2	10	3
cyst	0	0	0	0	0	0	1	0	0	0
hydronephrosis	0	0	1	0	1	1	0	0	0	0
hyperplasia of pelvic epithelium	0	0	2	0	0	0	0	0	0	0
interstitial lymphocytic infiltrates	3	2	0	1	0	0	0	0	0	0
mineralization	1	1	0	3	1	2	0	1	0	0
hydronephrosis	0	0	1	0	1	1	0	0	0	0
pyelonephritis, pyelitis	1	0	0	0	1	2	0	2	0	0
concretions, pelvis	0	1	0	0	0	0	0	0	0	0
hemorrhage	1	0	0	0	0	0	0	0	0	0
papillary necrosis	1	0	1	0	0	0	0	0	0	0

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

As no experimental data is available for this pigment, data of Na⁺(Pigment Red 57), Ca²⁺(Pigment Red 57:1 and 48:2) or Sr²⁺(Pigment Red 57-Sr) salts of the anion or its chlorinated analogue and on BaCl₂are taken into account. For all of these read-across substances, systemic availability was demonstrated during the course of the respective studies and the respective counter ions are of low toxicity.

 

 

Although the pigment is highly insoluble in water, it is expected that it becomes at least partly dissolved in the strongly acidic environment of the stomach. Therefore, effects both related to Ba²⁺and to the organic anion are used for hazard assessment. In the following section, effects for both are discussed separately.

 

The study with Pigment Red 57:1 for repeated dose oral gavage toxicity (MHLW 1993) was performed in Crj: CD (SD) rats with a product of 98% purity following OECD testing guideline 422 (MHLW, 1993a) 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. NOELs for repeat dose toxicity are considered to be 100 mg/kg/day for males and less than 100 mg/kg/day for females.

 

This toxicity profile is very similar to that observed for Pigment Red 48:2 which differs from Pigment Red 57:1 by a chlorine atom. In the subacute oral toxicity study (OECD 422, MHLW 2009) using 1% Tween 80 in water as vehicle, the NOEL was determined to be 40 mg/kg bw. 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-coloured stool (red) was observed in all animals of the 40 mg/kg group and higher. Abnormal contents (test substance-coloured reddish contents) the digestive organs such as coecum 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 behaviour test, body weight, food consumption, haematology, or blood chemistry.

 

Reversible kidney toxicity with a NOEL of 25 mg/kg bw and a LOAEL of 75 mg/kg bw was also observed in the subacute oral toxicity study (OECD 407) withPigment Red 57-Sr using olive oil as vehicle (DIC 2006). Absolute and relative kidney weights were increased after 4 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 in take compared to control rats. Clinical signs consisted of salivation at the highest dose group during treatment.

 

In the chronic feeding study with rats with the sodium salt of Pigment Red 57 exacerbation of spontaneous renal disease in aged rats was observed with a NOEL of 0.3% for females and 0.05% for males (CTFA 1981a). The dietary concentration of 0.05% corresponds to an average dose of 26 mg/kg bw for males and 31 mg/kg bw for females. During the study, histopathology was only performed for the high dose group (CTFA 1981a). Histopathology of the mid and low dose group was performed as a follow-up investigation and reported a year later (CTFA 1982). Usage of data from the sodium salt is justified because passage through the acidic environment of the stomach dissolves the divalent salt. Uptake of the sodium salt is confirmed by urine colouration. Throughout the study, significant body weight depressions were seen for males at the highest dose level of 2.0%. When compared to control means, these decreases were considered moderate by 52 weeks and marked by 78 weeks of study. When analyzed statistically, significance was seen (when compared to combined control means) at all intervals analyzed between study weeks 10-91. For rats that died between the interim 12-month and the terminal sacrifice, males of the highest dose group had a higher incidence of chronic nephritis, renal tubular epithelial hyperplasia, myocardial fibrosis, reticular hyperplasia of the spleen, atrophy/degeneration of the testicular tubules and pigment in the spleen. These lesions are common spontaneous or aging lesions in rats. There appeared to be an exacerbation of this spontaneous renal disease in aged rats in the mid and high-dose group male rats and in the high-dose group female rats. The sodium salt of Pigment Red 57 was eliminated in urine and faeces as indicated by a dose-dependent colouration. After 12 months, an interim sacrifice was performed with 10 rats per dose group and no test-item related histopathology findings were observed.

 

No indication of kidney toxicity was observed in the carcinogenicity study in mice with the sodium salt of Pigment Red 57 at doses of 0.5, 1 and 5% in the diet (CTFA 1981b). A few long-term feeding studies are mentioned in secondary sources (Anonymous 1993 and Scientific Opinion on the re-evaluation of Litholrubine BK (E 180) as a food additive, EFSA Journal 2010;8 (5):1586). As no original reports for these studies could be retrieved and findings appear to be consistent with the available data, these studies were not taken into further account.

Results for all analogue pigments are largely comparable. Kidney effects are clearly the most sensitive endpoint for the BONA metal laked pigments. It was not observed upon subacute testing of starting materials, so it is considered to be caused by the parent substance or a metabolite. For details it is referred to published information on 3-hydroxy-2-naphthoic acid (OECD SIDS 2004) and 4-aminotoluene-3-sulfonic acid (OECD SIDS 2003). Subacute testing by gavage resulted in comparable NOAELs regardless of whether a lipophilic or hydrophilic vehicle was used.

 

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 colouration 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, 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/day and no further adjustment is considered necessary. The kidney effects showed up very quickly after dosing e.g. in a 14-day dose-range finding study. The cytotoxic effect leads 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 (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.

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 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.

 

 

Toxicity of Barium chloride dehydrate was investigated in GLP compliant drinking water studies with rats and mice in the US National Toxicology Program (US NTP 1994). Detailed investigations were performed during the 13-week study and at the 15 months interim sacrifice of the 2-year carcinogenicity studies (US NTP 1994). Additional investigations on Barium plasma levels and incorporation in bone were performed.

In rats, 1000 ppm or 65 mg/kg bw of Ba²⁺in drinking water was the NOEL for 13 weeks (US NTP 1994). At 2000 ppm treatment resulted in increased levels of phosphorous in serum. At 4000 ppm, reduced body weight and mortality were observed in addition to renal tubule dilatation in the outer stripe of the outer medulla and cortex occurred. 4000 ppm corresponded to 200 mg/kg bw for males and 180 mg/kg bw for females. No treatment-related findings were found in investigations on cardiotoxicity (electrocardiogram and blood pressure).

In the subsequent 2-year study in rats, effects did not significantly increase with time and no adverse findings were noted at 1250 ppm. At the highest dose of 2500 ppm, reduction in drinking water consumption and body weight were recorded.

At the interim sacrifice at 15 months, there were no differences in haematology and clinical chemistry parameters as well as in histopathology findings. The plasma barium concentrations (mg/ml) were significantly increased in males receiving 1,250 and 2,500 ppm and in all exposed groups of females (male: 0 ppm, 0.98; 500 ppm, 1.00; 1,250 ppm, 1.23; 2,500 ppm, 1.68; female: 0 ppm, 0.74; 500 ppm, 0.99; 1,250 ppm, 0.97; 2,500 ppm, 1.43).

Barium levels in bone in rats from the 2,500 ppm groups were about 400 times greater than those in the controls.

At terminal sacrifice, no treatment-related histopathology findings were noted. As animals reduced their drinking water uptake with age, the mean daily consumption for a concentration in drinking water 1250 ppm was calculated as 30 mg/kg bw for males and 45 mg/kg bw for females.

 

Findings in mice were consistent with the findings in rat, taking into account that mice were exposed to higher concentrations and that these resulted in more pronounced effects at the highest dose group. After two-year exposure, the incidence of nephropathy was significantly increased in male and female mice receiving2500ppm (160 mg/kg bw for males and 200 mg/kg bw for females). The nephropopathywasmorphologically distinct from spontaneous degenerative lesions that are commonly observed in agingB6C3F,mice. The nephropathy is probably caused by crystals of insoluble Barium salts and was considered to be the cause of the higher morbidity. The NOEL was found to be 500 ppm (ca 30 - 35 mg/kg bw of Barium). In contrast to rats, mice do not reduce drinking water consumption.

 

Incorporation of Barium into the bone was not accompanied by adverse effects in rats and mice. The target organ for repeated dose toxicity was identified as kidney and this is considered to be linked to precipitates of insoluble Barium salts.

 

For both the anion and the cation, kidney was identified as the target organ for chronic toxicity at doses in the range of 30 mg/kg bw and above. The mode of action appears to be different. Whereas it is linked to precipitates in the case of Barium, the anion causes regeneration of the tubular epithelium.

Taking into account that Barium accounts for 25% of the molecular weight of the pigment, the NOEL for Barium effects would be in the range of 120 mg pigment /kg bw for chronic exposure. 

Significant synergistic effects from co-precipitation of Barium and the organic anion in the kidney are not expected because the organic will undergo azo reduction unless metabolism is overloaded and because Barium is principally eliminated in the faeces. This is supported by hazard data of Pigment Red 53:1 (5-Chloro-2-[(2-hydroxy-1-naphthalenyl)azo]-4-methyl benzenesulfonic acid, barium salt (2:1), CAS no 5160-02-1) For this organic Ba²⁺pigment, a NOEL of 25 mg/kg bw was established in a two-year feeding study and (OECD SIDS document of Pigment Red 53:1, 1999). Pigment Red 53:1 lacks the carbonic acid function on the naphthyl ring.

 

The NOAEL for chronic oral exposure is determined to be 25 mg/kg bw.

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 1272/2008. All kidney effects were completely reversible within tested recovery periods of 14 days. Subacute exposure with up to 300 mg/kg did not induce severe or serious damage, i.e., clear functional disturbance or morphological changes.

As a result the substance is not considered to be classified for repeated dose oral or dermal toxicity under Regulation (EC) No. 1272/2008.