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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1977 -1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study, partly performed prior to the introduction of GLP. Survival rate of males slightly reduced by bacterial infection. Little details on method for analytics.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1981
Reference Type:
other: Amendment to study report
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate
EC Number:
227-497-9
EC Name:
Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate
Cas Number:
5858-81-1
Molecular formula:
C18H14N2O6S.2Na
IUPAC Name:
disodium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate
Details on test material:
- Name of test material (as cited in study report): D&C no. 6, K-7034
- Molecular formula (if other than submission substance): CAS no. 5858-81-1
- Substance type: pigment (dark-orange red powder)
- Physical state: solid
- Analytical purity: 95% (Lot no. AA3473) and 96% (Lot no. AA 5169)
- Impurities (identity and concentrations): no information available
- Lot/batch No.: AA 3473 and AA 5169
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable
- Storage condition of test material: not indicated.
- Other: supplier H. Kohnstamm and Company Inc. New York (USA)

Test animals

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.


Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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).

Results and discussion

Results of examinations

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).

Effect levels

open allclose all
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 %

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion