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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2, 1977 - June 3, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2, 1977 - June 3, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Sixty rats/sex were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed. Two control groups were included. After 60 days, the rats were mated. From the litters, 70 rats/sex/ group were selected. After weaning at 21 days, these rats were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed for a life-time (sacrifice males in week 95 and sacrifice females in week 126). Reproductive parameters were evaluated to determine fertility index, gestation anomalies and effects on parturition and lactation. Indices for live birth and survival to weaning were calculated. During the study, mortality, clinical signs, body weight, eyes and urine were monitored regularly. Hematologial and biochemical evaluations and urinalyses were conducted for 10 rats/ sex/group at 3, 6, 12, 18 and 21 (males) or 24 (females) months of study. Feed intake was monitored for each individual rat. An interim sacrifice and necropsy of 10 rats/sex/group was conducted following 12 months of test substance exposure. After sacrifice, gross necropsy was performed, major organs were weighed and histopathogy was done.

GLP compliance:

no

Remarks:

Study performed before GLP principles were implemented.

Limit test:

no

Specific details on test material used for the study:

Concentrations of test substance in the diets were corrected for purity (correction factors 1.0526 and 1.0417 for the lots with purities of 95% and 96%, respectively).

Species:

rat

Strain:

other: CD

Details on species / strain selection:

The Charles River CD rat and their derived offspring were selected because these rats have been shown to be sensitive to the toxic effects of a variety of chemicals.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Masachusetts
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
P: shortly post weaning (weanlings)
- Weight at study initiation (P): 75-155 g (males), 75-114 g (females)
- Fasting period before study: no
- Housing: individually, in hanging wire mesh cages (except during mating and nusring periods)
- Diet: Purina Laboratory Chow (basal diet), ad libitum; during post-weaning: Purina Laboratory Chow, Rodent laboratory Chow #5001 and certified laboratory Chow #5002, ad libitum. Supplier: Ralston Purina Company. Food withheld overnight before blood collection (F1 only).
- Water: tap water, ad libitum except overnight before blood collection (F1 only).
- Acclimation period: 15 days

DETAILS OF FOOD QUALITY: Each new lot feed was analysed for the presence of pesticide residues, heavy metals and aflatoxins. From 1979 on, the water supply was analysed on a quaterly basis for the presence of heavy metals, pesticides and coliforms.

ENVIRONMENTAL CONDITIONS (weekly average after week 31)
- Temperature (°C): 21.7
- Humidity (%): 53%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 02 September 1977 To: 06 Januay 1978 (P); From: 06 Januay 1978 To: 03 June 1980 (F1)

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

DIET PREPARATION
- The appropriate amount of test substance for each treated group was added to the basal laboratory diet and mixed in a tin-shell blender. Test diets were mixed separately for each sex group. From 21 April 1978, test article was mixed to the basal diet in a twin-shell blender. The prepared test diet was then divided into two approximately equal portions; the first and second portions emptied from the blender were designated for use in male and female diets, respectively. Control and treated diest were stored under ambient conditions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

1. Periodic test diet analysis
The main batch of feed for each group was sampled (3 sub-samples representing three distinct areas of the batch) and assayed weekly during the first 13 weeks and every 4 weeks thereafter. On day 7 of weeks 1, 4 and 12 and every 3 months thereafter, a random sampe of feed remaining in the feeder jars was collected for each group from top, middle and bottom locations of selected animal raks and subsequently assayed.
2. Homogeneity
Homogeneity determinations were conducted on diets prepared for study week 12, 34 and 136.

Duration of treatment / exposure:

P: 18 weeks (including 60 days pre-mating)
F1: 95 weeks (males); 126 weeks (females); start exposure in utero

Frequency of treatment:

Continuous exposure (feeding study)

Dose / conc.:

0.05 other: %

Remarks:

Corresponding to 26 (males) and 31 (females) mg/kg bw/day (mean test substance exposure throughout the study, calculated on food consumption and body weight)

Dose / conc.:

0.3 other: %

Remarks:

Corresponding to 161 (males) and 189 (females) mg/kg bw/day (mean test substance exposure throughout the study, calculated on food consumption and body weight)

Dose / conc.:

2 other: %

Remarks:

Corresponding to 1117 (males) and 1315 (females) mg/kg bw/day (mean test substance exposure throughout the study, calculated on food consumption and body weight)

No. of animals per sex per dose:

70

Control animals:

yes

Details on study design:

-

Positive control:

-

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations
F0: Weekly. In addition, females were weighed on days 0, 6, 15 and 21 of gestation and on days 0, 4, 14 and 21 of lactation.
F1, pups: as a litter on days 0, 4 and 14 of lactation. The pups were weighed individually on day 21 of lactation.
F1, post-weaning: Weekly for the first 14 weeks, biweekly the next 12 weeks and once monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
F0: Individual food consumption measurements were recorded weekly for the first 18 weeks of the study.
F1: Individual food consumption measurements were recorded weekly for the first 14 weeks, biweekly (the second 7 days of every two weeks) the next 12 weeks and once monthly (7 days during the last week of each month) thereafter.
Compound consumptions were calculated from the diet consumption and body weight measurements.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
F0: All rats, at 15 weeks;
F1: All rats, once during the pretest period, and at 3, 6, 12, 18, 21 (males) and 24 months (females) of the study.
Ophthalmoscopic examination was performed following pupillary dilation with 1% tropicamide solution. The binocular indirect ophthalmoscope was utilized with a positive 20-diopter focusing and magnifying lens. The clarity of the ocular media (precornal tear film, cornea, aqueous, lens and vitreous) and fundic reflex were initially evaluated. The ocular adnexa and iris were viewed under magnification and the lens focal distance changed to view the fundic tissues (approximately the equator of the globe).

HAEMATOLOGY: Yes (F1)
- Time schedule for collection of blood: At 3, 6, 12, 18 and 21 (males) or 24 (females) months
- Anaesthetic used for blood collection: Not specified, blood obtained via orbita puncture.
- Animals fasted: Yes, overnight (food and water was withheld)
- How many animals: 10 rats/ sex/ group (in addition, blood was collected from 5 unfasted rats/ sex/ group at 20 months)
- Parameters: hemoglobin, hematocrit, total and differential blood counts, total erythrocyte count and total reticulocyte count. In addition, from 20 months on, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration measurements were included.

CLINICAL CHEMISTRY: Yes (F1)
- Time schedule for collection of blood: At 3, 6, 12, 18 and 21 (males) or 24 (females) months
- Anaesthetic used for blood collection: Not specified, blood obtained via orbita puncture.
- Animals fasted: Yes, overnight (food and water was withheld)
- How many animals: 10 rats/ sex/ group (in addition, blood was collected from 5 unfasted rats/ sex/ group at 20 months)
- Parameters: glucose, blood urea nitrogen, serum glutamic oxaloacetic- and serum glutamic pyruvic-transaminase activities, serum alkaline phosphatase activity, serum total protein and creatinine.

URINALYSIS: Yes (F1)
- Time schedule for collection of blood: At 3, 6, 12, 18 and 21 (males) or 24 (females) months
- Anaesthetic used for blood collection: Not specified, blood obtained via orbita puncture.
- Animals fasted: Yes, overnight (food and water was withheld)
- How many animals: 10 rats/ sex/ group (in addition, blood was collected from 5 unfasted rats/ sex/ group at 20 months)
- Parameters: pH, specific gravity, qualitative tests for protein, glucose, bilirubin, ketones, and occult blood, description of color and appearance and microscopic examination of the sediment.

Sacrifice and pathology:

GROSS PATHOLOGY (all rats F1): Yes, complete necropsy after sacrifice by carbon dioxide asphyxiation.

ORGAN WEIGHTS (all rats F1): Brain, kidney, liver, spleen, testes, thyroid, heart, uterus, ovaries, adrenals

HISTOPATHOLOGY (all animals F1 in the control and high dose groups): Abdominal aorta, adrenals, bone and bone marrow (femur), blood smear, brain (3 sections including frontal cortex and basal ganglia, parietal cortex and thalamus, cerebellum and pons), esophagus, eye, gonads (ovaries/ testes with epididymides), heart (with coronary vessels), intestine (cecum, colon, duodenum, ileum), kidneys (2), liver (2 sections), lung and mainstem bronchi, lymph nodes (mediastinal and mesenteric), mammary gland, mandibular salivary gland, sciatic nerve, pancreas, pituitary, seminal vesicles, skeletal muscle (rectus femoris), skin, spinal cord, spleen, stomach, thymus, trachea, thyroid/ parathyroid, urinary bladder, uterus/ prostate. Gross lesions of uncertain nature and all tissue masses or suspect tumors and regional lymp nodes were microscopically examined in all animals. Kidneys of F1 animals from the low and mid dose (0.05 and 0.3%) were also examined.

Statistics:

See "Any other information on materials and methods inc. tables".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F0/F1:
In treated rats, a dose-related colouration was observed of the hair and exposed skin (light-red to red), of urine (dark yellow to orange) and faeces (light-red to red). No other changes in general behaviour and appearance considered to be related to the test substance were seen.
F1:
At the 66-week interim period, a yellow or red material on the anogenital region was noted more frequently for treated rats. The most frequent incidental findings seen at this time for control and treated rats were hair loss, soft stool, red material around the eye, redness and swelling of one or both ears and rales.
During the interval 66 to 90 weeks of study, particularly after week 85, the animals began to exhibit several findings at a greater frequency than previously noted; these findings which usually preceded death or moribundity included yellow or red material on the anogenital region, red material around the eyes, excessive lacrimation, rales and labored breathing.
During the interval 91-116 weeks of study, females had rales and accumulation of yellow or red material on the anogenital area slightly more often for treated animals than the controls. The other observations (previously noted) were still evident in similar numbers of treated and control animals.
Through 126 weeks of study (95 weeks for the males), masses (abdominal, anogenital, thoracic, inguinal and axillary) were noted. The incidence of masses was simlar for the treated and control groups and was within limits that would be expected for animals of this age and strain.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

F0:
Three control rats (2 males (weeks 3 and 12) and 1 female (week 11)), 3 rats dosed at 0.05% (3 females, weeks 1, 9 and 18) and one male dosed at 0.3% (week 18) died during the study. In absence of a dose-relationship, these mortalities were not considered to be related to the test substance.
F1:
Survival indices were similar for control and treated pups with all survival indices being at least 98%.
Survival at termination for males (week 95) was 48%, 28%, 30%, 40% and 15% for control groups 1 and 2 and the groups exposed to 0.05, 0.3 and 2.0%, respectively (for females (week 126) 25%, 27%, 27%, 25% and 18% for control groups 1 and 2 and the groups exposed to 0.05, 0.3 and 2.0%, respectively). The higher mortality rate for males in the highest dose group was noticeable starting week 78 (with survival rates of 77% (all controls) and 73%, 73% and 55% for the groups dosed at 0.05%, 0.3% and 2.0%, respectively).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F0, F1 (pups):
Changes in body weights were similar for control and treated rats.
F1:
Throughout the study, significant body weight depressions were seen for males at the highest dose. When compared to control means, these decreases were considered moderate by 52 weeks and marked by 78 weeks of study (-11% and -17.6% for high dose males compared to controls, respectively). At study termination (week 91), the relative difference in body weight compared to controls (both control groups combined) was -8.1%, -6.1% and -19.4% for males dosed at 0.05, 0.3 and 2.0%, respectively. For the high dose males only, the difference was statistically significant for all intervals analysed between study weeks 10-91.
For females, the relative difference in body weight compared to controls (both control groups combined) at study termination (week 121) was -4.5%, -1.7% and -7.0% for rats dosed at 0.05, 0.3 and 2.0%, respectively. The difference was statistically different for females in the high dose group at study weeks 2-4, 8, 9, 11-14 and 65, however the effect was not considered to be adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F0:
Food consumption was similar for control and treated rats.
F1:
Throughout the study, mean food consumption values were similar for control and treated rats. Occasional statistically significant differences were noted (when compared to combined control means) for females dosed at 0.05% (weeks 69-78 and 82-91) and 0.3% (weeks 82-91). These differences reflected an increase in food consumption of the treated rats. At termination, food consumption was equal among the groups.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F0/1:
No changes considered to be related to the test substance were seen during the week 15 ophthalmoscopic examination.
At 6 months of study (F1), findings seen occasionally for control and treated rats included ocular discharge, keratitis, anterior synechia, chorioretinal degeneration and coloboma of the optic nerve. rats with chorioretinal degeneration showed localized linear areas of choroidal hyperreflectivity with normal overlying retinal vessels. Coloboma of the optic nerve was considered a congenital, non-progressive malformation of the sclera and optic nerve.
At 21 months of study, some treated rats were reported as having posterior segment inflammation. This lesion was described as being confined to the fundic structure and appeared as a generalized change but most pronounced in the peripapillary zone. the lesion was characterized by a cottony irregular texture yo the retinal and choroid area; appearing as localized areas of infiltration but not associated with the vasculature. The significance of this lesion could not be determined.
At pretest and 3, 12, 18, 21 (males) and 24 (females) months of study, the observations noted for control and treated trats were considered representative of pathology that would be expected for these animals given age, sex and strain; no obvious trends in pathology suggestive of test substance-related reactions were noted.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes considered to be related to the test substance were seen in the hematological studies. Although some statistically significant differences were found between the mean values of treated rats when compared to the combined control values, none of these differences was considered biologically significant. Incidental findings noted for a few control and treated rats during the study at single occasions included elevated total leucocyte and reticulocyte counts and/or decreased hemoglobin, hematocrit and erythrocyte values. In absence of a trend (during the study, exposure-related) these findings were considered not adverse.

At 20 months of study, one low dose male and one high dose male had markedly to moderately elevated leucocyte counts. Erythrocyte counts, hemoglobin and hematocrit values for a few of the control and treated mice were markedly increased. This effects were found not to be related to test substance exposure.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes considered to be related to the test substance were seen in the biochemical studies. Incidental findings noted for a few control and treated rats included elevations in serum glutamic oxaloacetic- and serum glutamic pyruvic-transaminase activities, alkaline phosphatase, blood urea nitrogen, and/or creatinine.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

F0/F1:
The urine of rats at the 0.05% dosage level appeared to be slightly darker in color when compared to the urine of control rats. The urine of rats at the 0.3% dosage level generally appared to be yellow-orange in color, and the urine for rats dosed at 2.0% was yellow-dark orange. This effect was a direct effect of presence of the non-metabolized substance in the urine and considered non-adverse.
No other differences were seen in the urinalysis values between control and treated rats.

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):

F1: At final sacrifice, liver, kidney and heart weights of high dose males were increased relative to body weight compared to control group (+9%, +38% and +23%, for respectively liver, kidneys and heart). Since the effect in liver were ,10%, this was not considered adverse. In females, kidney weights for the high dose group were also increased relative to body weight compared to control group (+16.5%). Since no histopathological effects were seen in kidney and heart, this effect was also considered non-adverse. In mid and high dose females, absolute and relative adrenal weights were increased compared to controls (-30.6% and -9.4% (absolute); -33% and -10.2% (relative)). In absence of a dose-response, this effect on adrenals is not considered to be adverse. For all exposed females, uterine weight was increased (absolute: +119%, +77.7%, 194.6% for groups exposed to respectively 0.05%, 0.3% and 2.0%; relative: +103.8%, +92.3%, 138.5% for groups exposed to respectively 0.05%, 0.3% and 2.0%). It is noted that the standard deviations were high for these effects, no clear dose response was seen and no histopathological effects were noted in the uteri, therefore this observation was considered not adverse.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound-related macroscopic changes present for animals which died during the study or which were sacrificed at 12 months or terminally. The changes observed were related to spontaneous disease and were agonal or non-specific.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was a higher incidence of chronic nephritis, renal tubular epithelial hyperplasia, myocardial fibrosis, reticular tubules and pigment in the spleen, atrophy/degeneration of the testicular tubules and pigment in the spleen for high dose males when compared to controls for animals that died on study from 12 months to termination (higher mortality rate was seen at high dose males compared to other groups). The nature of the lesions found in the high dose males was not specific, but rather representative for aging males of this strain.
There was no significant increased incidence of the above lesions in males at terminal sacrifice. No other lesions were found that were attributed to the test substance. The lesions that were present were those of spontaneous disease and they were not unusual for animals of this species and strain.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no compound related neoplastic lesions. A summary table is included under "Any other information on results incl. tables.

Other effects:

not examined

Description (incidence and severity):

No test substance-related effects were seen in fertility, gestation, parturition or lactation indices.

Details on results:

Average test substance consumption throughout the study was calculated at the end of the study as being 26, 161 and 1117 mg/kg bw/day for males and 31, 189 and 1315 mg/kg bw/day for females for the groups dosed at 0.05%, 0.3% and 2.0%, respectively.

At 20 months of study, blood samples from an additional two rats/sex/group were drawn for viral analysis. The PVM and KRV titers were found to be higher than expected for rats of this age. In lung and trachea tissue of three rats, no Sendai virus was isolated. The trachea samples were all positive for Mycoplasma pulmonis. Tissue samples from these rats were also found to be positive for Pseudomonas.

The concentration of the test substance in the diet was shown to be 95-99% of the target concentration. Homogeneity analyses showed that the test substance concentrations varied less than 5% from the mean at all levels (study weeks 33 and 34), or were within ± 10% of the respective sample mean (week 136). The test substance content of samples of feed batches stored at room temperature for 0, 7, 14 or 21 days was 86 to 109% of the initial mean test substance concentration. The test substance content of samples of feed batches stored at 37°C for 0 or 7 days was 88 to 109% of the initial mean test substance concentration.

The feed (12 feed lots) was found not to contain 11 pesticides or PCB above 0.05 ppm. Presumptive residues of malathion residues ranging 0.10 – 0.12 ppm were found in three lots. The heavy metal content ranged from <0.2 to 1.13 ppm arsenic, 0.08 to 0.19 ppm cadmium, 0.29 to 1.37 ppm lead and < 0.05 ppm mercury. Aflatoxins were not detected in any feed lots (<0.02 ppm).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 0.3 other: %

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Correlates to 161 mg/kg bw/day

Dose descriptor:

NOAEL

Effect level:

>= 2 other: %

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects seen at highest dose tested.

Remarks on result:

other: Correlates to 1315 mg/kg bw/day

Key result

Critical effects observed:

no

Summary neoplastic findings

	Control 1		Control 2		0.05%		0.3%		2.0%
Observation	M	F	M	F	M	F	M	F	M	F
Number examined microscopically	70	70	70	70	*	*	*	*	70	70
Total tumor bearing animals	18	56	25	49	18	52	17	48	19	43
Total number of tumors	22	99	34	80	22	101	20	70	22	73
Total benign tumor bearing animals	17	48	24	47	15	45	12	44	1	41
Total number of benign tumors	20	75	31	64	16	71	13	60	16	62
Total malignant tumor bearing animals	2	23	3	16	5	26	6	18	6	10
Total number of malignant tumors	2	24	3	16	6	30	7	19	6	11

 

Conclusions:

Based on the results of a chronic feeding study, the NOAEL for males was found to be 0.3% in feed (corresponding to appr. 161 mg/kg bw/day). For females, the NOAEL was found to be 2.0% in feed (corresponding to 1315 mg/kg bw/day).

Executive summary:

A chronic feeding study was conducted with D&C Red #6. Sixty rats/sex were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed. Two control groups were included. After 60 days, the rats were mated. From the litters, 70 rats/sex/ group were selected. After weaning at 21 days, these rats were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed for a life-time (sacrifice males in week 95 and sacrifice females in week 126). Reproductive parameters were evaluated to determine fertility index, gestation anomalies and effects on parturition and lactation. Indices for live birth and survival to weaning were calculated. During the study, mortality, clinical signs, body weight, eyes and urine were monitored regularly. Hematologial and biochemical evaluations and urinalyses were conducted for 10 rats/ sex/group at 3, 6, 12, 18 and 21 (males) or 24 (females) months of study. Feed intake was monitored for each individual rat. An interim sacrifice and necropsy of 10 rats/sex/group was conducted following 12 months of test substance exposure. After sacrifice, gross necropsy was performed, major organs were weighed and histopathogy was done.

All exposed rats showed direct effects of the test substance: coloured fur, urine and feces. The in utero exposure did not result in adverse effects on body weights of maternal animals or pups, food consumption, ophthalmoscopic examinations, fertility of gestation and lactation indices. For the post-weaning part, males at the high dose group showed significantly decrease in body weight gain compared to controls, with similar feed intake for exposed and control rats. No changes considered to be related to test substance exposure were observed during ophthalmoscopy. No adverse effects were seen on hematology and biochemistry, no test substance-related changes were present at macroscopy. No significant increased incidence of these lesions was seen in males at terminal sacrifice. For females, histopathology did not reveal differences between control and exposed animals. There were no test substance related neoplastic lesions in males or females. Based on these results, the NOAEL for males was found to be 0.3% in feed (corresponding to appr. 161 mg/kg bw/day). For females, the NOAEL was found to be 2.0% in feed (corresponding to 1315 mg/kg bw/day).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2, 1977 - June 3, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Sixty rats/sex were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed. Two control groups were included. After 60 days, the rats were mated. From the litters, 70 rats/sex/ group were selected. After weaning at 21 days, these rats were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed for a life-time (sacrifice males in week 95 and sacrifice females in week 126). Reproductive parameters were evaluated to determine fertility index, gestation anomalies and effects on parturition and lactation. Indices for live birth and survival to weaning were calculated. During the study, mortality, clinical signs, body weight, eyes and urine were monitored regularly. Hematologial and biochemical evaluations and urinalyses were conducted for 10 rats/ sex/group at 3, 6, 12, 18 and 21 (males) or 24 (females) months of study. Feed intake was monitored for each individual rat. An interim sacrifice and necropsy of 10 rats/sex/group was conducted following 12 months of test substance exposure. After sacrifice, gross necropsy was performed, major organs were weighed and histopathogy was done.

GLP compliance:

no

Remarks:

Study performed before GLP principles were implemented.

Limit test:

no

Specific details on test material used for the study:

Concentrations of test substance in the diets were corrected for purity (correction factors 1.0526 and 1.0417 for the lots with purities of 95% and 96%, respectively).

Species:

rat

Strain:

other: CD

Details on species / strain selection:

The Charles River CD rat and their derived offspring were selected because these rats have been shown to be sensitive to the toxic effects of a variety of chemicals.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Masachusetts
- Females nulliparous and non-pregnant: yes
- Age at study initiation, P: shortly post weaning (weanlings)
- Weight at study initiation, P: 75-155 g (males), 75-114 g (females)
- Fasting period before study: no
- Housing: Individually, in hanging wire mesh cages (except during mating (co-housing of one male and one female) and nursing periods (females housed in plastic boxes containing ground corncob bedding)). After weaning at 21 days after birth, pups of a litter were housed together for approximately 2 weeks.
- Diet: Purina Laboratory Chow (basal diet), ad libitum; during post-weaning: Purina Laboratory Chow
, Rodent laboratory Chow #5001 and certified laboratory Chow #5002, ad libitum. Supplier: Ralston Pu
rina Company. Food withheld overnight before blood collection (F1 only).
- Water: tap water, ad libitum except overnight before blood collection (F1 only).
- Acclimation period: 15 days

DETAILS OF FOOD QUALITY: Each new lot feed was analysed for the presence of pesticide residu
es, heavy metals and aflatoxins. From 1979 on, the water supply was analysed on a quaterly basis for
the presence of heavy metals, pesticides and coliforms.
ENVIRONMENTAL CONDITIONS (weekly average after week 31)
- Temperature (°C): 21.7
- Humidity (%): 53%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 02 September 1977 To: 06 Januay 1978 (P); From: 06 Januay 1978 To: 03 June 1980 (F1)

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

DIET PREPARATION
- The appropriate amount of test substance for each treated group was added to the basal laboratory diet and mixed in a tin-shell blender. Test diets were mixed separately for each sex group. From 21 April 1978, test article was mixed to the basal diet in a twin-shell blender. The prepared test diet was then divided into two approximately equal portions; the first and second portions emptied from the blender were designated for use in male and female diets, respectively. Control and treated diest were stored under ambient conditions.

Details on mating procedure:

Rats were housed in units of one male and one female of the same dose group. A maximum period of 7 days was allowed for mating. During this period, vaginal smears were examined until sperm and/or a copulatory plug was observed (gestation day 0).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

1. Periodic test diet analysis
The main batch of feed for each group was sampled (3 sub-samples representing three distinct areas of the batch) and assayed weekly during the first 13 weeks and every 4 weeks thereafter. On day 7 of weeks 1, 4 and 12 and every 3 months thereafter, a random sampe of feed remaining in the feeder jars was collected for each group from top, middle and bottom locations of selected animal raks and subsequently assayed.
2. Homogeneity
Homogeneity determinations were conducted on diets prepared for study week 12, 34 and 136.

Duration of treatment / exposure:

P: 18 weeks (including 60 days pre-mating)
F1: 95 weeks (males); 126 weeks (females); start exposure in utero

Frequency of treatment:

Continuous exposure (feeding study)

Dose / conc.:

0.05 other: %

Remarks:

Corresponding to 33 (males) and 48 (females) (F0)/ 26 (males) and 31 (females) mg/kg bw/day (F1) (mean test substance exposure throughout the study, calculated on food consumption and body weight)

Dose / conc.:

0.3 other: %

Remarks:

Corresponding to 202 (males) and 294 (females) (F0)/ 161 (males) and 189 (females) mg/kg bw/day (F1) (mean test substance exposure throughout the study, calculated on food consumption and body weight)

Dose / conc.:

2 other: %

Remarks:

Corresponding to 1341 (males) and 1987 (females) (F0)/ 1117 (males) and 1315 (females) (F1) mg/kg bw/day (mean test substance exposure throughout the study, calculated on food consumption and body weight)

No. of animals per sex per dose:

P: 60
F1: 70 (selected from at least 35 litters), 10 of these were sacrificed after 12 months

Control animals:

yes

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (for pups at least once)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
BODY WEIGHT: Yes
- Time schedule for examinations
Weekly. In addition, females were weighed on days 0, 6, 15 and 21 of gestation and on days 0, 4, 14 and 21 of lactation.
FOOD CONSUMPTION AND COMPOUND INTAKE:
Individual food consumption measurements were recorded weekly for the first 18 weeks of the
study.
Compound consumptions were calculated from the diet consumption and body weight measurements.
OPHTHALMOSCOPIC EXAMINATION: Yes
All rats, at 15 weeks.

Litter observations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
BODY WEIGHT: Yes
- Time schedule for examinations
Pups: as a litter on days 0, 4 and 14 of lactation. The pups were weighed individually on day 21 of lactation.
Post-weaning: Weekly for the first 14 weeks, biweekly the next 12 weeks and once monthly thereafter.
FOOD CONSUMPTION AND COMPOUND INTAKE:
F1: Individual food consumption measurements were recorded weekly for the first 14 weeks, biweekly (the second 7 days of every two weeks) the next 12 weeks and once monthly (7 days during the last week of each month) thereafter. Compound consumptions were calculated from the diet consumption and body weight measurements.
OPHTHALMOSCOPIC EXAMINATION: Yes
All rats, once during the pretest period, and at 3, 6, 12, 18, 21 (males) and 24 months (females) of the study.
Ophthalmoscopic examination was performed following pupillary dilation with 1% tropicamide solution. The binocular indirect ophthalmoscope was utilized with a positive 20-diopter focusing and magnifying lens. The clarity of the ocular media (precornal tear film, cornea, aqueous, lens and vitreous) and fundic reflex were initially evaluated. The ocular adnexa and iris were viewed under magnification and the lens focal distance changed to view the fundic tissues (approximately the equator of the globe).
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 3, 6, 12, 18 and 21 (males) or 24 (females) months
- Anaesthetic used for blood collection: Not specified, blood obtained via orbita puncture.
- Animals fasted: Yes, overnight (food and water was withheld)
- How many animals: 10 rats/ sex/ group (in addition, blood was collected from 5 unfasted rats/ sex/group at 20 months)
- Parameters: hemoglobin, hematocrit, total and differential blood counts, total erythrocyte count and total reticulocyte count. In addition, from 20 months on, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration measurements were included.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 3, 6, 12, 18 and 21 (males) or 24 (females) months
- Anaesthetic used for blood collection: Not specified, blood obtained via orbita puncture.
- Animals fasted: Yes, overnight (food and water was withheld)
- How many animals: 10 rats/ sex/ group (in addition, blood was collected from 5 unfasted rats/ sex/group at 20 months)
- Parameters: glucose, blood urea nitrogen, serum glutamic oxaloacetic- and serum glutamic pyruvictransaminase activities, serum alkaline phosphatase activity, serum total protein and creatinine.
URINALYSIS: Yes
- Time schedule for collection of blood: At 3, 6, 12, 18 and 21 (males) or 24 (females) months
- Anaesthetic used for blood collection: Not specified, blood obtained via orbita puncture.
- Animals fasted: Yes, overnight (food and water was withheld)
- How many animals: 10 rats/ sex/ group (in addition, blood was collected from 5 unfasted rats/ sex/ group at 20 months)
- Parameters: pH, specific gravity, qualitative tests for protein, glucose, bilirubin, ketones, and occult blood, description of color and appearance and microscopic examination of the sediment.

Postmortem examinations (offspring):

GROSS PATHOLOGY (all rats F1): Yes, complete necropsy after sacrifice by carbon dioxide asphyxiation.
ORGAN WEIGHTS (all rats F1): Brain, kidney, liver, spleen, testes, thyroid, heart, uterus, ovaries, adrenals
HISTOPATHOLOGY (all animals in the control and high dose groups F1): Abdominal aorta, adrenals, bone and bone marrow (femur), blood smear, brain (3 sections including frontal cortex and basal ganglia, parietal cortex and thalamus, cerebellum and pons), esophagus, eye, gonads (ovaries/ testes with epididymides), heart (with coronary vessels), intestine (cecum, colon, duodenum, ileum), kidneys (2), liver (2 sections), lung and mainstem bronchi, lymph nodes (mediastinal and mesenteric), mammary gland, mandibular salivary gland, sciatic nerve, pancreas, pituitary, seminal vesicles, skeletal muscle (rectus femoris), skin, spinal cord, spleen, stomach, thymus, trachea, thyroid/ parathyroid, urinary bladder, uterus/ prostate. Gross lesions of uncertain nature and all tissue masses or suspect tumors and regional lymp nodes were microscopically examined in all animals. Kidneys of F1 animals from the low and mid dose (0.05 and 0.3%) were also examined.

Statistics:

See "Any other information on materials and methods inc. tables".

Reproductive indices:

Fertility index: Percentage pregnancies per matings for males and females)

Offspring viability indices:

Gestation survival index: Percentage live pups per total pups born, directly after birth, and at post natal day 4;
Fourteen day survival index: Percentage surviving pups on post natal day 14 per surviving pups on post natal day 4;
Twenty-one day survival index: Percentage surviving pups on post natal day 21 per surviving pups on post natal day 4

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Orange coloration of hair, exposed skin, feet, tails, urine and/or faeces was observed for all exposed rats in a dose-related manner. No other changes in general behaviour and appearance considered to be related to the test substance were seen.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three control rats (2 males (weeks 3 and 12) and 1 female (week 11)), 3 rats dosed at 0.05% (3 females, weeks 1, 9 and 18) and one male dosed at 0.3% (week 18) died during the study. In absence of a dose-relationship, these mortalities were not considered to be related to the test substance.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Changes in body weights were similar for control and treated rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was similar for control and treated rats.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No changes considered to be related to the test substance were seen during the week 15 ophthalmoscopic examination.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The urine of rats at the 0.05% dosage level appeared to be slightly darker in color when compared to the urine of control rats. The urine of rats at the 0.3% dosage level generally appared to be yellow-orange in color, and the urine for rats dosed at 2.0% was yellow-dark orange. This effect was a direct
effect of presence of the non-metabolized substance in the urine and considered non-adverse. No other differences were seen in the urinalysis values between control and treated rats.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The fertility indices for females and males were 83% and 95% for the two control groups, and 93% for all three test substance groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at highest dose tested.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical effects were seen in the F1 rats before weaning. In treated rats, a dose-related colouration was observed of the hair and exposed skin (light-red to red), of urine (dark yellow to orange) and faeces (light-red to red). At the 66-week interim period, a yellow or red material on the anogenital region was noted more frequently for treated rats. The most frequent incidental findings seen at this time for control and treated rats were hair loss, soft stool, red material around the eye, redness and swelling of one or both ears and rales. During the interval 66 to 90 weeks of study, particularly after week 85, the animals began to exhibit several findings at a greater frequency than previously noted; these findings which usually preceded death or moribundity included yellow or red material on the anogenital region, red material around the eyes, excessive lacrimation, rales and labored breathing.
During the interval 91-116 weeks of study, females had rales and accumulation of yellow or red material on the anogenital area slightly more often for treated animals than the controls. The other observations (previously noted) were still evident in similar numbers of treated and control animals. Through 126 weeks of study (95 weeks for the males), masses (abdominal, anogenital, thoracic, inguinal and axillary) were noted. The incidence of masses was simlar for the treated and control groups and was within limits that would be expected for animals of this age and strain.
The observed effects are correlated to chronic exposure and not considered to be related to reproduction or developmental effects of test item exposure.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Survival indices were similar for control and treated pups with all survival indices being at least 98%. Substance-related effects on mortality were not seen up to week 52 and therefore not considered to be a developmental effect, but rather related to chronic exposure.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Changes in body weights of pups were similar for control and treated rats. Substance-related effects on body weight were not seen up to week 52 and therefore not considered to be a developmental effect.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No effects were seen in the first 6 months of the F1-generation. Effects at a later timepoint were not considered to be related to exposure to the substance (for details, see section 7.5.1).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes considered to be related to the test substance were seen in the hematological studies. Although some statistically significant differences were found between the mean values of treated rats when compared to the combined control values, none of these differences was considered biologically significant. Incidental findings noted for a few control and treated rats during the study at single occasions included elevated total leucocyte and reticulocyte counts and/or decreased hemoglobin, hematocrit and erythrocyte values. In absence of a trend (during the study, exposure-related) these findings were considered not adverse. At 20 months of study, one low dose male and one high dose male had markedly to moderately elevated leucocyte counts. Erythrocyte counts, hemoglobin and hematocrit values for a few of the control and treated mice were markedly increased. This effects were found not to be related to test substance exposure.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes considered to be related to the test substance were seen in the biochemical studies. Incidental findings noted for a few control and treated rats included elevations in serum glutamic oxaloacetic- and serum glutamic pyruvic-transaminase activities, alkaline phosphatase, blood urea nitrogen, and/or creatinine.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No effects were observed in the pups. The urine of weaned rats at the 0.05% dosage level appeared to be slightly darker in color when compared to the urine of control rats. The urine of rats at the 0.3% dosage level generally appared to be yellow-orange in color, and the urine for rats dosed at 2.0% was yellow-dark orange. This effect was a direct effect of presence of the non-metabolized substance in the urine and considered non-adverse. No other differences were seen in the urinalysis values between control and treated rats.

Sexual maturation:

no effects observed

Description (incidence and severity):

No effects were noted on the (development of) the sexual organs of the F1-generation.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The pups were not examined. At final sacrifice, liver, kidney and heart weights of high dose males were increased relative to body weight compared to control group (+9%, +38% and +23%, for respectively liver, kidneys and heart). Since the effect in liver were ,10%, this was not considered adverse. In females, kidney weights for the high dose group were also increased relative to body weight compared to control group (+16.5%). Since no histopathological effects were seen in kidney and heart, this effect was also considered non-adverse. In mid and high dose females, absolute and relative adrenal weights were increased compared to controls (-30.6% and -9.4% (absolute); -33% and -10.2% (relative)). In absence of a dose-response, this effect on adrenals is not considered to be adverse. For all exposed females, uterine weight was increased (absolute: +119%, +77.7%, 194.6% for groups exposed to respectively 0.05%, 0.3% and 2.0%; relative: +103.8%, +92.3%, 138.5% for groups exposed to respectively 0.05%, 0.3% and 2.0%). It is noted that the standard deviations were high for these effects, no clear dose response was seen and no histopathological effects were noted in the uteri, therefore this observation was considered not adverse.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The pups were not examined. There were no test substance-related macroscopic changes present for animals which died during the study or which were sacrificed at 12 months or terminally. The changes observed were related to spontaneous disease and were agonal or non-specific.

Histopathological findings:

no effects observed

Description (incidence and severity):

The pups were not examined. There was a higher incidence of chronic nephritis, renal tubular epithelial hyperplasia, myocardial fibrosis, reticular tubules and pigment in the spleen, atrophy/degeneration of the testicular tubules and pigment in the spleen for high dose males when compared to controls for animals that died on study from 12 months to termination (higher mortality rate was seen at high doe males compared to other groups). The nature of the lesions found in the high dose males was not specific, but rather representative for aging males of this strain. There was no significant increased incidence of the above lesions in males at terminal sacrifice. No other lesions were found that were attributed to the test substance. The lesions that were present were those of spontaneous disease and they were not unusual for animals of this species and strain.

Other effects:

no effects observed

Description (incidence and severity):

There were no compound related neoplastic lesions. A summary table is included under "Any other information on results incl. tables" of section 7.5.1.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 2 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen on reproduction up to and including the highest dose tested (2% in feed).

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Based on the results of a chronic feeding study, which included exposure of two generations, the parental, reproduction and developmental NOAEL was found to be 2.0%, which correlates to appr. 1117 mg/kg bw/day for males and for females 1315 mg/kg bw/day.

Executive summary:

A chronic feeding study, covering two generations, was conducted with D&C Red #6. Sixty rats/sex were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed. Two control groups were included. After 60 days, the rats were mated. From the litters, 70 rats/sex/ group were selected. After weaning at 21 days, these rats were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed for a life-time (sacrifice males in week 95 and sacrifice females in week 126). Reproductive parameters were evaluated to determine fertility index, gestation anomalies and effects on parturition and lactation. Indices for live birth and survival to weaning were calculated. During the study, mortality, clinical signs, body weight, eyes and urine were monitored regularly. Hematologial and biochemical evaluations and urinalyses were conducted for 10 rats/ sex/group at 3, 6, 12, 18 and 21 (males) or 24 (females) months of study. Feed intake was monitored for each individual rat. An interim sacrifice and necropsy of 10 rats/sex/group was conducted following 12 months of test substance exposure. After sacrifice, gross necropsy was performed, major organs were weighed and histopathogy was done.

All exposed rats showed direct effects of the test substance: coloured fur, urine and feces. The in utero exposure did not result in adverse effects on body weights of maternal animals or pups, food consumption, ophthalmoscopic examinations, fertility or gestation and lactation indices. For the postweaning part, males at the high dose group showed significant decrease in body weight gain compared to controls, with similar feed intake for exposed and control rats. No changes considered to be related to test substance exposure were observed during ophthalmoscopy. No adverse effects were seen on hematology and biochemistry, no test substance-related changes were present at macroscopy. Histopathology did not reveal adverse effects in exposed animals. There were no test substance related neoplastic lesions in males or females. As the observed adverse effects in males on body weight gain were not expected to influence their ability to reproduce and were considered to be related to chronic exposure instead of in utero exposure, this was not included in the paternal NOAEL. Based on these considerations, the parenteral, reproduction and developmental NOAEL was found to be 2.0% (corresponding to appr. 1117 mg/kg bw/day for males and for females 1315 mg/kg bw/day).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Sixty rats/sex were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed. Two control groups were included. After 60 days, the rats were mated. From the litters, 70 rats/sex/ group were selected. After weaning at 21 days, these rats were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed for a life-time (sacrifice males in week 95 and sacrifice females in week 126). Reproductive parameters were evaluated to determine fertility index, gestation anomalies and effects on parturition and lactation. Indices for live birth and survival to weaning were calculated. During the study, mortality, clinical signs, body weight, eyes and urine were monitored regularly. Hematologial and biochemical evaluations and urinalyses were conducted for 10 rats/ sex/group at 3, 6, 12, 18 and 21 (males) or 24 (females) months of study. Feed intake was monitored for each individual rat. An interim sacrifice and necropsy of 10 rats/sex/group was conducted following 12 months of test substance exposure. After sacrifice, gross necropsy was performed, major organs were weighed and histopathogy was done.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

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:

C18H12N2Na2O6S

IUPAC Name:

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

Test material form:

solid: particulate/powder

Remarks:

dark orange-red powder

Specific details on test material used for the study:

Concentrations of test substance in the diets were corrected for purity (correction factors 1.0526 and 1.0417 for the lots with purities of 95% and 96%, respectively).

Test animals

Species:

rat

Strain:

other: CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Masachusetts
- Females nulliparous and non-pregnant: yes
- Age at study initiation, P: shortly post weaning (weanlings)
- Weight at study initiation, P: 75-155 g (males), 75-114 g (females)
- Fasting period before study: no
- Housing: Individually, in hanging wire mesh cages (except during mating (co-housing of one male
and one female) and nursing periods (females housed in plastic boxes containing ground corncob b
edding)). After weaning at 21 days after birth, pups of a litter were housed together for approximately
2 weeks.
- Diet: Purina Laboratory Chow (basal diet), ad libitum; during post-weaning: Purina Laboratory Chow
, Rodent laboratory Chow #5001 and certified laboratory Chow #5002, ad libitum. Supplier: Ralston
Pu
rina Company. Food withheld overnight before blood collection (F1 only).
- Water: tap water, ad libitum except overnight before blood collection (F1 only).
- Acclimation period: 15 days
DETAILS OF FOOD QUALITY: Each new lot feed was analysed for the presence of pesticide residu
es, heavy metals and aflatoxins. From 1979 on, the water supply was analysed on a quaterly basis for
the presence of heavy metals, pesticides and coliforms.
ENVIRONMENTAL CONDITIONS (weekly average after week 31)
- Temperature (°C): 21.7
- Humidity (%): 53%
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES:
From: 02 September 1977 To: 06 Januay 1978 (P); From: 06 Januay 1978 To: 03 June 1980 (F1)

Administration / exposure

Route of administration:

oral: feed

Details on analytical verification of doses or concentrations:

DIET PREPARATION
- The appropriate amount of test substance for each treated group was added to the basal laboratory diet and mixed in a tin-shell blender. Test diets were mixed separately for each sex group. From 21 April 1978, test article was mixed to the basal diet in a twin-shell blender. The prepared test diet was then divided into two approximately equal portions; the first and second portions emptied from the blender were designated for use in male and female diets, respectively. Control and treated diest were stored under ambient conditions.

Details on mating procedure:

Rats were housed in units of one male and one female of the same dose group. A maximum period of 7 days was allowed for mating. During this period, vaginal smears were examined until sperm and/or a copulatory plug was observed (gestation day 0).

Duration of treatment / exposure:

P: 18 weeks (including 60 days pre-mating)
F1: 95 weeks (males); 126 weeks (females); start exposure in utero

Frequency of treatment:

Continuous exposure (feeding study)

Duration of test:

P: 18 weeks (including 60 days pre-mating)
F1: 95 weeks (males); 126 weeks (females); start exposure in utero

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.05 other: %

Remarks:

Corresponding to 33 (males) and 48 (females) (F0)/ 26 (males) and 31 (females) mg/kg bw/day (F1) (mean test substance exposure throughout the study, calculated on food consumption and body weight)

Doses / concentrations 2

Dose / conc.:

0.3 other: %

Remarks:

Corresponding to 202 (males) and 294 (females) (F0)/ 161 (males) and 189 (females) mg/kg bw/day (F1) (mean test substance exposure throughout the study, calculated on food consumption and body weight)

Doses / concentrations 3

Dose / conc.:

2 other: %

Remarks:

Corresponding to 1341 (males) and 1987 (females) (F0)/ 1117 (males) and 1315 (females) (F1) mg/kg bw/day (mean test substance exposure throughout the study, calculated on food consumption and body weight)

No. of animals per sex per dose:

P: 60
F1: 70 (selected from at least 35 litters), 10 of these were sacrificed after 12 months

Control animals:

yes

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (for pups at least once)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
BODY WEIGHT: Yes
- Time schedule for examinations
Weekly. In addition, females were weighed on days 0, 6, 15 and 21 of gestation and on days 0, 4, 14 and 21 of lactation.
FOOD CONSUMPTION AND COMPOUND INTAKE:
Individual food consumption measurements were recorded weekly for the first 18 weeks of the study. Compound consumptions were calculated from the diet consumption and body weight measurements.
OPHTHALMOSCOPIC EXAMINATION: Yes All rats, at 15 weeks.

Fetal examinations:

Not applicable, F1 generation observed entire lives (for details, see section 7.8.1).

Statistics:

Summarized in section 7.8.1.

Indices:

Summarized in section 7.8.1.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Orange coloration of hair, exposed skin, feet, tails, urine and/or faeces was observed for all exposed rats in a dose-related manner. No other changes in general behaviour and appearance considered to be related to the test substance were seen.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three control rats (2 males (weeks 3 and 12) and 1 female (week 11)), 3 rats dosed at 0.05% (3 females, weeks 1, 9 and 18) and one male dosed at 0.3% (week 18) died during the study. In absence of a dose-relationship, these mortalities were not considered to be related to the test substance.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Changes in body weights were similar for control and treated rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was similar for control and treated rats.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The urine of rats at the 0.05% dosage level appeared to be slightly darker in color when compared to the urine of control rats. The urine of rats at the 0.3% dosage level generally appared to be yelloworange in color, and the urine for rats dosed at 2.0% was yellow-dark orange. This effect was a direct
effect of presence of the non-metabolized substance in the urine and considered non-adverse. No other differences were seen in the urinalysis values between control and treated rats.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

not specified

Description (incidence and severity):

The fertility indices for females and males were 83% and 95% for the two control groups, and 93% for all three test substance groups.

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not examined

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 other: %

Based on:

test mat.

Basis for effect level:

other: No adverse effects seen at highest dose tested.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No clinical effects were seen in the F1 rats before weaning. Changes in body weights of pups were similar for control and treated rats.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Survival indices were similar for control and treated pups with all survival indices being at least 98%.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Survival indices were similar for control and treated pups with all survival indices being at least 98%.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at highest dose tested.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of a chronic feeding study, which included exposure of two generations, the parental, reproduction and developmental NOAEL was found to be 2.0%, which correlates to appr. 1117 mg/kg bw/day for males and for females 1315 mg/kg bw/day.

Executive summary:

A chronic feeding study, covering two generations, was conducted with D&C Red #6. Sixty rats/sex were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed. Two control groups were included. After 60 days, the rats were mated. From the litters, 70 rats/sex/ group were selected. After weaning at 21 days, these rats were exposed to 0.05%, 0.3% or 2.0% of the test substance in feed for a life-time (sacrifice males in week 95 and sacrifice females in week 126). Reproductive parameters were evaluated to determine fertility index, gestation anomalies and effects on parturition and lactation. Indices for live birth and survival to weaning were calculated. During the study, mortality, clinical signs, body weight, eyes and urine were monitored regularly. Hematologial and biochemical evaluations and urinalyses were conducted for 10 rats/ sex/group at 3, 6, 12, 18 and 21 (males) or 24 (females) months of study. Feed intake was monitored for each individual rat. An interim sacrifice and necropsy of 10 rats/sex/group was conducted following 12 months of test substance exposure. After sacrifice, gross necropsy was performed, major organs were weighed and histopathogy was done.

All exposed rats showed direct effects of the test substance: coloured fur, urine and feces. The in utero exposure did not result in adverse effects on body weights of maternal animals or pups, food consumption, ophthalmoscopic examinations, fertility or gestation and lactation indices. For the postweaning part, males at the high dose group showed significant decrease in body weight gain compared to controls, with similar feed intake for exposed and control rats. No changes considered to be related to test substance exposure were observed during ophthalmoscopy. No adverse effects were seen on hematology and biochemistry, no test substance-related changes were present at macroscopy. Histopathology did not reveal adverse effects in exposed animals. There were no test substance related neoplastic lesions in males or females. As the observed adverse effects in males on body weight gain were not expected to influence their ability to reproduce and were considered to be related to chronic exposure instead of in utero exposure, this was not included in the paternal NOAEL. Based on these considerations, the parenteral, reproduction and developmental NOAEL was found to be 2.0%

(corresponding to appr. 1117 mg/kg bw/day for males and for females 1315 mg/kg bw/day).