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Description of key information

A chronic feeding study performed with a substance analogue in rats is available (Klimisch 2). Based on the 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). This result is read across to the registered ubstance.

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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Limit test:
no
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 D&C Red #6 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). This result is read across to PR63:1.
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). This result is read across to PR63:1.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
161 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The study was found to be reliable (Klimisch 2). The results of the study are supported by similar findings in a comparable chronic feeding study in mice.
System:
other: No specific toxicity seen, NOAEL based on effects on body weight gain.

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 study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data, PR63:1 is not classified for adverse effects after repeated dose toxicity according to Regulation (EC) 1272/2008.