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Description of key information

Three studies are suitable to provide information on toxicity after repeated dose administration. The subacute toxicity study in rats at dose levels up to 5 % (unknown purity) revealed formation of Heinzbodies, changes in hematology and pathological and histopathological changes in spleen and kidney. The 90d study at dose levels up to 10000 ppm which served as range finder for a cancer study confirms the findings of the 28d study. Additionally, congestion of the spleen and hemosiderosis in the liver was observed. A chronic study in mice at dose levels up to 1000 ppm revealed no adverse effects than changes in hematology.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
march 1978 - march 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
no
Species:
mouse
Strain:
CD-1
Remarks:
Charles River
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: 36d
- Weight at study initiation:
- Housing: in groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 1978-03-15 To: 1980-03-21
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): appropriate quantity by weight of D&C Red No. 9 (corrected for purity) was manually mixed with 1 kg of basal diet
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the first batch were analyzed after storage at room temperature for 8, 16 and 21 days and at 37°C for 8 days. In addition, the stability of D&C Red No. 9 in the diet under animal room conditions was determined by the analysis of diet remaining in three feed jars from each dietary level at the end of Weeks l, 4, 13 and quarterly thereafter. Starting with Week 4 and thereafter, bedding and feces which contaminated the feed in the feed containers were removed prior to analysis by sifting the feed through a sieve. In addition, samples of feed kept in the animal room but not exposed to mice were also analyzed at some intervals. Beginning January, 1979, and quarterly thereafter, samples of diet and test material were sent to a representative of the sponsor to confirm analytical results and demonstrate stability of the test material. The analytical method, results.and discussion of the analyses performed by LBI were described in the Analytical Chemistry Report
Duration of treatment / exposure:
18 month / 105 weeks
Frequency of treatment:
daily
Dose / conc.:
50 ppm
Dose / conc.:
250 ppm
Dose / conc.:
1 000 ppm
No. of animals per sex per dose:
60 per sex and dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: no data
- Section schedule rationale (if not random): no data
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT and food consumption: Yes
- Time schedule for examinations: Body weights and food consumption (measured over one-week periods) were obtained at weekly intervals through Week 14, at bi-weekly intervals Weeks 16-26, and at monthly intervals thereafter. Additionally, body weights were obtained at Week 104 and from the survivors on the day of termination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at months 3, 6, 12 and 18 of study
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: ten mice per sex per dose group were randomly selected from survivors
- Parameters checked: Hematology evaluation consisting of hematocrit, red blood cell count, white blood cell count (total and differential), hemoglobin value and reticulocyte count
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

abdominal aorta
adrenal glands
bone and bone marrow (femur)
brain (3 sections including
frontal cortex and basal
ganglia, parietal cortex
and thalamus; cerebellum
and pons)
cecum
colon
duodenum
esophagus
eye
gallbladder
heart (with coronary vessels)
ileum
jejunum
kidneys (2)
liver
lung and mainstem bronchi
mediastinal lymph node
mesenteric lymph node
mammary gland
mandibular salivary gland
nerve (sciatic)
ova ri es
pancreas
pituitary gland
prostate gland
seminal vesicles
ske~tai ~usci~e (~ctus femoris)
skin
cervical spinal cord
spleen
stomach
testes with epididymides
trachea
thymus
thyroid/parathyroids
uterus
urinary bladder
gross lesions of uncertain
nature, all tissue masses
or suspect tumor, or abnormal
regional lymph nodes
Statistics:
The controls were combined, weighted for the number of samples in each, for statistical analyses. Differences between mean values were analyzed using Dunnett’s t-test. Ratios were compared using a 2x2 contingency table with Yates’ correction. A probability of p< 0.05 was used as a basis to determine statistical significance. For body weight, organ weight and clinical pathology data, if a significant difference was observed between a dose and combined control group, the
two controls were compared to each other using a Student’s t-test at the p< 0.01 level. If the controls differ, then each control group was compared to the dose groups using Dunnett’s t-test at the p<0.05 level. Statistical analysis for tumor incidences was performed using the NCI program.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
high dose females: decrease in hemoglobin and hematocrit

At month 18, there was a significant decrease in the red blood cell count, hemoglobin and hematocrit values in the high dose females. There was also a significant decrease in the hematocrit values of the low dose females and a significant increase in the reticulocyte counts of the low dose males.The decrease in the red blood cell parameters of the high dose females indicated a possible compound related anemia. None of the other changes were judged to be compound related.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
haematology
Critical effects observed:
not specified

 Dietary Concentration (ppm of D&C Red No. 9) mg/kg/day (mean +/- S.E.)male  mg/kg/day (mean +/- S.E.)female
 50  7.4 +/- 0.34  11.5 +/- 0.54
 250  38.3 +/- 1.74  56.0 +/- 2.61
 1000  147.3 +/- 6.22  236.6 +/- 10,16

Table 1: Mean Hematology of Mice - Males

Parameter

Group number

Treatment [ppm]

Month

3

6

12

18

RBC

[mm³ x 106]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

7.557

7.852

7.705

7.239

7.744

7.731

7.882

7.717

7.800

7.567

7.625

7.465

7.725

7.323

7.524

7.321

7.662

7.388

8.247

7.401

7.824

7.451

8.183

7.755

PCV (HCT)

[%]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

49.75

48.10

48.93

48.05

48.85

49.67

48.40

50.50

49.45

49.30

48.65

47.25

45.85

43.65

44.75

43.45

44.7

44.45

44.80

41.72

43.26

41.33

45.55

43.90

Hemoglobin [GM%]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

15.15

15.25

15.20

14.74

15.13

15.07

14.91

15.23

15.07

14.97

14.69

13.98*

13.98

13.25

13.62

12.94

13.73

13.60

13.98

13.32

13.65

12.95

14.20

13.28

Retic. Count [%]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

4.27

3.85

4.06

4.52

4.55

4.41

2.58

2.53

2.56

2.61

3.59

3.26

3.57

3.57

3.57

3.68

4.20

3.54

1.94

1.74

1.84

2.63*

1.36

1.56

WBC

[mm³ x 10³]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

6.56

5.66

6.11

5.92

5.94

5.36

5.99

7.15

6.57

7.82

7.82

5.40

6.83

8.28

7.55

8.71

7.95

8.26

6.60

8.08

7.34

7.89

7.68

7.26

*Significant at p < 0.05 as compared to combined controls (Groups 1 and 2): Dunnett’s t-test.

Table 2: Mean Hematology of Mice - Females

Parameter

Group number

Treatment [ppm]

Month

3

6

12

18

RBC

[mm³ x 106]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

7.566

7.127

7.347

7.103

7.545

8.435*

7.116

7.539

7.328

7.407

6.864

7.116

7.465

7.403

7.434

7.575

7.534

7.785

8.101

8.359

8.230

7.772

8.094

7.233*

PCV (HCT)

[%]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

49.05

48.50

48.78

47.90

48.00

49.50

48.70

48.75

48.73

50.85

47.05

49.80

45.20

44.80

45.00

45.75

44.90

46.35

46.10

46.55

46.33

42.56*

43.95

41.75*

Hemoglobin [GM%]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

15.66

15.63

15.65

15.11

15.16

15.55

15.02

15.03

15.03

15.35

14.42

15.86

13.67

13.68

13.68

14.08

13.63

14.25

14.43

14.60

14.52

13.51

14.04

12.78*

Retic. Count [%]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

4.57

4.24

4.41

4.51

3.84

3.06

2.98

2.87

2.93

3.84

3.41

2.78

4.00

3.87

3.93

4.19

3.67

2.86

1.31

1.99

1.65

1.78

1.74

2.13

WBC

[mm³ x 10³]

1

2

1 and 2

3

4

5

0

0

Combined controls

50

250

1000

5.89

6.94

6.42

6.13

5.64

5.42

5.16

6.84

6.00

6.61

5.83

6.75

7.11

7.01

7.06

6.31

5.67

6.13

3.96

5.24

4.60

5.04

4.62

5.29

*Significant at p < 0.05 as compared to combined controls (Groups 1 and 2): Dunnett’s t-test.

Table 3: Mean organ weights in mice - Terminal kill

Sex

Dose level [ppm]

Body weight [g]

Kidneys [g]

Liver [g]

Spleen [g]

Male

Control I

Control II

Combined Controls

50

250

1000

38.2

38.0

38.1

35.2*

35.5*

36.2

0.9330

0.9526

0.9416

0.8482

0.8583

0.8192*

2.2361

2.2168

2.2277

2.2702

2.3626

2.1000

0.1347

0.1570

0.1446

0.2063

0.1392

0.1208

Females

Control I

Control II

Combined Controls

50

250

1000

30.8

31.0

30.9

31.3

32.3

29.7

0.5414

0.6394

0.5896

0.5723

0.5681

0.5388

1.6491

1.7311

1.6894

1.8094

1.7708

1.7133

0.1520

0.1922

0.1718

0.1798

0.1747

0.1885

*Significant at p < 0.05 as compared to combined controls (Groups 1 and 2): Dunnett’s t-test.

Executive summary:

D&C Red No. 9 was administered to mice for two years by incorporation into the diet at concentrations of 50, 250 and l000 ppm. The high dose level animals were colored slightly orange. No adverse effect of treatment was evident based on survival, observations, body weight and food intake. A decrease in the value of red blood cell parameters in the high dose females at month 18 indicated a possible compound-related anemia. The gross and histopathologic evaluation and tumor incidence analyses did not reveal any compound related effects.

All tumors encountered in the current study were common spontaneously occurring tumors of. mice or uncommon tumors that affected only individual mice without regard to treatment group. There was no apparent treatment-related increase in tumor incidence. There was no apparent difference between treated and control mice with respect to non-tumorous lesions encountered in the study. The histopathologic findings indicate that D&C Red No 9 administered per os at the dose levels employed and under conditions of this study did not produce morphologically identifiable evidence of toxigenic effect on CD-1 mice. In addition, the study yielded no evidence of carcinogenicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
147.3 mg/kg bw/day
Study duration:
chronic
Species:
mouse
System:
haematopoietic
Organ:
blood

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

Procedure and observations

The repeated dose toxicity of the test material was evaluated by subacute, subchronic and by a combined chronic/cancer study.

In the course of the subacute toxicity study (equivalent to OECD 407), the test material was fed in diet at doses of 0, 0.2, 1 and 5% to rats (10/sex/dose) for 32 consecutive days (Hoechst 1973). In all treatment groups discoloration of feces and urine was observed. A strong decrease in body weight was recorded after one week at the high dose group which tends to normalize but was still lower than control at the end of the study. The body weight of the mid dose males was also lower than in control animals at the end of the study. Development of Heinz bodies (high dose group 100%, mid dose 30 % and low dose 10 % Heinz bodies in erythrocytes) and a dose dependend increase in leucocytes and a decrease in erythrocytes in all treatment groups was determined. A significant and dose dependent increase in spleen weight, enlarged and blackish colored spleen and brownish colored kidneys at mid and high dose animals was observed; enlarged spleen was seen at the low dose group. Histopathology revealed general disturbances of iron metabolism and iron storage which was evident by a dose dependent increase in iron storage in liver, in kidney tubular epithelium (not in low dose group) and a moderate to strong increase of iron levels in spleen in all treatment groups.

A subchronic study was conducted to determine repeated dose toxicity of D & C Red No. 9 and to estimate the concentrations to be used in the chronic studies (NTP 1982). The test substance was administrated in diet at concentrations of 3000 - 50.000 ppm to rats or 600 - 10.000 ppm to mice (10/sex/dose) for 91 days. One male rat receiving 6,000 ppm and one female rat receiving 3,000 ppm died. Mean body weight gains were comparable among all groups. Treatment of rats caused a dark discoloration of the spleen as well as enlargement, congestion and hyperplasia. Pigment deposition in the renal tubular epithelium of the kidneys was observed in all dosed animals and hyperplasia of the thymic lymph nodes was found in 75%-100% of all rats, exept the low dose animals. Hemosiderosis of the liver was found in all female rats and in male rats in a dose dependent manner. All mice survived until scheduled necropsy. Congestion of the spleen was observed in 55 of 60 mice receiving 2,500 ppm or more. Deposits of hemosiderin were present to a greater extent in all animals receiving 600 ppm or more.

D&C Red No. 9 was administered to mice for two years by incorporation into the diet at concentrations of 50, 250 and l000 ppm (CFTA 1982). The high dose level animals were colored slightly orange. No adverse effect of treatment was evident based on survival, observations, body weight and food intake. A decrease in the value of red blood cell parameters in the high dose females at month 18 indicated a possible compound-related anemia. The gross and histopathologic evaluation and tumor incidence analyses did not reveal any compound related effects. All tumors encountered in the current study were common spontaneously occurring tumors of mice or uncommon tumors that affected only individual mice without regard to treatment group.

Discussion

Performance and analysis of the subacute toxicity study is very briefly described, information on substance purity are not mentioned, individual and historical data are not given. Therefore, a NOAEL is not derived. However, it should be mentioned that remarkable effects, e.g. Heinz body formation, changes in red blood count and disturbances in iron metabolism and storage occured in all treatment groups which are equivalent to 10, 50 and 250 mg/kg bw/day.

The subchronic toxicity study revealed effects comparable to the subacute study, e.g. at spleen, liver and iron metabolism. Though, this study was conducted as range finder for a cancer study and methods and results are described briefly. Hemosiderosis of the liver was rated as mild at 3,000 ppm (90 - 120 mg/kg bw); thus doses of 1,000 and 3,000 ppm were selected for rats in the chronic study. The occurrence of these lesions, hemosiderosis, and congestion was not considered life threatening per se; however, it was considered indicative of potentially severe toxicity in a chronic study. Doses of 1,000 and 2,000 ppm were selected for mice in the chronic study to avoid possible toxic effects.

The histopathologic findings of the 2 year chronic/cancer study indicate that D&C Red No 9 did not produce morphologically identifiable evidence of toxigenic effect on mice. In addition, the study yielded no evidence of carcinogenicity.

In conclusion, the substance causes remarkable effects in rats from app. 100 mg/kg bw onward. These effects include hematotoxicity, disturbances in iron metabolism as well as changes and damages at spleen, liver and kidney. The symptoms in mice are less severe than in rat. Most likely, metabolites of the test article (1-amino-2-naphtol) cause methemoglobinamia leading to disturbances of iron metabolism and subsequently increased iron deposition in liver and spleen (hemosiderosis). Moreover, the substance or metabolites are attached at methemoglobin and transported via red blood cells into the spleen which acts as a filter for old or damaged erythrocytes. During degradation of methemoglobin, the substance or metabolite is released and affects spleenic mesenchymal tissue. In the course of this explanation it must be considered that humans are more sensitive to met-hemoglonformation than rats and, therefore, effects in humans may occur even at lower doses.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

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

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