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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform
Details on test material:
D&C Red No. 9
Desert Red C15-0-10 Batch #547530
Not less than 76 % pure
FDA Certified Lot #AA-3779
received from The Cosmetic, Toiletry and Fragrance Association, Inc. (CTFA), by Litton Bionetics, Inc. (LBI).
Three drums were received on February 28, 1978, and designated as LBI No. 2521, and the other four drums were received on March 13, 1978 and designated as LBI No. 2521a.

Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.

Test animals

Species:
mouse
Strain:
CD-1
Remarks:
Charles River
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
haematology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

 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.

Applicant's summary and conclusion

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.