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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
the study was not conducted as a one-generation study but meets basic principles of this test (exposure duration F0 animals, dose level, observations); histopathology of parental target organs and analysis of oestrous-cycle/spermatogenesis was not performed
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
red powder
Desert Red - D&C Red No. 9 Ba. Lake
Batch #547530, C-15-I01
Not less than 76% Pure Dye
FDA Certification Lot #AA3779

received from The Cosmetic, Toiletry and Fragrance Association, Inc. (CTFA), by Litton Bionetics, Inc. (LBI)
on August 12, 1977
designated as LBI No. 1643. FDA certification reported 76% purity.

Test animals

Species:
rat
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: (P) x 40d
- Housing: singly until mating (8 weeks), 1:1 for mating 7d, again separated for pregnancy, delivery and lactation (21d), F1 in groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 18d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/- 1
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): The appropriate quantity (by weight) of D&C Red No. 9 (corrected for purity) was manually mixed with 5 kg of basal diet. This premix was mixed with approximately one-half the final volume of basal feed for 15 minutes in a twin shell blender. The remaining basal feed was then added, and mixing was continued for 15 minutes. From Weeks 1-15, 36 kg of diet were prepared at each dose level. From Week 15 until weaning of the pups, the diet was prepared in 54 kg batches as frequently as needed to supply adequate feed for the rats.
- Storage temperature of food: room temperature
Details on mating procedure:
- M/F ratio per cage: one to one
- Length of cohabitation: 7d
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): singly for pregnancy, delivery and lactation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity of the preparations was determined by analyzing 9 discrete samples from each dietary level of the first batch. Additionally, samples of the first batch were analyzed after storage at room temperature (25°C) for 7, 14 and 21 days and 7 days at 370C. During the remainder of the in utero phase of the study, the test diet was analyzed approximately weekly. The stability of D&C Red No. 9 in the diet under animal room conditions was determined by the analysis of diet remaining in 3 feed jars from each dietary level at the end of Weeks 2, 5, and 12. At Week 10 only, samples of the 100 ppm diet were analyzed to check for low values found in Weeks 2 and 5.
Duration of treatment / exposure:
8 weeks before mating and during mating (P), 30 month (whole life time) of the F1 generation
Frequency of treatment:
daily
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 9.5 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
Dose / conc.:
500 ppm (nominal)
No. of animals per sex per dose:
60
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment: random
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: physical appearance, signs of toxicity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
in weekly intervals until mating was initiated

days of gestation
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
bodyweight: The pups were sexed and weighed as a litter and the means were calculated on Days 0, 4 and 14. On Day 21, they were sexed and individual body weights were recorded. Body weights and food consumption were determined weekly for Weeks 1-14, subsequently at biweekly intervals over one week for Weeks 16-26, and at monthly intervals thereafter. Body weights were taken for all animals prior to necropsy

food consumption: Food consumption for dam and litter was determined for Days 0-4, 5-14, and 15-21.

clinical observation: Detailed observations were performed on all pups on Days 0, 4, 14 and 21 of lactation. After weaning and throughout the chronic phase, the animals were observed at least twice daily for general physical appearance, signs of toxicity and mortality.

ophthalmoscopic observation: The eyes of all surviving rats were examined at the initiation of the chronic phase, and at Months 3, 6, 12, 18 and 2

pathologic evaluation: Clinical pathologic evaluation was performed at Months 3, 6, 12, 18 and 24 of the chronic phase of the study. Rats scheduled for clinical pathology were fasted overnight prior to collection of the samples.

Hematology: hb, hc, RBC, white blood cell count, reticulocyte count. Specimens were collected by orbital sinus bleeding for blood chemistry and hematology samples

Clinical chemistry: glucose, BUN, SGOT, SGPT, alkaline phophatase, creatinine, total protein

urinalysis: color, appearance, specific gravity, protein, pH, ketones, bilirubin, glucose, occult blood and microscopic sediment was performed at the same intervals as the clinical pathologic testing. Urine was collected in stainless steel collection cages.

STANDARDISATION OF LITTERS
One male and i female pup were randomly selected from each litter when possible. To provide 70 animals per sex per dose group, litters were selected randomly to contribute an additional male and/or female pup which was selected randomly from the remaining pups

GROSS EXAMINATION OF DEAD PUPS:
no

PARAMETERS EXAMINED
The following parameters were examined: live pubs, dead pubs, sex, mean pub weight, on days 0, 4, 14 and 21
Postmortem examinations (parental animals):
SACRIFICE
Pups not selected for the chronic study, and the F0 generation rats were discarded

GROSS NECROPSY
- no

HISTOPATHOLOGY / ORGAN WEIGHTS
no
Postmortem examinations (offspring):
SACRIFICE
All animals dying on study or killed (via carbon dioxide) were necropsied. For the 12 month interim kill, 10 animals/sex/dose group were randomly
selected from the survivors and all surviving animals were killed at the 30 month terminal kill.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
At the interim and terminal kills organ weights were determined for the adrenal glands, brain, heart, kidneys, liver, spleen, thyroids, testes, ovaries, and uterus. The organ weight/body weight percentages were calculated.

abdominal aorta
adrenal gland (2)
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
heart (with coronary vessles)
ileum
jejunum
kidneys (2)
liver
lung and mainstem bronchi
mediastinal lymph node
mesenteric lymph node
mammary gland
mandibular salivary gland
nerve (sciatic)
ovaries
pancreas
pituitary gland
prostate
seminal vesicles
skeletal muscle (rectus femoris)
skin
spinal cord (cervical)
spleen
stomach
testes with epididymides
thymus
trachea
thyroid/parathyroid
urinary bladder
uterus
gross lesions of uncertain
nature and all tissue
masses or suspect tumors and
abnormal regional lymph nodes

A blood smear (air dried/methanol fixed) was taken from the animals at the scheduled kills for possible histopathologic evaluation. Blood smears
were also taken from moribund animals starting with those after the interim kill.

Tumor incidence analysis.
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 [I]. 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 differed, then each control group was compared to the dose groups using Dunnett’s t-test at the p<0.05 level. Statistical analyses for tumor incidences where values of p<0.0l were considered significant was performed by using the NCI program developed by Thomas, et. al.
Reproductive indices:
reproductive performance and gesatation period
Offspring viability indices:
Pup viability was determined as gestation viability (live birth vs total birth), neonate viability (live pups Day 4 vs live pups Day 0), early lactation viability (live pups Day 14 vs live pups Day 4), and late lactation viability (live pups Day 21 vs live pups Day 14). The overall viability (live pups Day 21 vs live pups Day O) has also been calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Sporadic statistically significant differences have occurred, but were judged to be unrelated to treatment.
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: see table below

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
The decrease in total pups represents pups which were found dead, cannibalized, or sacrificed because they had escaped and could not be returned to the proper litter. The above data did not reveal any compound related effect.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The decreased mean body weight of female pups of the 500 ppm treatment level at Day 21 of lactation was the only value statistically different from the combined controls. However, the decrease was less than 10% of the control weight and was judged not to represent a compound related effect.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
For the 12 month interim kill, significantly high values for both the spleen weight and the spleen weight-body weight percentages was observed in the high dose females. The spleen weight and spleen weight-body weight percentages for the high dose males were elevated as compared to the combined control but this was not statistically different. Spleen weight and spleen weight-body weight percentages values for the high dose of both sexes were not statistically significant at the 30 month terminal kill but the values were elevated as compared to the combined values.
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
no effects observed
Description (incidence and severity):
The hemosiderosis observed in the spleens reported for the high dose females in the 12 Month Interim Pathology Report was subjectively graded to be more severe than similar lesions in the control animals, but the pathologist judged that this effect was unrelated to the test material. However, the hematology data reveal.ed a significant decrease in erythrocyte count and a significant increase in the spleen weight in the high dose females (500 ppm) at the 12-month interim kill. At this interval the apparent slight increase in hemosiderin of the spleen in the high dose females tends to corroborate the hematologic data and the increase of the spleen weight.
The histopathologic evaluation of the control and high dose animals in addition to grossly noted lesions from mid and low dose animals after the 12-month interim kill did not reveal any obvious compound related effect. The lesions observed in the spleens of the high dose females at the 12 Month interim kill were not apparent at the time of the terminal sacrifice.

Details on results (F1)

sex ratio: No statistically significant differences between treated and combined controls were observed.

pub deaths: distribution of pup deaths was uniform among all 5 groups, for both the treated and the control. Therefore, it was judged that the deaths which occurred were not compound related, and the absence of gross necropsy data did not affect the scientific integrity of the study.

The tumor incidence analysis did not suggest any compound related effect,

Effect levels (F1)

Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The mean intake (mg/kg/day) of test material of the F1 generation has been calculated as the mean daily food

consumption multiplied by the intended dietary concentration divided by the mean body weight. The mean intake of test

material per animal over the course of the FI generation study was as follows.

 Dietary Concentration(ppm of D&C Red No. 9)  mg/kg/day (Mean ± SE) males  mg/kg/day (Mean ± SE) females
 100  5.08 + 0.41  6.36 + 0.39
 200  10.02 + 0.82  12.53 + 0.80
 500  25.89 + 2.12  32.42 + 2.12

mean material intake F0 generation

The food consumption values during lactation were excluded from the mean intake of test material per animal during the course of the F0 phase, because the food consumption values during this period reflected the food consumed by both the pups and the dams

 Dietary Concentration(ppm of D&C Red No. 9)  mg/kg/day (Mean ± SE) males  mg/kg/day (Mean ± SE) females
 100  8.3 ± 0.03  8.5 ± 0.21
 200  17.4 ± 0.73  16.9 ± 0.44
 500  42.5 ± 1.54  42.2 ± 1.05

Table 1: Cumulative deaths (percent) and numbers of survivors in parentheses by week in F0 rats

Week

Sex

Dose [ppm of Red No. 9]

0 (Control 1)

0 (Control 2)

100

200

500

1

2

3

4

5

6

7

8

12

Male

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (59)

0 (59)

0 (59)

0 (59)

0 (59)

0 (59)

0 (59)

0 (59)

0 (59)

1

2

3

4

5

6

7

8

Female

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

1.7 (59)

1.7 (59)

1.7 (59)

1.7 (59)

1.7 (59)

1.7 (59)

1.7 (59)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (60)

0 (61)

0 (61)

0 (61)

0 (61)

0 (61)

0 (61)

0 (61)

0 (61)

Day of gestation

0

6

15

21

 

0 (60)

0 (60)

0 (60)

0 (60)

 

0 (60)

0 (60)

0 (60)

0 (60)

 

1.7 (59)

1.7 (59)

1.7 (59)

1.7 (59)

 

0 (60)

0 (60)

0 (60)

0 (60)

 

0 (61)

0 (61)

0 (61)

0 (61)

Day of lactation

0

4

14

21

 

0 (60)

0 (60)

0 (60)

0 (60)

 

0 (60)

0 (60)

0 (60)

0 (60)

 

1.7 (59)

1.7 (59)

1.7 (59)

1.7 (59)

 

0 (60)

0 (60)

0 (60)

0 (60)

 

0 (61)

0 (61)

0 (61)

0 (61)

Table 2: Mean body weight [g] in F0 rats

Week

Sex

Dose [ppm of Red No. 9]

0 (Control 1)

0 (Control 2)

100

200

500

0

1

2

3

4

5

6

7

8

12

Male

183

229

280

311

347

377

406

427

446

481

186

225

280

314

350

376

402

425

440

481

179

223

275

308

345

374

402

425

438

480

184

224

253*

311

348

373

397

422

436

482

179

223

267*

304

345

371

399

422

438

483

0

1

2

3

4

5

6

7

8

Female

147

166

183

201

218

230

242

250

254

147

166

190

204

219

231

242

249

256

148

167

191

207

220

232

244

251

258

147

169

190

206

221

231

245

250

258

146

169

187

199

216

225

240

245

256

Day of gestation

0

6

15

21

 

253

271

317

397

 

256

274

313

386

 

258

277

316

397

 

256

276

312

391

 

253

272

313

393

Day of lactation

0

4

14

21

 

300

317

335

302

 

291

307

323

295

 

299

308

324

299

 

297

307

325

292

 

295

304

322

303

*Significant at p < 0.05 as compared to controls: Dunnett’s t-test

Table 3: Pup viability

Indices [%]

Dose [ppm of Red No. 9]

0 (Control 1)

0 (Control 2)

Combined Controls

100

200

500

Gestational viability (Live pups / total pups day 0)

98

(670 / 682)

99

(651/650)

99

(1321/1340)

98

(624/635)

99

(663/667)

99

(651/660)

Neonate viability (Day 4 / Day 0)

98

 (659 / 670)

96

(626/651)

97

(1285/1321)

99

(619/624)*

99

(655/663)

99

(645/651)*

Early lactation viability (Day 14 / Day 4)

99

(649 / 659)

97

(605/626)

98

(1254/1285)

97

(599/619)

98

(642/655)

98

(631/645)

Late lactation viability (Day 21 / Day 14)

97

(629/649)

97

(584/605)

97

(1213/1254)

95

(569/599)

97

(623/642)

98

(617/631)

Overall viability (Day 21 / Day 0)

94

(629/670)

90

(584/651)

92

(1213/1321)

91

(569/624)

94

(623/663)

95

(617/651)

*Significant at p < 0.05 as compared to controls: Chi-Square test with Yates‘ correction

Table 4: Mean Pup Body Weight [g]

Day (mean)

Dose [ppm of Red No. 9]

0 (Control 1)

0 (Control 2)

Combined Controls

100

200

500

0

4

14

21 (males)

21 (females)

6.5

10.3

23.6

36.9

35.0

6.5

10.1

24.8

36.0

34.7

6.5

10.2

24.2

36.5

34.9

6.7

10.0

23.4

35.0

32.7

6.6

10.3

23.7

34.0

32.4

6.4

10.0

22.8

34.4

32.1*

*Significant at p < 0.05 as compared to controls: Dunnett’s t-test

Table 5: Male / female pups ratio

Day (mean)

Dose [ppm of Red No. 9]

0 (Control 1)

0 (Control 2)

Combined Controls

100

200

500

0

4

14

21

332 / 337

332 / 327

324 / 325

306 / 323

300 / 351

295 / 331

285 / 320

277 / 307

632 / 688

627 / 658

609 / 645

583 / 630

294 / 329

299 / 320

284 / 315

281 / 290

314 / 347

320 / 335

308 / 334

289 / 334

326 / 325

335 / 310

323 / 308

319 / 298

Table 6: Percent cumulative deaths and numbers of survivors in parentheses in F1 rats

Month

Sex

Dose [ppm of Red No. 9]

0 (Control 1)

0 (Control 2)

100

200

500

0

6

12

18C

24

30

Male

0 (71)d

2.8 (69)

2.8 (69)

16.9 (59)

38.9 (44)

78.9 (15)

0 (70)

0 (70)

0 (70)

24.3 (53)

44.3 (39)

78.5 (15)

0 (70)

0 (70)

0 (70)

21.4 (55)

45.7 (38)

78.5 (15)

0 (70)

0 (70)

1.4 (69)

22.9 (54)

37.1 (44)

74.3 (18)

0 (70)

1.4 (69)

1.4 (69)

24.3 (53)

44.3 (39)

78.5 (15)

0

6

12

18C

24

30

Female

0 (69)d

0 (69)

2.9 (68)

20.3 (55)

43.5 (39)

75.4 (17)

0 (70)

2.9 (68)

2.9 (68)

21.4 (55)

51.4 (34)

74.3 (18)

0 (70)

1.4 (69)

1.4 (69)

22.9 (54)

40.0 (42)

72.9 (19)

0 (70)

1.4 (69)

5.7 (66)

25.7 (52)

51.4 (34)

70.0 (21)

0 (70)

1.4 (69)

2.8 (68)

22.9 (54)

38.6 (43)

68.6 (22)

Cincludes interim kill

done animal originally mis-sexed

Applicant's summary and conclusion

Executive summary:

The test material, D&C Red No. 9, was administered to rats in the diet at concentrations of 100, 200 and 500 ppm for 8 weeks prior to mating. The treatment was continued during gestation and lactation. The test material was judged not to have an effect on body weight, food consumption, or fertility of the F0 generation rats. Similarly, no effect was evident on the viability or growth of F1 pups from birth to weaning. Rats from parents treated with graded dietary concentrations of D&C Red No. 9 (0, 100, 200 and 500 ppm) have been continued on treatment for 30 months after weaning. The only difference between the treated groups and the control (0 ppm) groups now judged to be related to the test material is the increase in the spleen weight in the high dose females at the 12 month interval. The apparent decreased values of red cell parameters seen at 12 months were not evident at 18 or 24 months. The gross and histopathologic evaluation and the tumor incidence analyses of the animals did not reveal any compound related effect.