Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

Toxicity to reproduction

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

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

Test material information

Constituent 1

Reference substance name:

Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

EC Number:

225-935-3

EC Name:

Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

Cas Number:

5160-02-1

Molecular formula:

C17H13ClN2O4S.1/2Ba

IUPAC Name:

barium(2+) bis(5-chloro-2-[(1E)-2-(2-hydroxynaphthalen-1-yl)diazen-1-yl]-4-methylbenzene-1-sulfonate)

Test material form:

solid: nanoform

Details on test material:

red powder
Desert Red - D&C Red No. 9 Ba. Lake
Batch #547530, C-15-I01
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 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:

rat

Strain:

CD-1

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: (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 / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

200 ppm (nominal)

Doses / concentrations 3

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 18^C 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 18^C 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.