Registration Dossier

Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1972/73
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions; no GLP

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1973
Report Date:
1973
Reference Type:
publication
Title:
Toxicology of Indigo - a review
Author:
Ferber KH
Year:
1987
Bibliographic source:
J. Environ. Pathol. Toxicol. Oncol. 7, 73 -84 (1987)
Reference Type:
publication
Title:
Multi-generation Reproduction Studies with Certified Colors in Rats
Author:
Pierce E, Agersborg H, Borzelleca J, Burnett C, Eagle E, Ebert A, Kirschman J, Scala R
Year:
1974
Bibliographic source:
Toxicol  Appl Pharmacol. 1974;29:121-122

Materials and methods

Principles of method if other than guideline:
Final version of Kirschman and Carpenter "A multi-generation study rats", dated January 7, 1972, amended on January 15 by Kirschman
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name: D&C Blue #6
- Substance type: active ingredient
- Physical state: solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan
Age at arrival: (P) 75 days
- Age at study initiation: (P) 11 - 12 weeks; (F1) 4 weeks
- Weight at study initiation: (P) Males: approx. mean 320 g; Females: approx. mean 238 g ; (F1) Males: approx. mean 164 g; Females: approx. mean 140 g
- Housing: before mating: 2/cage; during mating: 1 male:2 females; thereafter - females: single
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: Purina Formulab TM Chow ad libitum
- Water: ad libitum
- Acclimation period: 8 days

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks
- Mixing appropriate amounts with (Type of food): Purina Formulab TM Chow
- Concentration in food: percentage color in diet = (dose level [mg/kg bw/day] x mean body weight [kg]) / (10 x mean food consumption [g/rat/day])
Details on mating procedure:
- M/F ratio per cage: 1:2
- Age at mating: (P) 1 - ca. 99 days; 2 - ca 169 days
(F1b) 1 - ca 100 days; 2 - ca 169 days; 3 - ca. 242 days
(F2b) 1 - ca. 102 days
- Treatment prior mating: 2 weeks
- Length of cohabitation: 15 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
F0: 2-weeks prior mating until death - approx. 100 to 130 days
F1b/2b: from weaning until death - approx. 265 to 295/ 125 to 155 days
Frequency of treatment:
continuously
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: (P) 1 - ca. 99 days; 2 - ca 169 days
(F1b) 1 - ca 100 days; 2 - ca 169 days; 3 - ca. 242 days
(F2b) 1 - ca. 102 days
Doses / concentrations
Remarks:
Doses / Concentrations:
5;50;150;500 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
10 males + 20 females
Control animals:
yes, concurrent no treatment
Details on study design:
3 parallel control groups from different color testings were used as comparators
Positive control:
no

Examinations

Parental animals: Observations and examinations:
BEHAVIOUR AND APPEARENCE: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes
- Compound intake calculated from the consumption and body weight gain data: Yes

-OTHER:
- Male and female fertility
- Gestation length
- F1b - third mating: half of dams per group were killed on Day 19: examination of
- uterus and contents examined: no of embryos, empty implantation sites, late and early resorption sites, any abnormal condition
- ovaries: no of corpora lutea
- necropsy in 5 rats/sex/group: (also true for F3a litter)
- marcoscopic examination and preservation of all tissues listed below for all dose groups
- adrenals, colon, heart, ileum, jejunum, kidney, liver, lung, ovary, uterus, spleen, stomach, testes, thyroid, urinary bladder, aorta, esophagus, eyes, mesenteric lymph node, pancreas, peripheral nerve, prostate, salivary gland, seminal vesicles, skeletal muscle, thymus, trachea
- microscopic examination of selected tissues in rats from the control and high dose groups: adrenals, colon, heart, ileum, jejunum, kidney, liver, lung, ovary, uterus, spleen, stomach, testes, thyroid, urinary bladder

Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter randomized; excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: necropsy see above


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
see above: Observations and examinations
Postmortem examinations (offspring):
- F1b - third mating: half of dams per group were killed on Day 19: examination of
- uterus and contents examined: no of embryos, empty implantation sites, late and early resorption sites, any abnormal condition
- ovaries: no of corpora lutea
- necropsy in 5 rats/sex/group: (also true for F3a litter)
- marcoscopic examination and preservation of all tissues listed below for all dose groups
- adrenals, colon, heart, ileum, jejunum, kidney, liver, lung, ovary, uterus, spleen, stomach, testes, thyroid, urinary bladder, aorta, esophagus, eyes, mesenteric lymph node, pancreas, peripheral nerve, prostate, salivary gland, seminal vesicles, skeletal muscle, thymus, trachea
- microscopic examination of selected tissues in rats from the control and high dose groups: adrenals, colon, heart, ileum, jejunum, kidney, liver, lung, ovary, uterus, spleen, stomach, testes, thyroid, urinary bladder


SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age (except those of F2c generation delivered by cesarean section on Day 19 of pregnancy).
- These animals were subjected to postmortem examinations macroscopic and/or microscopic examination as follows:


NECROPSY
- Gross necropsy consisted of external and internal examinations
- F3a litter: necropsy in 5 rats/sex/group:
- marcoscopic examination and preservation of all tissues listed below for all dose groups
- adrenals, colon, heart, ileum, jejunum, kidney, liver, lung, ovary, uterus, spleen, stomach, testes, thyroid, urinary bladder, aorta, esophagus, eyes, mesenteric lymph node, pancreas, peripheral nerve, prostate, salivary gland, seminal vesicles, skeletal muscle, thymus, trachea
- microscopic examination of selected tissues in rats from the control and high dose groups: adrenals, colon, heart, ileum, jejunum, kidney, liver, lung, ovary, uterus, spleen, stomach, testes, thyroid, urinary bladder
Statistics:
Quantitative variables of 3 control and 4 dosage groups: intercomparison with Bartlett's homogeneity of variances, analysis of variance, Duncan's multiple range tests
In case of inhomogeneity: F-test --> if not significant: Student's t-test
--> if significant: Cochran t-test or sum of ranks

Levels of significance: a - 0.05 > P > 0.01
b - 0.01 > P > 0.001
c - P < 0.001

Statistical methods used are described in "Selection of the valid number of sampling units and a consideration of their combination in toxicological studies involving reproduction, teratogenesis or carcinogenesis! Weil CS. Food and Cosmetics Toxicology 8:177-182; 1970
Reproductive indices:
Fertility index
Gestation index
Gestation survival index
Offspring viability indices:
4-day survival index
14-day survival index
21-day survival index
Lactation index

Results and discussion

Results: P0 (first parental animals)

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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

No test item-related effects were observed

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; body weight; gross pathology; histopathology
Remarks on result:
other: Generation: F3 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

No test item-related effects were observed

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOEL
Generation:
F2
Effect level:
500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No test item-related effects were observed, only random, not deleteriuous effects were associated with the inclusion of indigo in the diet of rats for 3 generations.
Indigo does neither have adverse effects on the reproductive system and fertility of parental rats and theit offspring, nor on the development of the offspring.
Executive summary:

10 males and 20 females of each generation were dosed with 0, 0, 0, 5, 50, 150, 500 mg/kg bw/day of D&C Blue #6 and mated. The parent generation (F0) rats were bred twice, the F1b were bred thrice, and the F2b were bred once. The criteria of effect included the following indices: fertility, gestation, gestation survival, 4 -/14 -/21 -day survival. Furthermore, the diet consumption and body weights of the parent rats and their progeny data were examined as were the number of resorption sites and corpora lutea at the 19-day kill of the third mating of half of the F1b dams.

The differences between dosed and control groups were neither dose-related, nor progressive, nor were they indicative of mean or median values exceeding those of the controls. It is therefore concluded that only random, not deleteriuous effects were associated with the inclusion of indigo in the diet of rats for 3 generations.