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Effects on fertility

Link to relevant study records
Reference
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
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
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
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
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
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
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: oestrous cycle:
not specified
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
No test item-related effects were observed
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)
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
No test item-related effects were observed
Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Generation:
F2
Effect level:
500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Reproductive effects observed:
not specified
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
chronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Three Generation Study With Indigo In Rats:
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 deleterious effects were associated with the inclusion of indigo in the diet of rats for 3 generations.


Short description of key information:
No effects on reproduction process observed

Effects on developmental toxicity

Description of key information
No effects on intra-uterine development observed
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1971/1972
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
no maternal toxicity observed
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (obtained as weanlings)
- Age at study initiation: satisfactory age for mating (about 8 weeks)
- Weight at study initiation: -
- Fasting period before study: -
- Housing: single
- Diet: Purina Laboratory Chow ad libitum
- Water: tap ad libitum
- Acclimation period: ca. 5 weeks
Route of administration:
oral: gavage
Vehicle:
other: methylcellulose 0.5%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
suspension in 0.5% methylcellulose


VEHICLE
- Dow Methocel 90 HG, 15000 CPS
- Justification for use and choice of vehicle (if other than water): TS is insoluble in water
- Concentration in vehicle: 50 mg/mL
- Amount of vehicle: 10 mL/kg (control and high dose), 3.2 mL/kg, 1 mL/kg
- Lot/batch no.: MM 101012K

POSITIVE CONTROL
- Acetylsalicylic acid (Aspirin)
- Supplier: Mallinckrodt
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- 1 M/1 F per cage
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 through 15 of gestation
Frequency of treatment:
daily
Duration of test:
21 days
Remarks:
Doses / Concentrations:
50, 160, 500 mg/kg/day
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
20 pregnant females
Control animals:
yes, concurrent vehicle
other: positive control: aspirin
Details on study design:
3 dosage levels were run concurrently with 2 negative (vehicle) and one positive (Aspirin) control groups
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: -

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 15, 20 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes
- Number viable and dead fetuses: Yes
- Distribution by sex: Yes
- Mean litter size: Yes
Fetal examinations:
- Mean fetal weight
- Determination of sex
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one-third per litter
- Skeletal examinations: Yes: two-third per litter
Statistics:
Frequency of occurrence and seriousness of anomaly compared to control groups
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
-
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
-
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
D&C Blue No. 6 administered by gavage to groups of 20 female rats each during days 6 through 15 of gestation at dose levels of 50, 160, and 500 mg/kg/day was without effect on reproductive performance, maternal weight gain, and fetal development.
Executive summary:

Groups of 20 pregnant female Charles River CD rats received suspensions of D&C Blue No. 6 by stomach tube at doses of 500, 160 and 50 mg/kg/day from day 6 through day 15 of gestation. Comparable groups received either the methylcellulose suspending vehicle as negative controls or aspirin, 250 mg/kg/day as positive controls. On day 20 of gestation Caesarean section was performed, the uterus examined and fetuses processed for subsequent examination for skeletal or visceral anomalies.

On the basis of numbers of viable and dead fetuses, resorption sites, mean fetal weight, distribution by sex, mean litter size, frequency of skeletal and visceral or structural anomalies; weight gain of pregnant females: Indigo was without effect on reproductive performance, maternal weight gain and fetal development.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Teratogenicity Indigo Rat:
Groups of 20 pregnant female Charles River CD rats received suspensions of D&C Blue No. 6 by stomach tube at doses of 500, 160 and 50 mg/kg/day from day 6 through day 15 of gestation. Comparable groups received either the methylcellulose suspending vehicle as negative controls or aspirin, 250 mg/kg/day as positive controls. On day 20 of gestation Caesarean section was performed, the uterus examined and fetuses processed for subsequent examination for skeletal or visceral anomalies.
On the basis of numbers of viable and dead fetuses, resorption sites, mean fetal weight, distribution by sex, mean litter size, frequency of skeletal and visceral or structural anomalies; weight gain of pregnant females: Indigo was without effect on reproductive performance, maternal weight gain and fetal development.

Teratogenicity Indigo Rabbit:
Groups of 10 pregnant female New Zealand white rabbits received suspensions of D&C Blue No. 6 by stomach tube at doses of 500, 160 and 50 mg/kg/day from day 6 through day 18 of gestation. Comparable groups received either the methylcellulose suspending vehicle as negative controls or thalidomide at 100 mg/kg/day as positive controls. On day 29 of gestation Caesarean section was performed, the uterus examined and fetuses autopsied for detection of visceral anomalies. Subsequently, the eviscerated carcasses were processed for examination of skeletal anomalies.
On the basis of numbers of viable and dead fetuses, resorption sites, mean fetal weight, distribution by sex, mean litter size, frequency of skeletal and visceral or structural anomalies; weight gain of pregnant females: Indigo was without effect on reproductive performance, maternal weight gain and fetal development.

Justification for classification or non-classification

No effects on reproduction observed, no classification necessary