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EC number: 274-356-2 | CAS number: 70161-20-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to reproduction
Administrative data
- Endpoint:
- three-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data from peer reviewed journal
Data source
Reference
- Reference Type:
- publication
- Title:
- Multigeneration Study OF FD & C Blue NO. 2 in Rats
- Author:
- J. F. BORZELLECA, E. I. GOLDENTHAL and F. X. WAZETER
- Year:
- 1 986
- Bibliographic source:
- Fd Chem. Toxk'. Vol. 24, No. 2, pp. 159-163, 1986
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: As mentioned below
- Principles of method if other than guideline:
- Three-generation reproduction toxicity study of FD & C Blue NO. 2 in Rats
- GLP compliance:
- not specified
- Limit test:
- no
- Justification for study design:
- No data available
Test material
- Reference substance name:
- Disodium 5,5'-(2-(1,3-dihydro-3-oxo-2H-indazol-2-ylidene)-1,2-dihydro-3H-indol-3-one)disulphonate
- EC Number:
- 212-728-8
- EC Name:
- Disodium 5,5'-(2-(1,3-dihydro-3-oxo-2H-indazol-2-ylidene)-1,2-dihydro-3H-indol-3-one)disulphonate
- Cas Number:
- 860-22-0
- Molecular formula:
- C16H10N2O8S2.2Na
- IUPAC Name:
- disodium 3,3'-dioxo-1,1',3,3'-tetrahydro-2,2'-biindole-5,5'-disulfonate
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
- Details on test material:
- - Name of test material (as cited in study report): FD & C Blue NO. 2
- Molecular formula : C16H8N2O8S2.2Na
- Molecular weight : 466.3572 g/mole
- Substance type: Organic
- Physical state: Powder
- Impurities (identity and concentrations): 7% volatile matter
Constituent 1
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): FD & C Blue NO. 2
- Molecular formula : C16H8N2O8S2.2Na
- Molecular weight : 466.3572 g/mole
- Substance type: Organic
- Physical state: Powder
- Impurities (identity and concentrations): 7% volatile matter
Test animals
- Species:
- rat
- Strain:
- other: Charles River CD
- Details on species / strain selection:
- No data available
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (Wilmington, MA)
- Age at study initiation: (P) x wks; (F1) x wks: P- 80-85 days
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: No data available
- Fasting period before study: No data available
- Housing: Animals were housed individually in stainless-steel wire-mesh cages, except during the mating, lactation and post-weaning periods when the females were placed in plastic shoe-box cages containing bedding. Each rat was identified with a metal ear tag. If the tag was lost, the animal was re-tagged and/or toe-clipped.
- Diet (e.g. ad libitum): Basal diet (Purina Rodent Chow from Ralston Purina Co., Inc., St Louis, MO), ad libitum
- Water (e.g. ad libitum): Water, ad libitum
- Acclimation period: No data available
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21°C
- Humidity (%):40-60%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle
Administration / exposure
- Route of administration:
- oral: feed
- Type of inhalation exposure (if applicable):
- not specified
- Vehicle:
- other: Basal diet
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: FD & C Blue No. 2 was mixed with the powdered chow in a twin shell blender to provide theoretical dose levels of 2.5, 25, 75 and 250 mg/kg/day.
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Basal diet (Purina Rodent Chow from Ralston Purina Co., Inc., St Louis, MO)
- Storage temperature of food: Stored in environmentally controlled room with limited access.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet
- Concentration in vehicle: 0, 2.5, 25, 75 and 250 mg/kg/day.
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available - Details on mating procedure:
- - M/F ratio per cage: 1:2 ratio
- Length of cohabitation: 15 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Vaginal smears were performed daily until sperm or a copulatory plug was found.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)] No data available
- After successful mating each pregnant female was caged (how): Female was placed on corn-cob bedding in an individual plastic shoe-box cage and remained there until it was remated.
- Any other deviations from standard protocol: Each female was rested for a minimum of 10 days after lactation before being mated again.
Each mating was with a different male from the same dosage group. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Prior to initiation of the study, assays were performed to determine the homogeneity and stability of FD & C Blue No. 2 in the prepared diets. The concentrations of the test compound in the diets were determined weekly during the first 13 wk of the study and monthly thereafter. All lots of basic feed used in the study were analysed for heavy metals, chlorinated hydrocarbons and aflatoxin. These analyses demonstrated that the basic feed contained acceptably low levels of contaminants, that the diets were prepared properly and that the dietary content of the test material was stable
- Duration of treatment / exposure:
- 224 days
- Frequency of treatment:
- Daily
- Details on study schedule:
- F0 geneartion:
F0 animals were meated twice to give F1a and F1b
F1a - on 21-day examined for external abnormalities and killed.
F1b - random selections were made of ten male and 20 female pups from the control and treated groups of the F1b litters to serve as the second generation (F0 parental rats. The remaining F1b pups were examined for external abnormalities, killed and discarded.
F1 geneartion:
F0 animals were meated twice to give F2a and F2b
F2a - examined for external abnormalities
and killed at the end of the 21-day lactation
period.
F2b – selections were made of ten male and 20 female pups from the control and treated groups to serve as the third generation (F2) parental rats.
F2 geneartion:
F0 animals were meated twice to give F3a , F3b and F3c
F3a – On 21 days old and necropsied and tissues were collected for histopathology.
F3b - F2 animals were remated to give F3a, On 21 days necropsied and tissues were collected for histopathology.
F3c - F2 animals were remated to give F3c. On day 19 of gestation, half of the dams from the control and treated groups were killed.
Doses / concentrations
- Remarks:
- Doses / Concentrations:
0, 2.5, 25, 75 and 250 mg/kg/day
Basis:
- No. of animals per sex per dose:
- Total: 1410
F0 geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female
F1a geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female
F1b geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female
F2a geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female
F2b geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female
F2c geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female
F3a geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female
F3b geneartion:
0 mg/kg bw/day: 10 male, 20 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female - Control animals:
- yes, concurrent vehicle
- Details on study design:
- No data available
- Positive control:
- No data available
Examinations
- Parental animals: Observations and examinations:
- .CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table [No.?] were included.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):No data
- Time schedule for examinations:
OTHER:Specific parameters for the reproductive phase of this study included survival of pups to weaning and growth of the pups.Body weights of pups aged 4 days (before and after reduction of litters to ten pups) and aged 14 days were obtained by weighing as litters. The pups were weighed individually at 21 days of age. - Oestrous cyclicity (parental animals):
- No data available
- Sperm parameters (parental animals):
- No data available
- Litter observations:
- Morbidity and mortality, body weights, food consumption and food efficiency of F1 and F2 geneation were examined.
- Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: yes (The F0 parent rats were killed with chloroform and necropsied.The F1 parent rats were then killed. Five male and five female rats from the control and each of the treated groups were necropsied.On day 19 of gestation, half of the F2 dams from control and treated groups were killed by chloroform anesthesia. Remaining F2 parent rats were then killed and discarded.
GROSS NECROPSY: yes
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
HISTOPATHOLOGY / ORGAN WEIGHTS
The thyroid, adrenals, lung. heart, spleen, stomach, jejunum, ileum, colon, liver, kidneys, urinary bladder, testes or ovaries and uterus
from five male and five female rats from the F1 and F3, generations of the control and 250-mg/kg/day groups were embedded in paraffin, sectioned, stained
with haematoxylin and eosin and examined microscopically. - Postmortem examinations (offspring):
- .SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY: yes
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
HISTOPATHOLOGY / ORGAN WEIGTHS: yes
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively. - Statistics:
- Fertility indices were compared using the chisquare test criterion with Yates' correction for 2 × 2 contingency tables (Steel & Torrie, 1960) to judge the significance of differences. Gestation, 4-day, 14-day and 21-day survival indices were compared by the rank sum tests described by Snedecor & Cochran (1967) and Weil (1970) to judge the significance of differences. The numbers of pups born alive were compared by analysis of variance and the t test, as described by Steel & Torrie (1960), using the multiple comparison tables of Dunnett (1964) to judge the significance of differences.
- Reproductive indices:
- Fertility indices, gestation and lactation indices were examined.
- Offspring viability indices:
- Yes, viability on day 4, 14 and 21 were examined.
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Slightly bluish-coloured fur was noted in rats of the 250-mg/kg/day dosage group. Bluish-green-coloured faeces were produced by rats in the 75- and 250-mg/kg/day groups. No other changes considered to be related to FD & C Blue No. 2 were noted in general behaviour or appearance. Signs noted with similar frequency in control and treated rats included occasional ocular or nasal porphyrin discharge, soft tools, respiratory congestion and slight alopecia.
- Dermal irritation (if dermal study):
- not specified
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One control female rat (F0) and one female (F0) in the 2.5-mg/kg/day group) died during the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No effect on body weight of treated rats was observed as compared to control.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Although variations in food consumption were recorded for all groups, no marked or consistent differences were observed between control and treated groups
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not specified
- Urinalysis findings:
- not specified
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- not specified
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- not specified
Reproductive function / performance (P0)
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- not specified
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- No significant change was observed in F0 fertility index. The fertility indices for female rats given 2.5 or 25mg/kg/day weresignificantly lower than those for control females during the production of the F2 litters. However, no reduction in fertility for female rats was observed in the F 2 litters in the two higher dosage groups, 75 and 250mg/kg/day, nor in the F1 and F3 litters at any dosage level. Because of the lower fertility indices observed at the lower dosage levels in the F2 litters, the study was extended to the F3b litter. Although fertility indices were also reduced for some groups of male rats in the F2b and F2c litters, the changes were considered to represent variation rather thancompound-related effects.
There was no evidence of any impairment of reproductive performance.
Details on results (P0)
Clinical signs: When treated with 250 mg/kg body weight/day, Slightly bluish-coloured fur and Bluishgreen- coloured faeces were observed in treated rats as compared to control.
When treated with 75 mg/kg body weight/day, Bluishgreen-
coloured faeces were observed in treated rats as compared to control.
Occasional ocular or nasal porphyrin discharge, soft tools, respiratory congestion and slight alopecia were observed in treated rats which were in similar frequency with control.
Body weight: No effect on body weight of treated rats was observed as compared to control.
Food consumption: Although variations in food consumption were recorded for all groups, no marked or consistent differences were observed between control and treated groups.
During the mating period, the diets fed were prepared on the basis of the females' body weights and food consumption. Therefore, the amount of colouring consumed by male rats during this period was slightly lower than the required dosage level.
Test substance intake: During the weaning period, the pups were not prevented from eating the food provided for the dams; the consumption of colouring by female rats during the gestation and weaning periods therefore appeared to be slightly higher than anticipated.
Reproductive function: estrous cycle
F2 parentes: No effect on live and dead foetuses, resorptions and corpora lutea of treated rats were observed as compared to control.
Reproductive function: sperm measures: No data available
Reproductive performance
F1 parentes: When treated with 2.5 and 250 mg/kg body weight/day, significant decrase in fertility indices of treated female rats were observed as compared to control.
Because of the lower fertility indices observed at the 2.5 mg/kg bw/day dosage group in the F 2 litters, the study was extended to the F3b litter.
No effect on F0 and F3 generation fertility indices were observed as compared to control.
The changes were considered to represent variation rather than compound-related effects.
Organ weights No effect on organ weight of treated rats were oberved as compared to control.
Gross pathology: No effect on Gross pathology of treated rats were oberved as compared to control.
Histopathology: No data available
other findings
Food efficiency: No effect on food efficiency of treated rast were observed as compared to control.
Effect levels (P0)
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effect on survival, body weights, food consumption, food efficiency, Estrous cyclicity, Foetal development, organ weights and gross pathology
- Remarks on result:
- other: No toxic effects on reproductive parameters were observed
Target system / organ toxicity (P0)
- Critical effects observed:
- not specified
- System:
- other: not specified
- Organ:
- not specified
- Treatment related:
- not specified
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
Results: F1 generation
General toxicity (F1)
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Slightly bluishcoloured fur was noted inpups given 250mg/kg/day and bluish-greencoloured faeces were produced by pups given 75 or 250 mg/kg/day. No other changes considered to be compound related were noted in general behaviour or appearance. Changes noted occasionally in control or treated pups included alopecia, respiratory congestion and runting.
- Dermal irritation (if dermal study):
- not specified
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The average body weights of the pups were similar in control and treated groups. Although the average number of pups born per litter varied among the groups, no dose relationship was apparent.
- Food consumption and compound intake (if feeding study):
- not specified
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not specified
- Urinalysis findings:
- not specified
- Sexual maturation:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- There were no compound-related effects on organ weights.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No compound-related gross pathological change in any of the rats that were killed and necropsied
- Histopathological findings:
- no effects observed
- Description (incidence and severity):
- The incidence of skeletal abnormalities in pups dying during the study and examined was very low in all the groups. Skeletal variations that tend to occur in rat pups (accessory ribs, hypoplastic skull bones, absent sternebrae and absent, bipartite, hemi- or misshapen centra) were seen in this study but were considered to be frequently occurring variations that were not biologically significant. FD & C Blue No. 2 had no effect on the number of pups with visceral abnormalities in any group.
- Other effects:
- not specified
Developmental neurotoxicity (F1)
- Behaviour (functional findings):
- not specified
Developmental immunotoxicity (F1)
- Developmental immunotoxicity:
- not specified
Details on results (F1)
Clinical signs: When treated with 250 mg/kg body weight/day, Slightly bluish-coloured fur and Bluishgreen- coloured faeces were observed in treated pups as compared to control.
When treated with 75 mg/kg body weight/day, Bluishgreen-
coloured faeces were observed in treated pups as compared to control.
Respiratory congestion and runting were observed in treated rats which were in similar frequency with control.
Body weight:
F1 and F2 pups:
No effect on body weight of treated pups was observed as compared to control.
Food consumption:
F1 and F2 pups:
No effect on food consumption of treated pups was observed as compared to control.
Organ weights: No effect on organ weight of treated pups were oberved as compared to control.
Gross pathology: No effect on Gross pathology of treated pups were oberved as compared to control.
Histopathology: No gross anatomical abnormalities were observed in fetouse of treated rats as compared to control.
Effect levels (F1)
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effect on survival, body weights, food consumption, food efficiency, Estrous cyclicity, Foetal development, organ weights, Gross pathology and Histopathology
- Remarks on result:
- other: overall no developmental toxic effects were observed
Target system / organ toxicity (F1)
- Critical effects observed:
- not specified
- System:
- other: not specified
- Treatment related:
- not specified
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
Results: F2 generation
General toxicity (F2)
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Slightly bluishcoloured fur was noted inpups given 250mg/kg/day and bluish-greencoloured faeces were produced by pups given 75 or 250 mg/kg/day. No other changes considered to be compound related were noted in general behaviour or appearance. Changes noted occasionally in control or treated pups included alopecia, respiratory congestion and runting.
- Dermal irritation (if dermal study):
- not specified
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The average body weights of the pups were similar in control and treated groups. Although the average number of pups born per litter varied among the groups, no dose relationship was apparent.
- Food consumption and compound intake (if feeding study):
- not specified
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not specified
- Urinalysis findings:
- not specified
- Sexual maturation:
- not specified
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- There were no compound-related effects on organ weights.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No compound-related gross pathological change in any of the rats that were killed and necropsied
- Histopathological findings:
- not specified
- Description (incidence and severity):
- The incidence of skeletal abnormalities in pups dying during the study and examined was very low in all the groups. Skeletal variations that tend to occur in rat pups (accessory ribs, hypoplastic skull bones, absent sternebrae and absent, bipartite, hemi- or misshapen centra) were seen in this study but were considered to be frequently occurring variations that were not biologically significant. FD & C Blue No. 2 had no effect on the number of pups with visceral abnormalities in any group.
- Other effects:
- not specified
Developmental neurotoxicity (F2)
- Behaviour (functional findings):
- not specified
Developmental immunotoxicity (F2)
- Developmental immunotoxicity:
- not specified
Effect levels (F2)
- Dose descriptor:
- NOAEL
- Generation:
- F2
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effect on survival, body weights, food consumption, food efficiency, Estrous cyclicity, Foetal development, organ weights, Gross pathology and Histopathology
- Remarks on result:
- other: overall no developmental toxic effects was observed
Target system / organ toxicity (F2)
- Critical effects observed:
- not specified
- System:
- other: not specified
- Organ:
- not specified
- Treatment related:
- not specified
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
Overall reproductive toxicity
- Reproductive effects observed:
- not specified
- Treatment related:
- not specified
- Relation to other toxic effects:
- not specified
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
Any other information on results incl. tables
Summary of reproduction and lactation data for F1 litters
Dosage group (mg/kg/day) |
Fertility indexɫ |
Gestation survival index‡ |
Survival index§ |
|||||||||
Females |
Males |
4-day |
14-day |
21 –day |
||||||||
|
F1a litters |
|||||||||||
0 |
14/20 |
70 |
8/10 |
80 |
184/184 |
100 |
182/184 |
99 |
137/137 |
100 |
137/137 |
100 |
0 |
18/20 |
90 |
10/10 |
100 |
247/249 |
99 |
243/247 |
98 |
179/179 |
100 |
168/179 |
94 |
2.5 |
20/20 |
100 |
10/10 |
100 |
257/264 |
97 |
256/257 |
100 |
192/193 |
99 |
192/193 |
99 |
25 |
18/20 |
90 |
10/10 |
100 |
245/248 |
99 |
242/245 |
99 |
180/180 |
100 |
176/180 |
98 |
75 |
17/20 |
85 |
10/10 |
100 |
217/218 |
100 |
217/217 |
100 |
164/164 |
100 |
164/164 |
100 |
250 |
17/20 |
85 |
10/10 |
100 |
212/214 |
99 |
191/212 |
90 |
152/152 |
100 |
17152/152 |
100 |
|
F1b litters |
|||||||||||
0 |
13/20 |
65 |
7/10 |
70 |
157/159 |
99 |
157/157 |
100 |
126/126 |
100 |
126/126 |
100 |
0 |
19/19 |
100 |
10/10 |
100 |
242/244 |
99 |
240/242 |
99 |
190/190 |
100 |
189/190 |
99 |
2.5 |
18/19 |
95 |
10/10 |
100 |
228/238 |
96 |
225/228 |
99 |
178/178 |
100 |
178/178 |
100 |
25 |
19/20 |
95 |
10/10 |
100 |
257/263 |
98 |
252/257 |
98 |
187/188 |
99 |
187/188 |
99 |
75 |
18/20 |
90 |
10/10 |
100 |
220/222 |
99 |
218/220 |
99 |
172/172 |
100 |
172/172 |
100 |
250 |
15/20 |
75 |
8/10 |
80 |
182/187 |
97 |
181/182 |
99 |
143/143 |
100 |
143/143 |
100 |
ɫ Figures tabulated are (F) no. of pregnancies/no, of matings, followed by the index, and (M) no. of fertile males/total no. of males mated, followed by the index.
‡ No. of newborn pups alive/total no. born, followed by the index.
§The 4-day, 14-day and 21-day survival indices are preceded, respectively, by no. of pups surviving to day 4/no. liveborn, no. of pups surviving to day 14/no. retained at day 4, and no. of pups surviving 'to day 21/no. retained at day 4.
Summary of reproduction and lactation data for F2 litters
Dosage group (mg/kg/day) |
Fertility indexɫ |
Gestation survival index‡ |
Survival index§ |
|||||||||
Females |
Males |
4-day |
14-day |
21 –day |
||||||||
|
F2a litters |
|||||||||||
0 |
16/20 |
80 |
9/10 |
90 |
184/184 |
100 |
184/184 |
100 |
156/158 |
99 |
153/158 |
97 |
0 |
17/20 |
85 |
9/10 |
90 |
194/199 |
97 |
190/194 |
98 |
163/164 |
99 |
158/164 |
96 |
2.5 |
10/20 |
50* |
7/10 |
70 |
100/100 |
100 |
100/100 |
100 |
91/91 |
100 |
90/91 |
99 |
25 |
10/20 |
50* |
6/10 |
60 |
109/109 |
100 |
109/109 |
100 |
97/98 |
99 |
94/98 |
96 |
75 |
17/20 |
85 |
9/10 |
90 |
177/178 |
99 |
177/177 |
100 |
155/158 |
98 |
146/158 |
92 |
250 |
13/20 |
65 |
8/10 |
80 |
177/123 |
95 |
117/117 |
100 |
109/110 |
99 |
106/110 |
96 |
|
F2b litters |
|||||||||||
0 |
18/20 |
90 |
10/10 |
100 |
211/222 |
95 |
209/211 |
99 |
161/161 |
100 |
161/161 |
100 |
0 |
16/20 |
80 |
9/10 |
90 |
178/179 |
99 |
178/178 |
100 |
147/147 |
100 |
147/147 |
100 |
2.5 |
8/20 |
40** |
5/10 |
50* |
83/84 |
99 |
79/83 |
95 |
65/66 |
98 |
65/66 |
98 |
25 |
12/20 |
60 |
8/10 |
80 |
129/130 |
99 |
128/129 |
99 |
104/105 |
99 |
104/105 |
99 |
75 |
16/20 |
80 |
9/10 |
90 |
197/203 |
97 |
196/197 |
99 |
153/153 |
100 |
153/153 |
100 |
250 |
15/20 |
75 |
8/10 |
80 |
135/138 |
98 |
134/135 |
99 |
114/114 |
100 |
114/114 |
100 |
|
F2c litters |
|||||||||||
0 |
13/20 |
65 |
9/10 |
90 |
53/53 |
100 |
52/53 |
98 |
44/45 |
98 |
44/45 |
98 |
0 |
16/20 |
80 |
9/9 |
100 |
81/82 |
99 |
78/81 |
96 |
61/61 |
100 |
61/61 |
100 |
2.5 |
8/20 |
40* |
5/10 |
50* |
31/31 |
100 |
29/31 |
94 |
24/27 |
89 |
24/27 |
89 |
25 |
8/20 |
40* |
6/10 |
60 |
33/33 |
100 |
33/33 |
100 |
28/28 |
100 |
28/28 |
100 |
75 |
11/20 |
55 |
9/10 |
90 |
84/92 |
91 |
83/84 |
99 |
70/70 |
100 |
69/70 |
99 |
250 |
10/20 |
50 6 |
6/10 |
60 |
47/47 |
100 |
46/47 |
98 |
42/42 |
100 |
42/42 |
100 |
ɫ Figures tabulated are (F) no. of pregnancies/no, of matings, followed by the index, and (M) no. of fertile males/total no. of males mated. followed by the index.
‡ No. of newborn pups alive/total no. born, followed by the index.
§The 4-day, 14-day and 21-day survival indices are preceded, respectively, by no. of pups surviving to day 4/no. liveborn, no. of pups surviving to day 14/no. retained at day 4, and no. of pups surviving to day 21/no. retained at day 4.
Values marked with asterisks are significantly lower than those for the concurrent control groups: *P < 0.05; **P < 0.01.
Summary of reproduction and lactation data for F3 litters
Dosage group (mg/kg/day) |
Fertility indexɫ |
Gestation survival index‡ |
Survival index§ |
|||||||||
Females |
Males |
4-day |
14-day |
21 –day |
||||||||
|
F3a litters |
|||||||||||
0 |
12/20 |
60 |
9/10 |
90 |
114/114 |
100 |
112/114 |
98 |
105/106 |
99 |
102/106 |
96 |
0 |
9/20 |
45 |
6/10 |
60 |
90/91 |
99 |
90/90 |
100 |
79/79 |
100 |
78/79 |
99 |
2.5 |
14/20 |
70 |
8/10 |
80 |
144/150 |
96 |
143/144 |
99 |
114/124 |
92 |
113/124 |
91 |
25 |
11/20 |
55 |
7/10 |
70 |
108/110 |
98 |
106/108 |
98 |
87/97 |
90 |
86/97 |
89 |
75 |
11/20 |
55 |
7/10 |
70 |
95/96 |
99 |
94/95 |
99 |
88/88 |
100 |
86/88 |
98 |
250 |
14/20 |
70 |
8/10 |
80 |
153/153 |
100 |
153/153 |
100 |
132/132 |
100 |
131/132 |
99 |
|
F3b litters |
|||||||||||
0 |
14/20 |
70 |
8/10 |
80 |
116/119 |
97 |
104/116 |
90 |
94/95 |
99 |
94/95 |
99 |
2.5 |
15/20 |
75 |
8/10 |
80 |
137/142 |
96 |
136/137 |
99 |
121/121 |
100 |
120/121 |
99 |
25 |
15/20 |
75 |
9/10 |
90 |
140/141 |
99 |
139/140 |
99 |
125/126 |
99 |
124/126 |
98 |
75 |
11/20 |
55 |
7/10 |
70 |
101/104 |
97 |
101/101 |
100 |
88/89 |
99 |
88/89 |
99 |
250 |
15/20 |
75 |
8/10 |
80 |
146/146 |
100 |
145/146 |
99 |
127/128 |
99 |
127/128 |
99 |
ɫ Figures tabulated are (F) no. of pregnancies/no, of matings, followed by the index, and (M) no. of fertile males/total no. of males mated, followed by the index.
ɫ No. of newborn pups alive/total no. born, followed by the index.
§The 4-day, 14-day and 21-day survival indices are preceded, respectively, by no. of pups surviving to day 4/no. liveborn, no. of pups surviving to day 14/no. retained at day 4, and no. of pups surviving to day 21/no. retained at day 4.
Applicant's summary and conclusion
- Conclusions:
- NOAEL was considered to be 250 mg/kg body weight/day for F0, F1 and F2 generation, when CD male and female rats were treated with FD & C Blue NO. 2
- Executive summary:
In a three generation reproduction study, groups of ten males and twenty female Charles River CD rats were fed FD&C Blue No 2 at dietary levels providing intakes of 0, 2.5, 25, 75 and 250 mg/kg bw/day. Two females were placed in a male's cage for the entire mating period (15 days). Vaginal smears were performed daily until sperm or a copulatory plug was found; this was designated as day 0 of pregnancy. At the end of the mating period, each
female was placed on corn-cob bedding in an individual plastic shoe-box cage and remained there until it was remated. Each female was rested for a minimum of 10 days after lactation before being mated again. Each mating was with a different male from the same dosage group.
The control and treated F0 rats were maintained on their respective diets for 2 wk prior to the first mating period. They were then mated, when approximately 100 days old. The rats (F0) were mated twice. The pups (F1a) from the first mating were examined for external abnormalities and killed at the end of the 21-day lactation period. After the second mating, random selections were made of ten male and 20 female pups from the control and treated groups of the F1b litters to serve as the second generation (F1) parental rats. The remaining F1b pups were examined for external abnormalities, killed and discarded. The F0 parent rats were also killed at this time.
After weaning, the F1b pups selected for the second generation continued to be fed on their control or appropriately treated diet for 80 days and were then mated. The pups from the first mating (F2a) were examined for external abnormalities and killed at the end of the 21-day lactation period. From the second mating (F2b), selections were made of ten male and 20 female pups from the control and treated groups to serve as the third generation (F2) parental rats. The remaining pups (F2b) were examined for external abnormalities, killed and discarded. Following the third mating, one-half of the mated females were killed on day 19 of gestation and the uteri and ovaries were examined. The remaining females were allowed to deliver. After weaning, the F2c pups were examined for external abnormalities, killed and discarded. The F1 parent rats were then killed. Five male and five female rats from the control and each of the treated groups were necropsied and their tissues were collected for histopathology.
After weaning, the F2b pups selected for the third generation continued to be fed on their control or appropriately treated diet for 80 days and were then mated. Five male and five female pups (F3a) from the control and each of the treated groups were killed when 21 days old and necropsied, and tissues were collected for histopathology. The parent rats (F2) were then re-mated and the pups from the second mating (F3b) were examined for external abnormalities and killed at the end of the 21-day lactation period. The F 2 parent rats were then re-mated for the third mating (F3c). On day 19 of gestation, half of the dams from the control and
treated groups were killed by chloroform anesthesia. Uteri were examined for any abnormal conditions and the presence of live and dead foetuses and of resorptions were recorded. All ovaries were inspected and the corpora lutea were counted. Remaining F2 parent rats were then killed and discarded.
The rats were observed twice daily for changes in behaviour or appearance and for morbidity and mortality. Individual body weights and food consumption were recorded weekly. Specific parameters for the reproductive phase of this study included observations of fertility, litter size, numbers of male and female pups, viability of the newborn, survival of pups to weaning and growth of the pups.All stillborn offspring and any progeny that died during the study were examined either by skeletal clearing or by necropsy. Body weights of pups aged 4 days (before and after reduction of litters to ten pups) and aged 14 days were obtained by weighing as litters. The pups were weighed individually at 21 days of age. The thyroid, adrenals, lung. heart, spleen, stomach, jejunum, ileum, colon, liver, kidneys, urinary bladder, testes or ovaries and uterus from five male and five female rats from the F1 and F3, generations of the control and 250-mg/kg/day groups were embedded in paraffin, sectioned, stained with haematoxylin and eosin and examined microscopically.
Fur and faeces were bluish-coloured in 75 mg/kg bw/day and 250 mg/kg bw/day groups. Gestation, viability and lactation indices of all litters from exposed animals did not differ from controls. Fertility indices were statistically significantly lower for F2female rats in the 2.5 and 25 mg/kg bw/day groups only and consequently considered as not being dose-related. Fertility indices were also reduced in F2b and F2cgroup of male rats. No statistically significant changes in the fertility index were observed in the F3generation. As effects on fertility indices in the F2generation were not dose-related and effects were not identified in the F1and F3generation this effect was not considered to be compound-related. Examination of the ovaries and uteri of F1dams killed on gestation day (GD) 19 revealed no gross anatomical abnormalities. No unusual changes were observed in the stillborn pups or in pups that died during the study. No compound-related gross or microscopic pathological lesions were noted in any of the F1or F3arats that were sacrificed and necropsied. Finally, no compound-related organ-weight variations were recorded in the F1rats. ThereforeNOAEL was considered to be250 mg /kg bw/day for FD & C BLUE NO. 2 (860-22-0) in Charles RiverCD male and female rats by oral administration (feed) in 3 generation study.(F0,F1 andF2 generation)
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