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Diss Factsheets

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun 1977 - Dec 1979
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Remarks:
Some phases of the study were conducted pre-GLP with no negative affection of the validity and integrity of the study.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Hilton-Davis Chemical Co. Div. of Sterling Drug Inc., Cincinnati, Ohio, USA
- Lot No.of test material: AA 2492
- Purity: not less than 90% pure color

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixed weekly with ground basal laboratory diet on a weight/weight basis to provide appropriate dosage levels
Species:
rat
Strain:
other: Charles River CD
Details on species / strain selection:
The Charles River derived rat has been used extensively to evaluate the toxicity and/or carcinogenicity of new chemicals; considerable control data have been accumulated for this strain of rat.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories,Wilmington, MA, USA
- Age at study initiation: weanlings
- Weight at study initiation: 108-155 g (males); 98-141 g (females); F1 generation started exposure at weaning
- Housing: individually (except during mating and lactation period); mating: in pairs
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approximately two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 °C
- Humidity (%): 40 - 60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Other: For F1-generation a maximum of two rats per sex from each litter were selected.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Diets were blended in a twin-shell blender.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 20-21 °C and a humidity range aof 40-60%.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability were analysed in the prepared diets before study initiation, weekly during the first 13 weeks of study and then monthly thereafter.
Duration of treatment / exposure:
30 months (plus in-utero phase) (with 10 animals per sex and dose for interim sacrifice after 12 months)
Frequency of treatment:
daily
Dose / conc.:
0.1 other: % (w/w)
Remarks:
equivalent to 50 mg/kg bw for males and 62 mg/kg bw for females (calculated, actual ingested).
Dose / conc.:
1 other: % (w/w)
Remarks:
equivalent to 514 mg/kg bw for males and 631 mg/kg bw for females (calculated, actual ingested).
Dose / conc.:
2 other: % (w/w)
Remarks:
equivalent to 1072 mg/kg bw for males and 1319 mg/kg bw for females (calculated, actual ingested).
No. of animals per sex per dose:
F0: 60 for treated groups and 120 for control group until week 11. After that large control group was split into two groups of 60 rats/sex
F1: 70
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: indicated to be based on existing studies
Two identical control groups were used to account for random biological variation.

F0 rats were mated to produce the F1 litters. Females were vaginally smeared daily until sperm or a copulatory plug was observed (= gestation day 0). The day all pups of a litter were delivered was defined as lactation day 0. F1 pups were counted, sexed and weighed. F1 males and Fa females were randomly selected and assigned to control or treatment group.
Positive control:
no positive control included.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were morbidity, mortality and overt signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly. In addition, F0 females weighed on gestation day 0, 6, 15, and 21 and on lactation day 0, 4, 14, and 21. Individual body weights were recorded weekly through the first 14 weeks, biweekly for week 16-26 and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Determined weekly through the first 14 weeks, biweekly for week 16-26 and monthly thereafter
- Compound consumption were calculated from the diet consumption measurement

FOOD EFFICIENCY:
No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: After the selection of the F1 rats was completed, and at 3, 6, 12, 18 and 24 months of study and prior to study termination weeks 111 (females) and 116 (males).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 months and before termination weeks 111 (females) and 116 (males)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes / No / No data
- How many animals: 10 per sex and dose group
- Parameters checked: haemoglobin, haematocrit, total eryrhrocyte count, total and differential leucocyte counts. and erythrocyte morphology

CLINICAL CHEMISTRY: Yes
- Time schedule: At 3, 6, 12, 18 and 24 months and before termination weeks 111 (females) and 116 (males)
- Animals chosen: 20 per sex from control and 10 per sex from treated groups
- Parameters analyzed: blood urea nitrogen, fasting blood glucose, total serum protein and creatinine, serum glutamic pyruvic transaminase activity (alanine aminotransferase), serum glutamic oxaloacetic transaminase activity (aspartate aminotransferase), serum alkaline phosphatase activity

URINALYSIS: Yes
- Time schedule: At 3, 6, 12, 18 and 24 months and before termination weeks 111 (females) and 116 (males)
- Parameters analyzed: specific gravity, pH and presence of protein, glucose, ketones, bilirubin and occult blood, appearance (gross and microscopic), microscopic examination of the sediment, urobilinogen and nitrite

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

- Organ weights: brain, gonads, kidneys, liver, spleen, testes, ovary and thyroid/parathyroid

HISTOPATHOLOGY: Yes (all animals trom the two control groups and from the high-dose (5.0%) group). Further groups were analysed in case of findings at the high dose groups.
Organs examined: adrenals (two), aorta (abdominal), bone and marrow (femur), blood smear (if anemia, enlarged thymus, lymphadenopathy, or hepatosplenomegaly was present), brain (three sections: frontal cortex and basal ganglia. parietal cortex and thalamus. and cerebellum and pons), oesphagus, eye, heart (with coronar vesels), intestine (caecum, colon, duodenum and ileum), kidneys (two), liver (two sections), lung and mainstem bronchi, lymph nodes (mesenteric and mediastinal), mammary gland, nerve (sciatic), ovaries, pancreas, pituitary, prostate, salivar gland (mandibular), seminal vesicles, skeletal muscle (rectus femoris), skin, spinal cord (cervical), spleen, stomach, testes with epididymides (two), thymus, thyroid with parathyraid, trachea, urinary bladder, uterus, any tissue with gross changes of an uncertain nature together with an apparentdy normal section of thc same tissue, and any tissue masses or suspect tumours rogether with regional lymph nodes.
Other examinations:
Test diet analyses: Samples were taken of each batch of basal laboratory feed for IRDC analysis of the pesticide, aflatoxic and heavy metal content.
Test sample analyses: Periodically throughout the study, samples were collected and analyzed for volatiles, pure dye content and HPLC purity. In addition, the test substance was tested for aerobic plate count, mold and yeast count and for the absence of Salmonella and E. Coli.
Statistics:
All statistical analyses were done for each sex seperately with significant differences determined by the probability level p<0.01, two-tailed.
Body weights, food consumption, clinical studies, and absolute and relative organ weights were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance of differences.
The two control groups were compared against each other. If no significant differences were identified, controls were combined and compared against treated groups, otherwise each control group was compared against each treated individually.
Survival data and time to neoplastic lesions were analyzed by the methods described by Thomas, Breslow, and Gart.
For the histopathologically proved neoplastic lesion incidences the two control groups were compared against each other. If no significant differences were identified, control groups were combined and tested against the high dose group. Otherwise, each control group was compared against the high dose group seperately.
The number of neoplastic lesion-bearing animals were analyzed at study termination using the chi-square test criterion.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings noted in similar numbers for control and treated rats included rales, red material around the eyes, nose or anogenital region, redness around the eyes, white areas in the internal eyes, corneal opacities, dilated pupils, increased distance between the pupil and the cornea and palpable masses primarily in the abdominal, thoracic or anogenital regions.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Four parental rats died during the study, one female in the 0.1% group, one male and one female in the 1.0% group and one male in the 2.0% group. These deaths were not attributed to treatment. Based on the mortality of the high-dose females, all females were sacrificed at 111 weeks. The males were sacrificed at week 116 when the mid-dose group mortality reached 80%.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional statistically significant differences were considered unrelated to the compound. After week 90, mean body weights of the 2% females began a downward trend. No statistically significant differences between the control and 2% group mean body weights were found at the intervals tested, and the difference was not considered biologically significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was slightly greater for the males and females at the 2.0% dosage level than for the control males and females. These differences were statistically significant for the males between weeks 27 and 66 and for the females between weeks 27 and 38 and between weeks 79 and 90 and can in all probability be attributed in part to the non-nutrient value of the compound at this dosage level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Chorioretinal degeneration (seen at 3 and 18 months), chorioidal hypoplasia (at 6 months), and chorioretinal hypoplasia (at 12 months). Conjunctivitis, keratitis, cataract were the most frequent findings. All changes in ophthalmic findigs during the study period were considered related to age rather than to compound administration.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Leucocyte mean values at the 2.0% level were slightly and very slightly increased for males and females, respectively, when compared to the combined control groups 1 and 2. The slight increase noted for the males involved slight-to-moderate elevations (i.e. 16.40 to 21.37x10³/cmm) for five of the ten individual values. The increase in the mean was statistically significant. The very slight increase noted for the females is due primarily to a moderately elevated increase for only one rat (21.54x10³ cmm).
Review of the hematologic profiles obtained during the conduct of the study did not indicate any changes which could be attributed to the test article. The statistically significant differences noted occurred at random and were not considered of biological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings (for 3 and 6 months) included slightly elevated blood urea nitrogen and creatinine levels for one rat at the 1.0% dosage level and one rat at the 2.0% dosage level at 6 months of the study. Mean SGPT value for the low-dose females was slightly higher than control means, but the increase was not considered to be physiologically significant. Elevated BUN values (one low-dose and two mid-dose males), slightly elevated glucose values (four control, three low-dose, two mid-dose and one high-dose male(s), two control, one low-dose and two-mid-dose females). No dosage relationship or definite compound relationship was evident in these changes.
Review of the biochemical values obtained during the conduct of this study did not indicate any changes whi'ch could be attributed to the test article, Some statistically significant differences were noted for a few biochemical parameters at some intervals, however, no biological significance was interpreted because there were no trends or consistent differences between control and treated groups.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The color of the urine samples collected from the treated rats was noted as light blue or green to dark blue or green in color in all mid- and high-dose treated rats and most low-dose treated rats. No other consistens differences were noted between the control and treated groups. Review of the urinalyses did not indicate any other changes which could be attributed to the test substance.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no toxicological significant variations in organ weights. There were no morphological correlations for the several statistcally significant variations.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Blue green discoloration of the stomach, small intestine and large intestine was observed in many of the compound treated animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no lesions which could be attributed to the compound. The lesions observed were those of spontaneous disease and they were not unusual in type or incidence for rats of this strain. There were no hyperplastic or neoplastic lesions which could be attributed to the compound.
Other effects:
no effects observed
Description (incidence and severity):
Male and female fertility indices, gestation length, pup survival indices through weaning, number of live and stillborn pups and pup weights at birth and weaning were similar for control and treated rats.
Rats at all three dosage levels had changes in the color of the hair, exposed skin and feces. Rats at 0.1% dosage level experienced light blue hair and skin returning to normal by week 4 and week 28, respectively, of the long-term phase of the study. Feces from these rats appeared green when sectioned and smeared. Rats fed at 1.0% and 2.0% had a blue hair and skin color at the initiation of the study which continued to termination. Feces collected from these rats appeared blue in color when sectioned and smeared.
Details on results:
However, some of the statistical analyses of life table curves and of the adjusted incidence were significant at the 0.01 level for total tumors and for all benign tumors in females. These analyses indicate that the tumors of high dose females occurred earlier in life than in controls although there was no increase in total numbers of tumors. There were no toxicologically significant analyses for any individual tumor type.
Relevance of carcinogenic effects / potential:
In summary, the analyses did not indicate an increase in the unadjusted incidence of tumors. In fact, a negative trend occurred.
Analyses of the adjusted incidence for total tumors in the female suggested that the neoplasms occurred earlier in the high dose animals.
The compound was not considered to be oncogenic for any of the individual tumor types and the statistical analyses of adjusted incidence for total tumors in the female were equivocal.
Dose descriptor:
NOAEL
Effect level:
>= 2 other: % in the diet (> 7000 mg/kg bw)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of carcinogenic effects at the highest tested dose
Dose descriptor:
NOEL
Effect level:
1 other: % (w/w) in the diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: adverse effects on body weights starting week 102 at 2%. No effects for males
Critical effects observed:
no

Results of tumor incidences in rats:

Neoplastic summary table

 

Control 1

0.1%

1.0%

2.0%

All tumors, female

84%

77%

77%

80%

All benign tumors, female

79%

71%

74%

70%

All tumors, male

57%

37%

46%

53%

All benign tumors, male

50%

24%

31%

47%

Moratlity data:

 

Survival/number initiated*

Treatment

Male**

Female***

Control 1

18/60

28/60

Control 2

13/60

24/60

0.1%

12/60

23/60

1.0%

12/60

28/60

2.0%

18/60

10/60

 

*Less interim sacrifice

**Survival at week 116

***Survival at week 116

Food consumption data:

Average basal food consumption (Control 1 & 2) and average food with compound consumption (g/rat/day)

Average compound consumption (mg/kg/day)

Treatment

Male*

Female**

Male*

Female**

Control 1

27.3

20.7

-

-

Control 2

27.1

21.0

-

-

0.1%

26.5

21.2

50

62

1.0%

28.0

21.7

514

632

2.0%

28.1

22.2

1073

1318

* week 114

** week 110

Conclusions:
The feeding of the test substance to rats at dose levels of 0.1, 1.0, and 2.0% beginning with the parents prior to mating and the F1 generation for 26 months, produced slight body weight decreases for treated rats. Although changes were seen in color of hair, skin and feces of rats, these signs were not considered toxicologically significant because they were due directly to the color of the dye. No treatment related effects were seen in behavior, survival, food consumption, hematologic, biochemical or urinalyses parameters. No compound related changes were noted in macroscopic or microscopic pathologic examinations. On visual inspection of the neoplastic incidence data, the test substance did not appear to be oncogenic. Statistical analyses did not indicate an increase in the unadjusted incidence of tumors. In fact, a negative trend was occurred. Analyses of the adjusted incidence for total tumors in the females suggested that the neoplasms occurred earlier in the high dose animals. The compound was not considered to be oncogenic for any of the individual tumor types and the statistical analyses of adjusted incidence for total tumors in the female were equivocal. While there is a slight possibility on statistical grounds that the test substance could be oncogenic, the only evidence is earlier appearance of tumors in the female and no definite relationship has been established in this study. The no-effect level of the test substance in this study was considered to be 2.0%.
Executive summary:

The test substance was offered at dosage levels of 0.1, 1.0, and 2.0% throughout a long-term feeding study in rats exposed in utero (F1 generation). Sixty animals/sex F0 rats and 70 animals/sex F1 rats were initiated at each dosage level and in each of two control groups. During the in utero portion of the study, the corresponding parental (F0) generation rats were fed identical dosage levels of the test substance. Rats were observed twice daily for signs of overt toxicity, moribundity and mortality. Detailed observations were recorded weekly. Individual body weights and food consumption values were recorded weekly during the F0 phase. These values were recorded weekly for the first 14 weeks, biweekly for the next 12 weeks, and once every 4 weeks thereafter during the F1 phase. Ophthalmoscopic examination of all rats were conducted at 3, 6, 13, 18, and 24 months and prior to termination at 25 and 26 months. Blood and urine samples were obtained from 10 males and 1o females randomly selected from each group at 3, 6, 12, 18, and 24 months and prior to termination at weeks 116 (males) and 111 (females) for hematologic and biochemical studies and urinalyses. Blue coloration of the hair and exposed skin was apparent for treated rats at the 1.0% and 2.0% levels throughout the study. Sectioned feces were green in color for rats at the 0.1% dosage level and blue in color for rats at the 1.0% and 2.0% dosage levels. Four parental rats died during the in utero phase. One hundred fifty seven (157) control and 257 treated F1 rats died or were sacrificed in extremis during 116 weeks of study for the males, or 111 weeks of study for the females. No specific compound-related pattern of mortality developed. Other than an indication of a partially-compensated anemia at 24 months (of unknown significance) and blue or green urine coloration, review of the clinical pathology data did not indicate any differences between the treated and control animals that were possibly attributable to the test article.

No compound-related gross or microscopic changes were observed. The inflammatory, degenerative and/or proliferative changes described were regarded of spontaneous nature, not uncommon among rats of this age and strain and unrelated to compound administration. On visual inspection of the neoplastic incidence data, the test substance did not appear to be oncogenic. Statistical analyses did not indicate an increase in the unadjusted incidence of tumors. In fact, a negative trend occurred. Analyses of the neoplams occurred earliert in the high dose animals. The compound was not considered to be oncogenic for any of the individual tumor types and the statistical analyses of adjusted incidence for total tumors in the female were equivocal. While there is a slight possibility on statistical grounds that the test sustance could be oncogenic, the only evidence is earliert appearance of tumors in the female and no definite relationship has been established in this study. The no-effect level of the test substance in this study was considered to be 2.0%.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Apr 1978 - Apr 1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Remarks:
Some phases of the study were conducted pre-GLP with no negative affection of the validity and integrity of the study.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Hilton-Davis Chemical Co. Div. of Sterling Drug Inc., Cincinnati, Ohio, USA
- Lot No.of test material: AA 2492
- Purity: not less than 90% pure color

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixed weekly with ground basal laboratory diet on a weight/weight basis to provide appropriate dosage levels
Species:
mouse
Strain:
CD-1
Details on species / strain selection:
The Charles River CD-1 mouse has been used extensively to evaluate the toxicity and/or carcinogenicity of new chemicals; considerable control data has been accumulated for this strain of mouse.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laborataries, Portage, MI., USA
- Age at study initiation: 4-weeks old
- Weight at study initiation: 24-31 g (males); 20-26 g (females)
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 °C
- Humidity (%): 40 - 60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE PERIOD: FROM 26 April 1978 TO: 28-30 April 1980
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Diets were blended in a twin-shell blender.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 20-21 °C and a humidity range aof 40-60%.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability were analysed in the prepared diets before study initiation, weekly during the first 13 weeks of study and then monthly thereafter.
Duration of treatment / exposure:
24 months
Frequency of treatment:
daily
Dose / conc.:
661 mg/kg bw/day (actual dose received)
Remarks:
Calculated for males. Equivalent to 0.5% (w/w)
Dose / conc.:
2 064 mg/kg bw/day (actual dose received)
Remarks:
Calculated for males. Equivalent to 1.5% (w/w).
Dose / conc.:
7 354 mg/kg bw/day (actual dose received)
Remarks:
Calculated for males. Equivalent to 5% (w/w).
Dose / conc.:
819 mg/kg bw/day (actual dose received)
Remarks:
Calculated for females. Equivalent to 0.5% (w/w).
Dose / conc.:
2 562 mg/kg bw/day (actual dose received)
Remarks:
Calculated for females. Equivalent to 1.5% (w/w).
Dose / conc.:
8 966 mg/kg bw/day (actual dose received)
Remarks:
Calculated for females. Equivalent to 5% (w/w).
No. of animals per sex per dose:
60
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: indicated to be based on existing studies

Two identical control groups were used to account for random biological variation.
Positive control:
no positive control included
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times daily (Mon-Fr) twice daily (weekends and holidays)
- Cage side observations were morbidity, mortality and overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through the first 14 weeks, biweekly for week 16-26 and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Determined weekly through the first 14 weeks, biweekly for week 16-26 and monthly thereafter
- Compound consumption were calculated from the diet consumption measurement

FOOD EFFICIENCY:
No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 months
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10 per sex and dose group
- Parameters checked: haemoglobin, haematocrit, total eryrhrocyte count, total and differential leucocyte counts

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes

- Organ weights: brain, thorax, and abdominal cavities

HISTOPATHOLOGY: Yes (all animals trom the two control groups and from the high-dose (5.0%) group). Further groups were analysed in case of findings at the high dose groups.
Organs examined: adrenal (two), aorta (abdominal), bone and marrow (femur), blood smear, brain (three sections: frontal cortex and basal ganglia. parietal cortex and thalamus. and cerebellum and pons), oesphagus. eye, (two. with optic nerve), gallbladder, gonads (ovaries (2), testes with epididymis (2)), heart (with coronar vesels), intestine (caecum, colon, duodenum and ileum), kidneys (two), liver (2 sections), lung and mainstem bronchi, lymph nodes (mesenteric and mediastinal), mammary gland, mandibular salivary gland, nerve (sciatic), pancreas, pituitary, prostate, seminal vesicles, skeletal muscle (rectus femoris), skin, spinal cord (cervical), spleen, stomach, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, any tissue with gross changes of an uncertain nature together with an apparently normal section of the same tissue, and any tissue masses or suspected tumours together with regional lymph nodes.
Other examinations:
Test diet analyses: Samples were taken of each batch of basal laboratory feed for IRDC analysis of the pesticide, aflatoxic and heavy metal content.
Test sample analyses: Periodically throughout the study, samples were collected and analyzed for volatiles, pure dye content and HPLC purity. In addition, the test substance was tested for aerobic plate count, mold and yeast count and for the absence of Salmonella and E. Coli.
Statistics:
All statistical analyses were done for each sex seperately with significant differences determined by the probability level p<0.01, two-tailed.
Body weights, food consumption, and clinical studies were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) as described by Steel and Torries using Dunnett's multiple comparison tables to judge significance of differences.
The two control groups were compared against each other. If no significant differences were identified, controls were combined and compared against treated groups, otherwise each control group was compared against each treated individually.
Survival data and time to neoplastic lesions were analyzed by the methods described by Thomas, Breslow, and Gart.
For the histopathologically proved neoplastic lesion incidences the two control groups were compared against each other. If no significant differences were identified, control groups were combined and tested against the high dose group. Otherwise, each control group was compared against the high dose group seperately.
The number of neoplastic lesion-bearing animals were analyzed at study termination using the chi-square test criterion.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings noted in similar low numbers of treated and control mice included tremors, labored breathing, localized hair loss, disrended abdomen, masses in the abdominal, inguinal and/or anogenital region, pale exposed skin and ocular changes (pale exes, excessive lacrimation, red and/or swollen eyelids, corneal opacity, dilated pupils, and white areas in the internal eye.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 104 weeks of study, the mean body weight values indicated an apparent compound-related decrease for the treated males and females.
Males: At 1.5% and 5.0%, significance at most intervals analyszed and occasional significance (0.5% dose level) during the first 14 weeks.
Females: Body weight decreases exhibited significance (primarily seen when values were compared to Control 2 or combined controls) at most intervals analyzed throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings noted during the study for a few control and treated mice included slight to marked decreases in hemoglobin, hematocrit and total erythrocytes and/or slight to marked increases in total leucocytes.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The hair color and exposed skin of the treated mice were blue. Feces of the treated mice appeared blue when sectioned and smeared. No other changes considered to be related to the test substance were seen.
Details on results:
There was no indication that the substance was carcinogenic in mice. With one exception (hemangioma, splee, female), there were no statistical analyses which were significant at the 0.01 level. This included analysis of all tumors, benign tumors, malignant tumors and all neoplasms with two or more observations in the high dose group. The actual incidence was 0%, 2%, 2%, and 5% in control, low dose, mid dose, and high dose groups, respectively. Hemangioma of the spleen is a common spontaneous tumor for this species and strain. It was unusual that none was observed in the controls and the incidence in the high dose animals was considered to be spurious. The single statistically significant value was not toxicologically significant and the compound was not oncogenic for hemangioma of the spleen.
The slight decrease in body weights after 24 months was considered to be related to the reduced nutritional value of the diet. The tested doses were extremely high.
Dose descriptor:
NOAEL
Effect level:
>= 5 other: % in the diet (> 7000 mg/kg bw)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of carcinogenic effects at the highest tested dose.
Critical effects observed:
no

Results of neoplastic findings in mice treated with the test substance:

Neoplastic summary table

 

Control 1

Control 2

0.5%

1.5%

5.0%

M

F

M

F

M

F

M

F

M

F

Number Animals Examined Microscopically

60

60

60

60

49

57

47

59

60

60

Total tumor bearing animals

20

28

17

29

11

23

10

21

18

23

Total number of tumors

21

33

18

40

12

23

10

22

19

29

Total benign tumor bearing animals

7

12

4

18

3

10

3

11

5

11

Total number of benign tumors

7

15

5

24

3

10

3

12

5

17

Total malignant tumor bearing animals

14

17

13

15

9

13

7

10

14

12

Total number of malignant tumors

14

18

13

16

9

13

7

11

14

14
Conclusions:
No treatment related effects were seen in behavior, survival, food consumption or hematological parameters. No compound related changes were noted in macroscopic or microscopic pathologic examinations. The test subtance was not found to be oncogenic in this study under the conditions chosen. The No-Effect Level is condidered to be 5%.
Executive summary:

The test substance was offered in the diet to Charles River CD-1 mice at dose levels of 0.5, 1.5, and 5.0% in a 24 -month long-term feeding study. Sixty mice/sex were randomly assigned to each of two control groups and each of three treatment groups. The mice were observed daily for signs of toxicity, moribundity and mortality. Detailed observations were recorded weekly. Individual body weights and food consumption values were recorded weekly during weeks 1 through 14, biweekly weeks 16 through 26, and once every 4 weeks thereafter. Hematologic studies were condcuted at 3, 6, 12, 18 and 24 months of study. The hair color and exposed skin of the treated mice were blue. The feces of treated mice appeared blue when sectioned and smeared. No other changes in general behavior and appearance or pharmatoxic signs, considered to be related to compound, were seen. Slightly lower mean body weight values were noted for the treated groups when compared to the controls. Food consumption values were similar for control and treated mice. All other parameters were within the expected range for this species at this age. There were no macroscopic or microscopic changes which could be attribited to the test compound. Statistical analysis indicated that the test compound was not oncogenic for animals of this species and strain under the conditions of this study.

Data source

Reference
Reference Type:
publication
Title:
Lifetime Toxicity/Carcinogenicity Studies of FD&C Blue no. 1 (Brilliant Blue FCF) in Rats and Mice
Author:
Borzelleca, J.F., Depukat, K., Hallagan, J.B.
Year:
1990
Bibliographic source:
Fd. Chem. Toxicol. 28, 221-234 (1990)

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Without DNT and DIT modules
Deviations:
yes
Remarks:
2 months pretreatment of F0; lifelong exposure of F1 to include investigation of carcinogenic potential
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt
EC Number:
223-339-8
EC Name:
Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt
Cas Number:
3844-45-9
Molecular formula:
C37 H34 N2 Na2 O9 S3
IUPAC Name:
disodium 2-({4-[ethyl(3-sulfonatobenzyl)amino]phenyl}{4-[ethyl(3-sulfonatobenzyl)iminio]cyclohexa-2,5-dien-1-ylidene}methyl)benzenesulfonate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Name of test material (as cited in study report): FD&C Blue No. 1 (CAS 3844-45-9)
- Supplier: Hilton Davis Co., Cincinnati, Ohio, USA
- Analytical purity: 90%
- Impurities (identity): subsidiary colourings, volatile chlorides and suiphates, and uncombined intermnediates
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data, but storage conditions not considered critical
- Storage condition of test material: no data
- Other: The compound was certified by the US FDA

Test animals

Species:
rat
Strain:
other: Charles-River CD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories,Wilmington, MA, USA
- Age at study initiation: 38 days at beginning of F0-phase
- Weight at study initiation: F1 generation started exposure at weaning
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 °C
- Humidity (%): 40 - 60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Other: For F1-generation a maximum of two rats per sex from each litter were selected.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Diets were blended in a twin-shell blender.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 20-21 °C and a humidity range aof 40-60%.
Details on mating procedure:
Cohabitation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability were analysed in the prepared diets before study initiation, weekly during the first 13 weeks of study and then monthly thereafter.
Duration of treatment / exposure:
30 months F1 (plus in-utero phase) (with 10 animals per sex and dose for interim sacrifice after 12 months)
2 months (F0-generation)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0.1 other: % in the diet
Remarks:
males 50 ± 18 mg/kg bw; females 62 ± 18 mg/kg bw
Dose / conc.:
1 other: % in the diet
Remarks:
males 514 ± 179 mg/kg bw; females 631 ± 173 mg/kg bw
Dose / conc.:
2 other: % in the diet
Remarks:
males 1072 ± 381 mg/kg bw; females 1319 ± 345 mg/kg bw
No. of animals per sex per dose:
60 (F0)
70 (F1)
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on existing repeated-dose toxicity data

Examinations

Parental animals: Observations and examinations:
Female rats were weighed an gestation days 0, 4, 14 and 21
Clinical signs, mortality
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
no
Litter observations:
from each litter, at least one rat was exposed for liftime and examined for systemic toxicity similart to OECD guideline 453

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were morbidity, mortality and gross clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through die first 14 weeks, biweekly for week 16-26 and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) and mean daily diet consumption calculated as g food/kg body weight/day: Yes:
- Determined weekly through die first 14 weeks, biweekly for week 16-26 and monthly thereafter

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes

FOOD EFFICIENCY:
No data


OPHTHALMOSCOPIC EXAMINATION: Yes (after 3, 6, 12, 18 and 24 months af the chronic phase.)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 months and before termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes / No / No data
- How many animals: 10 per sex and dose group
- Parameters checked: haemoglobin, haematocrit, total eryrhrocyte count, total and differential leucocyte counts. and erythrocyte morphology

CLINICAL CHEMISTRY: Yes (3, 6, 12, 18 and 24 months and before termination)
aspartate aminatransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, fasting glucose, total protein and creatinine

URINALYSIS: Yes (3, 6, 12, 18 and 24 months and before termination)
specific gravity, pH and presence of protein, glucose, ketones, bilirubin and occult blood, appearance (gross and microscopic)

NEUROBEHAVIOURAL EXAMINATION: No
Postmortem examinations (parental animals):
no data
Postmortem examinations (offspring):
- Organ weights: brain, gonads, kidneys, liver, spleen and thyroid

HISTOPATHOLOGY: Yes (all animals trom the two control groups and from the high-dose (5.0%) group). Further groups were analysed in case of findings at the high dose groups.
Organs examined: adrenal (twa), aorta (abdominal), bone and marrow (femur). blood smear, brain (three sections: frontal cortex and basal ganglia. parietal cortex and thalamus. and cerebellum and pons), oesphagus. eye, (two. with optic nerve), heart (with coronar vesels), intestine (caecum, colon, duodenum and iLeum), kidneys (two). liver, lung and mainstem, subbronchi (lungs inflated with formalin), lymph nodes, mesenteric and mediastinal), mammary gland, (inguinal), nerve (sciatic). ovaries pancreas, pituitary, prostate, salivar gland (mandibular), seminal vesicles (two), skeietal muscle (biceps femoris), skin.spinal cord (cervical), spieen stomach, testes, with epididymides, tbymus. thyroid with parathyraid, trachea, urinary bladder (inflated with formalin), uterus, any tissue with gross changes of an uncertain nature together with an apparentdy normal section of thc same tissue, and any tissue masses or suspect tumours rogether with regional lymph nodes.
Statistics:
The variances of thde two groups were tested tor equality using the F test (Gull 1978). lt the variances were equal, a standard independent two-sample test was used to -determiüne equality ot mneans. lt the variances differed, Welch's t-test was used to determine equal ity of means, using the Smith-Satterthwaite correction for unequal variances (Gill 1978). All tests were conducted at the 1.0% two-sided risk level. More detailed information is provided in Fd. Chem. Toxicol. 28, 221-234 (1990)
Reproductive indices:
fertility, gestation lengths, parturition, lactation
Offspring viability indices:
Number of live and stillborn pups
Pup survival through weaing

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in die 0.1% group, one male and one female in the 1.0% group, and one male in die 2.0% group died.
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

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

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 072 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

Details on results (F1)

Examinations on offspring performed after 12 months and at the end of lifetime exposure.
The F1 generation developed normally during their lifetime. In females, exposure of 2% in the diet caused effects on body weight gain and survival rate after 90 - 102 weeks of treatment, males were not affected. F1 animals showed no histopathology findings in the gonads or other organs.

A specific target organ was not identified.

Effect levels (F1)

open allclose all
Dose descriptor:
NOEL
Generation:
F1
Effect level:
631 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: NOEL = 1% in the diet
Remarks:
No adverse effects on reproductive organs
Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 072 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Remarks on result:
other: NOEL = 2% in the diet
Remarks:
No adverse effects on reproductive organs

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
1 319 mg/kg bw/day (actual dose received)
System:
other: none identified
Organ:
other: none identified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion