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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1974 - 1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
48 instead of 50 animals/sexe/dose were used, which is not considered to have affected the study. In addition clinical chemistry parameters were not measured, also considered to have no effect on the study (see results for explanation).
GLP compliance:
no
Remarks:
Study was undertaken before implementation of GLP principles
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Physical appearance: lemon yellow crystals

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Scientific Agribusiness Consultants International, Braintree, Essex, UK
- Weight at study initiation: 40-60 g
- Housing: Group housing of 4 animals of same sex and same treatment group per cage (aluminium; 45x36x16; open mesh galvanised steel floors)
- Diet: Free access to powdered rodent diet (Spratt's Laboratory Animal Diet No.2 supplied by Spratts Patent Ltd, Barking, Essex, UK).
- Water: Free access to tap water.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 20
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15-20
- Photoperiod (hrs dark / hrs light): 10/14

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: test article was admixtured with the animal diet, which was given ad libitum.
Food pellets were ground to powder, the appropriate weight of the test article was added to the powdered diet and the mixture was blended using a commercial food mixer. Test-article-diet mixtures were prepared weekly.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analyses were carried out to verify concentrations in the diet test substance mixtures.
Duration of treatment / exposure:
Two-year study: 104-107 weeks
Interim I: 49-51 weeks
Interim II: 80-82 weeks
Frequency of treatment:
Continuous (food available ad libitum)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 500, 5000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Two-year study: 48
Interim I: 12
Interim II: 12
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
A literature search was conducted and based on these data, the dosages were choosen.
- Selection of animals for selected measurements:
Random selection.
Positive control:
Not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily for abnormalities of appearance and behaviour or signs of ill-health.

BODY WEIGHT: Yes
- Time schedule for examinations:
Two year study: starting at day 1, once more during first week of treatment, then once every fortnight during first year. Thereafter weighed at monthly intervals until week 99 (males) or 100 (females)
Interim I: starting at day 1, once more during first week of treatment, then once a fortnight until week 48 (males) or week 50 (females).
Interim II: starting at day 1, once more during first week of treatment, then once a fortnight until week 78.
All surviving rats were weighed before being killed.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: once a fortnight during the first year of treatment. Thereafter, food intakes were recorded at approx. monthly intervals up to week 99 (males) or week 100 (females). For the first interim study, water intake was recorded approx. once a fortnight for the first interim study up to week 46, and for the second interim study up to week 78.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 14, 26 and 54, and at necropsy
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 12 male and 12 female rats from groups 0, 500 and 5000 ppm
- Parameters checked: Total erythrocyte count/ total leucocyte count/ differential leucocyte count/ haemoglobin concentration/ packed cell volume/ reticulocyte count

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: 8 weeks after start, thereafter with two-monthly intervals. Urine was collected from all surviving rats (if possible) before sacrifice.
- Metabolism cages used for collection of urine: Yes
- Parameters: volume, specific gravity (as measured by refractive index), semi-quantitative analysis of pH, glucose, blood, bilirubin, ketones and protein using reagent strips or content of cells.
Three methods:
(1) Six hour urine samples: Urine collected over a period of 6 hours after normal overnight feeding and drinking.
(2) two hour urine samples: Urine collected for 2 hours after oral water load (25 ml/kg)
(3) Eighteen hour urine samples: Urine collected for 6 hours commencing 18 hours after oral water load (25 ml/ kg)

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were fasted overnight prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated. Animals that died during the study and animals in extremis that were killed during the study, were examined.
- Dose groups that were examined: all
- Organs weighed:
Adrenals, brain, caecum, gonads, heart, kidneys, liver, pituitary gland, small intestine, spleen, stomach, thyroid gland.
- Furthermore, tissues were collected and stored in formalin according to OECD guidelines.

HISTOPATHOLOGY: Yes
A microscopic examination was made of all the tissue samples.
Statistics:
Comparisons were done using either a two-sided least significant difference test, or a two-sided pooled two sample t test, or a two-sided two sample t test, or a Mann-Whitney U test, depending on the degree of normality and homoscedasticity (i.e. the homogeneity of the error variances) of the data. Data were considered significant with p<0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Description (incidence and severity):
not required
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Description (incidence and severity):
no adverse effects were observed on weight, macroscopically or microscopically of/on organs nor on urinalysis, therefore adverse effects on clinical chemistry are not expected
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Description (incidence and severity):
not required
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Findings were generally similar in both the control and treated groups. The percentage mortality of the control rats for both sexes was higher that that of the three treatment groups at the end of the study. Female rats showed a statistically significant dose related decrease in mortality rate for the cumulative mortality levels for each group in every week. However, this was not statistically significant in the heterogeneity of death rates between the groups. Survival in each group is no less than 50% at 24 months for all groups, except for the control group males with a survival just below this value, 48%.

BODY WEIGHT AND WEIGHT GAIN
Body weight of male and female rats in the control and treatment groups were similar throughout the course of the 2-y study. Female rats given 5000 ppm had a slightly lower mean body weight compared to control rats during the latter of the study, resulting in statistically significance during some weeks, 41-49 and 57-61 and week 92, but the weights were all within 8% of the control values for these periods. This is not considered to be toxicologically relevant.
Interim I and II study: Comparable trends

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption differed during the study, sometimes being less compared to controls, sometimes being more compared to controls. In some weeks this was statistically significant, but these differences were not considered to be related to treatment.
Interim I and II study: comparable trends.

The calculated intake (mg/kg bw/d) over the whole study period expressed for m/f:
2-y study; Interim I; Interim II;
50 ppm 4.4/6.2; 5.4/6.8; 4.7/6.8
500 ppm 45.0/62.5; 51.8/69.8; 46.8/63.8
5000 ppm 450.7/630.1; 519.5/692.5; 461.8/672.5

WATER CONSUMPTION
The pattern of water consumption by male and female rats in the control and 50 and 500 ppm treatement groups was similar throughout the study. Male and female rats administered 5000 ppm drank more than control animals during the first nine months of the study (up to twice the volume of the control animals, strong week-to-week variation). During the last year of the study water quantities were comparable between 5000 ppm group and controls. While the difference seen in the first year of the study must be attributed to treament with sodium ferrocyanide there is no indication that this is an adverse effect (see also Urinalysis results).
Interim I and II study: Comparable trends, however, the high water intakes seen in rats given 5000 ppm substance during the earlier part of the 2-y study were not repeated in the corresponding periods of the interim studies.

HAEMATOLOGY
Changes observed in treated animals were not dose-related or common to both sexes on any single occasion, and were not found consistently on different occasions. Therefore none of the effects seen are considered to be a consequence of treatment.

CLINICAL CHEMISTRY
Although clinical chemistry parameters were not included in this study, as no adverse effects on organs nor on haematology and urinalysis, this is not considered to have affected the outcome of the study.

URINALYSIS
Some differences were seen between the results of renal function tests for treated rats compared with control rats. No permanent disturbance of the renal system was apparent. In the first 26 weeks of exposure a dose-dependent, significant increase in the numbers of cells excreted via the urine per hour was observed, both in male and female rats. This effect became less clear over time, and was absent at the last time point (104 weeks). Overall, kidney functions were normal during the study, therefore, the increased water intake of 5000 ppm treated male and female rats in the first part of the 2-y study (which correlates with increased urine volume) is not expected to be an adverse effect of the substance.
A trend of increased protein excretion in the urine of both control and treated rats was found. This is a characteristic of ageing rats, which severity or onset was not accelerated by the substance.

ORGAN WEIGHTS
Although there was some suggestion of increased caecum weight in male rats given 5000 ppm substance in the interim studies, this increase was not seen in the corresponding group in the 2-y study. Other differences were seen (increased spleen weight, decreased intestinal weight both at 50 ppm in male rats, increased pituitary glands in female rats at 50 ppm), but never consistent over sexes or dose dependent. It was thus concluded that the increased organ weights were not a toxicological relevant adverse effect.

HISTOPATHOLOGY: NON-NEOPLASTIC
Occasionally a single histopathological finding was found with a higher prevalence in one or more groups (lung, liver). However, no clear dose trend is found, no consistent pattern is seen in the male and female groups, therefore it is unlikely that these differences can be attributed to the treatment. In addition, periartritis was seen, but not statistically significant, and this is commonly found in old rats. An increased number of fibrous polyps was seen in the uteri of high dose females, however this is commonly found in old female rats. Since no clear dose response is seen, this was considered not to be related to treatment.

HISTOPATHOLOGY: NEOPLASTIC
No dose related effect was seen in tumours of the pituitary, pancreas and thyroid, which are relatively common in aged rats. Basal cell carcinoma, lipoma, sarcoma, fiborsarcoma and squamous cell carcinoma were found either in the skin or subcutaneous tissues of several animals. However, these kind of tumours have frequently been reported in control rats and therefore, it is unlikely they result from treatment.

Effect levels

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Key result
Dose descriptor:
NOAEL
Effect level:
>= 630 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects were observed up to the highest dose level tested
Key result
Dose descriptor:
NOAEL
Effect level:
>= 450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects were observed up to the highest dose level tested.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a long term (two year) repeated dose toxicity study with rats performed mainly according to OECD 453, supplemented with two interim studies (49-51 weeks and 80-82 weeks respectively), the NOAEL was determined to be ≥ 630 and ≥ 450mg/kg/day for females and males, respectively, based on the absence of effects seen at the highest concentration (5000 ppm in the food).
Executive summary:

In a long term (two year) toxicity study with rats, supplemented with two interim studies (49-51 weeks and 80-82 weeks respectively), male and females rats (48/sexe) were daily exposed to sodium ferrocyanide, which was mixed in the food. During the studies, body weight and food and water intake was monitored at regular intervals. Furthermore, blood and urine samples were examined for several relevant parameters. At the end of the study, rats were killed and organs and tissues were examined macro- and microscopically. Rats exposed to the highest concentration (5000 ppm in food) showed elevated water consumption in the first nine months. The sodium ferrocyanide in the diet did not adversely affect growth or general health of the rats in the three treatment groups. No dose-related variations were found in the haematology or urine analyses. Neither were dose-related differences found in the weight of the organs, or in the (non-neoplastic and neoplastic) histopathology of organs or tissues between the exposed groups or differences with the non-exposed groups. From these data, the NOAEL was determined to be ≥ 630 and ≥ 450 mg/kg bw/day for females and males, respectively, based on the absence of effects seen at the highest concentration (5000 ppm in the food).