Bis(hydroxylammonium) sulphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

other: range-finding study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acc. to guidelines, but preliminary study with technical shortcomings concerning definite dosing.

Reason / purpose for cross-reference:

other: main study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

28-day range-finding oral toxicity study mostly according to OECD TG 407

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl Thomae, Biberach a.d. Riss, Germany
- Age at study initiation: 42 days
- Average weight at study initiation: males: 164.8 g; females 135.2 g
- Housing: individually, in V2A cages, type DK III (Becker & Co. Castrop-Rauxel, Germany)
- Diet: Kliba 343 Futter Ratte/Maus/Hamster "A" (Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Air changes: fully air-conditioned rooms
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

In a 28-day range-finding oral toxicity study mostly according to OECD TG 407 groups of five male and five female Wistar rats received the test material in the drinking water in doses of 0, 25, 100, 400 or 1600 ppm for 4 consecutive weeks. The analytical investigation of the test substance preparations are performed at the end of the study. But, the estimation of compound consumption based on water consumption by male and female rats could not definitely be done in all dose groups because of some study restrictions. There were some technical shortcomings concerning definite calculated achieved intake of BHAS from the drinking water in the 25, 100 and 400 ppm dose groups because of the instability of the test material in water. No test substance or only minor amounts were verifiable especially for the low dose groups. Therefore, the dose is given in ppm instead of mg/kg bw. Stability tests for the test material at the highest dose of 1600 ppm in the drinking water for a storage of 4 days resulted in an average of 1437 ppm. So the average concentration of the test material in drinking water of the 1600 ppm dose group was approx. 142 mg/kg bw/d for males and 149 mg/kg bw/d for females.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test material was checked by photometry over a period of 4 days, but not over a time period of 10 days (as intended).

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

ad libitum

Remarks:

Doses / Concentrations:
0, 25, 100, 400, and 1600 ppm
Basis:
nominal in water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

After arrival of the animals they were acclimized for 6 days. 4 days prior to start of the exposure period the animals were divided into the treatment groups. The randomization was computer-based.

The preparation of the test material in the drinking water was conducted twice weekly. The test material was mixed with the water and then subsequently stirred for 10 minutes to ensure homogenization. The bottling was conducted with a partially automatized dosage apparatus (Fortuna Optifix).

Observations and examinations performed and frequency:

- The animals were inspected twice daily for mortality/moribundity (monday to friday) and once daily on saturday, sunday and on public holidays.
- Feed consumption, drinking water consumption and body weight were determined once a week. The average feed consumption (in mg/kg bw/day) was calculated for the high-dose group (1600 ppm) only.
- The state of health was checked each day, and when the animals were weighed, they were additionally inspected and palpated. Approx. 14 days after study inititation and at the end of the study, a swim-test was conducted with the high-dose animals as well as with the controls. The swim test was conducted in a 200 l tank (80 cm x 54 cm x 50 cm) which was 50 % filled with water at 25 °C. The animals were set into the middle of the tank and the time period was documented from setting the animals into the water until the beginning of swimming-movements. For a good comparison, one animal of the high dose group was simultaneously inserted into the water with an animal of the control group. Approx. 30 sec. after insertion the animals were taken out of the water and their fur was dried, possible observations were noted.

- Urine was collected for urinalyses on day 23 after the beginning of administration.
The following parameters were determined semiquantitatively in urine using test stripes and a reflection photometer (Clini-Tek, Ames, Frankfurt, Germany):
- pH
- total protein
- glucose
- ketones
- bilirubin
- blood
- nitrite
- urobilinogen
The sediment analysis was conducted by microscopy.

- Blood samples were taken for clinicochemical and hematological examinations 7, 28 and 30 days after the beginning of administration. Blood was taken in the morning out of non-fasted animals.
The following clinico-chemical/hematological parameters were determined:

Hematological examinations:
The following parameters were determined in blood using a particle counter:
- leukocytes
- erythrocytes
- hemoglobin
- hematocrit
- mean corpuscular volume
- mean corpuscular hemoglobin
- mean corpuscular hemoglobin concentration
- platelets
- Heinz-bodies
- methaemoglobin
The data obtained were transferred to a computer. The differential blood count was evaluated visually. The data were transferred to the computer.

Clotting analyses:
The clotting analyses were carried out using a ball coagulometer and the results transferred off-line to the computer.
The following parameter was determined:
- thromboplastin time (Hepato Quick's test)

Clinicochemical examinations:
An automatic analyzer was used to examine the clinicochemical parameters. The values obtained were transferred to a computer.
The following parameters were determined:

Enzymes
- glutamate-pyruvate transaminase
- glutamate-oxalacetate transaminase
- alkaline phosphatase
- lactate dehydrogenase
- plasma-cholinesterase

Blood chemistry:
- sodium
- potassium
- chloride
- inorganic phosphate
- calcium
- urea
- creatinine
- glucose
- total bilirubin
- total protein
- albumin
- globulins
- triglycerides
- cholesterol

Liver homogenate:
- gamma-glutamyl transferase
- GSH
- hormones (trijodthyronine and thyroxine)

Sacrifice and pathology:

At the end of the administration period, all animals were sacrificed by decapitation after they had been anesthetized by CO2 and were assessed by gross pathology.
Subsequently, a histopathological examination was carried out.
The weight of the animals as well as the weights of liver, kidneys, adrenal glands, testes, brain and spleen from all animals sacrificed at scheduled dates was determined.
Subsequently the following organs or tissues were fixed in 4 % formaldehyde solution:
brain, thyroid, kidneys, spleen, adrenal glands, heart, testes, peripheral nerve, sternum with medulla, spinal cord, all macroscopic changes and target organs.

After the organs had been fixed, processing, the examination by light microscopy and the evaluation of findings was performed in all animals of the control and the high dose groups. Additionally, the spleen, liver and kidneys were examined fom all animals of the 100 ppm and 400 ppm dose group.

Statistics:

Means and standard deviations were calculated.
Statistical analyses were conducted by using the Dunnett´s- test and the ANOVA analysis, the William´s test or the t-test.

Details on results:

No deaths occurred during the study. Food intake of BHAS-treated rats was similar to that of the controls. At 1600 ppm, rats of both sexes showed decreased water consumption, cyanosis and discoloration (yellow-red) of the urine in the last treatment week. The relevant study results of hematology, clinical biochemistry, organ weight assessment, necropsy and microscopy from males and females receiving 1600, 400 and 100 ppm are summarized in the following.

1600 ppm:
- blood (m/f): ↓ RBC, ↓ HB, ↓ HCT, ↓ MCHC, ↑ MCH, ↑ MCV, ↑ RET, ↑ Heinz bodies, ↑ methaemoglobin, morphological changes of RBC: anisocytosis, poikilocytosis, and polychromasia (m/f), ↑ WBC (m/f), ↑ Neut (m/f), ↑ Eos (m/f), ↑ Lymph (m/f), ↑ Mono (m/f), ↓ PLT (m/f), ↑ bilirubin (m/f), ↓ AP (m/f), ↓ glucose (f), ↓ Ca (m), ↑ P (m), ↑ bilirubin in urine (f)
- spleen (m/f): ↑ weight (abs/rel), splenomegaly, hemosiderin deposits, extramedullary hematopoiesis
- liver: hemosiderin deposits in Kupffer cells (m/f), extramedullary hematopoiesis and erythrophagocytosis (m/f), single megacaryocytes in intrasinosidial space (m/f), iron pigment deposition in hepatocytes (f)
- kidney: ↑ weight, abs/rel (m), tubular hemosiderosis (m/f), iron-negative pigment deposition in proximal tubulus (m/f)
- bone marrow (m): reticuloid hypeplasia, necrosis

400 ppm:
- blood: ↓ RBC (m), ↓ HCT (m), ↓ HB (m/f), ↑ MCV (f), ↑ RET (m/f), ↑ Heinz bodies (m/f), morphological changes of RBC: anisocytosis, micro- and macrocytosis (m), polychromatophily (m/f), ↑ Neut (m), ↓ bilirubin (f),
- spleen (m/f): splenomegaly; extramedullary hematopoiesis
- liver (m/f): extramedullary hematopoiesis

100 ppm: :
- blood (m): anisocytosis, polychromatophily
- spleen (m): splenomegaly

footnote:
↑: statistically significant increase compared with controls;
↓: statistically significant decrease compared with controls;
m: male; f: female;
RBC: Erythrocyte count; HB: Haemoglobin; HCT: Hematocrit; MCV: Mean corpuscular volume; MCH: Mean corpuscular haemoglobin; MCHC: Mean corpuscular haemoglobin concentration; WBC: Leukocyte count; PLT: Platelet count; RET: Reticulocyte count; Neut: Neutrophiles; Lymph: Lymphocytes; Mono: Monocytes; Eos: Eosinophiles; AP: Alkaline phosphatase; Ca: Calcium; P: Inorganic phosphorus;

Dose descriptor:

NOAEL

Effect level:

25 ppm

Sex:

male

Dose descriptor:

NOAEL

Effect level:

100 ppm

Sex:

female

Critical effects observed:

not specified

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

; no recovery period

Principles of method if other than guideline:

similar/equivalent to OECD TG 408, but without recovery period.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(hydroxylammonium) sulphate

EC Number:

233-118-8

EC Name:

Bis(hydroxylammonium) sulphate

Cas Number:

10039-54-0

Molecular formula:

H3NO.1/2H2O4S

IUPAC Name:

bis(hydroxyammonium) sulfate

Details on test material:

- Name of test material (as cited in study report): Hydroxylammoniumsulfat (HAS)
- Analytical purity: >= 99 %
- Lot/batch No.: 84/389
- Stability under test conditions: was ensured during the study period

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach a.d. Riss, Germany
- Age at study initiation: 42 days
- Average weight at study initiation: males ca. 117 g; females ca. 136 g
- Housing: individually in V2A wire mesh cages, type DK III (Becker & Co. Castrop-Rauxel, Germany)
- Diet: Kliba-Haltungsdiaet Ratte/Maus/Hamster 343 Mehl (Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: Milli-Q-Reinstwasser, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Air changes: fully air-conditioned rooms
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

other: Mili-Q extra pure water

Details on oral exposure:

In a subchronic oral toxicity study similar to OECD TG 408 (but without recovery period), Hydroxylammonium sulfate was administered to 60 Wistar rats (30 males and 30 females ) via the drinking water (in Milli-Q extra pure water) for 90 consecutive days. For comparison, one group of untreated animals (10 males and 10 females) was used as control.
The doses in the drinking water were 10 ppm (test group 1), 50 ppm (test group 2) and 250 ppm (test group 3). The doses administered corresponded to a mean daily test substance intake of approx. 0.9, 4 and 21 mg/kg body weight. The average daily food intake in mg/kg bw was calculated for each animal at the time intervals when the water consumption was determined.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and concentration control analyses, each samples of all concentrations were drawn at the start of the study, after 6 weeks and towards at the end of the administration period after 12 weeks of the study.
The analysis of the amount of the test material in the drinking water was determined by photometry.

Duration of treatment / exposure:

3 months

Frequency of treatment:

ad libitum

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 50, 250 ppm, corresponding to approx. 0.9, 4 and 21 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The preparation of the test material in the drinking water was conducted twice weekly (on tuesdays and fridays). The test material was mixed with the water and then subsequently stirred for 10 minutes to ensure homogenization. The bottling was conducted with a fully automatized dosage apparatus (Fortuna Optimat MP).

Examinations

Observations and examinations performed and frequency:

- The animals were inspected twice daily for mortality/moribundity (monday to friday) and once daily on saturday, sunday and on public holidays.
- Feed consumption, drinking water consumption and body weight were determined once a week.
- The state of health was checked each day, and when the animals were weighed, they were additionally inspected and palpated.
- Before the beginning of test substance administration and toward the end of the study, ophthalmological examinations were carried out in the animals of the control and 250 ppm groups.

- Urine was collected for urinalyses 29 and 78 days after the beginning of administration.
The following parameters were determined semiquantitatively in urine using test stripes and a reflection photometer (Clini-Tek, Ames, Frankfurt, Germany):
- pH
- total protein
- glucose
- ketones
- bilirubin
- blood
- nitrite
- urobilinogen
The sediment analysis was conducted by microscopy.

- Blood samples were taken for clinicochemical and hematological examinations 36 and 85 days after the beginning of administration. Blood was taken in the morning out of non-fasted animals.
The following clinico-chemical/hematological parameters were determined:

Hematological examinations:
The following parameters were determined in blood using a particle counter (S Plus model, by Coulter, Krefeld, Germany):
- leukocytes
- erythrocytes
- hemoglobin
- hematocrit
- mean corpuscular volume
- mean corpuscular hemoglobin
- mean corpuscular hemoglobin concentration
- platelets
- Heinz-bodies
- methaemoglobin
The data obtained were transferred to a computer (VAX 11/780; supplied by DEC, Munich, FRG). The differential blood count was evaluated visually. The data were transferred to the computer.

Clotting analyses:
The clotting analyses were carried out using a ball coagulometer (KC 10 model, by Amelung, Lemgo, Germany) and the results transferred off-line to the computer.
The following parameter was determined:
- thromboplastin time (Hepato Quick's test)

Clinicochemical examinations:
An automatic analyzer (Hitachi 737; by Boehringer, Mannheim, Germany) was used to examine the clinicochemical parameters. The values obtained were transferred to a computer (VAX 11/780; by DEC, Munich, Germany).
The following parameters were determined:
Enzymes
- alanine aminotransferase
- aspartate aminotransferase
- alkaline phosphatase

Blood chemistry:
- sodium
- potassium
- chloride
- inorganic phosphate
- calcium
- urea
- creatinine
- glucose
- total bilirubin
- total protein
- albumin
- globulins
- triglycerides
- cholesterol

Sacrifice and pathology:

At the end of the 3-month administration period, after a fasting period, all animals were sacrificed by decapitation after they had been anesthetized by CO2 and were assessed by gross pathology.
Subsequently, a histopathological examination was carried out.

The weight of the animals as well as the weights of liver, kidneys, adrenal glands, testes, brain and spleen from all animals sacrificed at scheduled dates was determined.
Subsequently the following organs or tissues were fixed in 4 % formaldehyde solution:
brain, pituitary gland, thyroid, thymus, trachea, lungs, heart, aorta, liver, spleen, kidneys, adrenal glands, pancreas, testes/ovaries, uterus, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, bladder, mesenterial lymph nodes, peripheral nerve, sternum with medulla, all macroscopic changes.

After the organs had been fixed, processing, the examination by light microscopy and the evaluation of findings was performed in all animals of the control and the high dose groups. Additionally, the lung, liver and kidneys were examined fom all animals of any dose group.

In the liver, spleen of all dose groups as well as in the kidneys of high dose and control groups an iron analysis was also conducted.

Statistics:

For food consumption, water consumption, body weights, organ weights and substance consumption, as well as for clinico-chemical and haematological parameters means and standard deviations were calculated. Statistical analyses were conducted for body weight as well as for clinico-chemical and haematological parameters by using the Dunnett´s- test and the ANOVA analysis.
The individual parameters of the urinalysis were checked for statistical significance by using the chi square test.

Results and discussion

Results of examinations

Details on results:

Regarding mortality, general appearance and behaviour of the animals there was no difference between treated and untreated animals. No toxicologically relevant differences in mean feed consumption per animal/d or per kg bw/d as well as body weight and body weight development were detected in male and female rats. The following findings were obtained and assessed as substance-induced.

At 250 ppm, rats of both sexes showed dark coloration of the urine. This is considered to be due to the substance-related effects on the blood. The hematological examination revealed indications of an increased destruction of red blood cells.
At 250 ppm in both sexes and at 50 ppm in females there was a reduction of the erythrocytes and haemoglobin values. In addition there were an increase of the MCH values in both sexes at 250 ppm and in females at 50 ppm, furthermore reduced values of hematocrit in females and of the MCHC in males at 250 ppm. In males receiving 50 ppm, decreased counts of red blood cells and decreased values of haemoglobin were also apparent during the treatment period, although these did not attain statistical significance. These findings are considered to be related also to an increased decay of erythrocytes. The increase of the MCV, and reticulocyte counts at 250 ppm in males and females were assessed as sign of compensate increased erythropoiesis. As a consequence of an increased leaving of juvenile erythrocytes out of the bone marrow a reinforced polychromasia was seen dose-dependent at 250 and 50 ppm in both sexes. At 50 ppm, a slight increase of reticulocytes in male and female rats was noted, and moreover in females a marginal increase of the MCV values. Furthermore, an increase of bilirubin concentration in both sexes at 250 ppm was observed. In males and females receiving 50 ppm, increased values of bilirubin were also apparent during the treatment period, although these did not attain statistical significance. This finding appears to be due to the increased decay of erythrocytes. The elevated methaemoglobin concentration and the reinforced evidence of Heinz bodies in both sexes at 250 ppm are indicative for methaemoglobinemia. Increased absolute and relative spleen weights were seen at 250 ppm in male and female rats. Increase of relative liver weights were noted only in males. In males and females receiving 50 ppm, increased absolute and relative adrenal weights were noted.
Histopathological findings representing secondary effects to the anemia included increased hemosiderin deposits in the spleen and liver of both males and females given 250 ppm. At 50 ppm, moderate increased hemosiderin deposits in the spleen were revealed in both sexes. In addition, sinus dilatation together with congestion of the spleen were observed dosedependent in both sexes at 50 and 250 ppm. 10 ppm did not alter the blood parameters in rats of both sexes.

10 ppm (approx. 0.9 mg/kg bw/d):
No changes were found to be attributed to the test substance administered.

50 ppm (approx. 4 mg/kg bw/d):
- Clinical signs: none specific (m/f)
- Blood: ↓ RBC (f), (↓) RBC (m), ↓ Hb (f), (↓) Hb (m), ↑ MCV (f), ↑ MCH (f), ↑ polychromasia (m/f), ↑ RET (m/f), (↑) bilirubin (m/f)
- Effects on organs:
adrenal weight ↑, abs/rel (m/f); spleen: hemosiderin deposits (m/f), sinus dilatation together with congestion (2m/2f)

250 ppm (approx. 21 mg/kg bw/d):
- Clinical signs: dark coloration of the urine (m/f)
- Blood: ↓ RBC (m/f), ↓ Hb (m/f), ↓ HCT (f), ↓ MCHC (m), ↑ MCV (m/f), ↑ MCH (m/f), ↑ RET (m/f), ↑ Heinz bodies (m/f), ↑ MetHb (m/f), ↑ polychromasia (m/f), ↑ bilirubin (m/f)
- Effects on organs:
spleen: ↑ weight, abs (m/f), ↑ weight, rel (m), hemosiderin deposits (m/f), sinus dilatation together with congestion (10m/10f);
liver: ↑ weight, rel (m), hemosiderin deposits (m/f)

↑: statistically significant increase compared with controls; (↑): increase compared with controls, no statistically significant but possibly of toxicological relevance;
↓: statistically significant decrease compared with controls;
(↓): decrease compared with controls, no statistically significant but possibly of toxicological relevance;
m: male; f: female;
RBC: Erythrocyte count; Hb: Haemoglobin; HCT: Hematocrit; MCV: Mean corpuscular volume; MCH: Mean corpuscular haemoglobin; MCHC: Mean corpuscular haemoglobin concentration; MetHb: methaemoglobin; RET: Reticulocyte count

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

> 0.9 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: 10 ppm: no substance related effects on blood and organs

2

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

>= 21 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no substance-related effects seen

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Hematology data for MetHB, reticulocytes and Heinz-Bodies on day 36 and 85, respectively

Males (n = 10)	MetHB: day 36	MetHB: day 85	reticulocytes: day 36	reticulocytes: day 85	Heinz-Bodies: day 36	Heinz-Bodies: day 85
0 ppm	0.0 ± 0.0	0.1 ± 0.1	19 ± 4	20 ± 8	0 ± 0	0 ± 0
10 ppm	0.0 ± 0.1	0.1 ± 0.2	17 ± 5	22 ± 5	0 ± 0	0 ± 0
50 ppm	0.0 ± 0.1	0.2 ± 0.2	25 ± 5	31 ± 10 *	0 ± 0	2 ± 2
250 ppm	0.2 ± 0.3	0.6 ± 0.2 **	72 ± 10 **	76 ± 13 **	253 ± 64 **	408 ± 80 **
Females (n = 10)
0 ppm	0.0 ± 0.0	0.1 ± 0.2	14 ± 3	15 ± 4	0 ± 0	0 ± 0
10 ppm	0.0 ± 0.0	0.1 ± 0.1	18 ± 8	17 ± 10	0 ± 0	0 ± 0
50 ppm	0.1 ± 0.2	0.1 ± 0.2	25 ± 10	28 ± 9 *	0 ± 0	7 ± 13
250 ppm	0.3 ± 0.3 **	0.9 ± 0.4 **	79 ± 23 **	80 ± 15 **	362 ± 136 **	450 ± 89 **

Statistics: Anova + Dunnet´s tests: * = P<0.05, ** = P<0.01, two-sided (statistical unit = animal)

Table 2: Absolute (g) and relative (%) body weights and selected organ weights

Males (n = 10)	body weight (abs.) (g)	body weight (rel.) (%)	spleen (abs.) (g)	spleen (rel.) (%)	liver (abs.) (g)	liver (rel.) (%)	adrenal glands (abs.) (g)	adrenal gland (rel.) (%)
0 ppm	409.32 ± 26.35	100	0.784 ± 0.122	0.191 ± 0.021	11.998 ± 1.128	2.927 ± 0.126	0.081 ± 0.008	0.02 ± 0.002
10 ppm	424.87 ± 42.81	100	0.837 ± 0.156	0.196 ± 0.026	13.153 ± 2.522	3.082 ± 0.379	0.091 ± 0.01	0.021 ± 0.002
50 ppm	423.9 ± 36.25	100	0.872 ± 0.165	0.205 ± 0.029	13.261 ± 1.657	3.121 ± 0.151	0.099 ± 0.009 *	0.024 ± 0.003 *
250 ppm	432.93 ± 45.6	100	1.587 ± 0.247 **	0.369 ± 0.066 **	14.236 ± 3.01	3.26 ± 0.373 *	0.094 ± 0.021	0.022 ± 0.003
Females (n = 10)
0 ppm	235.16 ± 27.48	100	0.521 ± 0.047	0.223 ± 0.023	7.425 ± 1.004	3.154 ± 0.147	0.013 ± 0.015	0.048 ± 0.009
10 ppm	226.16 ± 15.13	100	0.486 ± 0.066	0.214 ± 0.021	7.149 ± 0.563	3.163 ± 0.171	0.11 ± 0.008	0.049 ± 0.004
50 ppm	237.61 ± 35.95	100	0.536 ± 0.072	0.227 ± 0.025	7.627 ± 1.111	3.215 ± 0.14	0.117 ± 0.015	0.05 ± 0.006
250 ppm	232.35 ± 12.07	100	1.164 ± 0.307 **	0.499 ± 0.124 **	7.533 ± 0.325	3.247 ± 0.154	0.113 ± 0.011	0.049 ± 0.006

Statistics: Dunnet´s test: * = P<0.05, ** = P<0.01, two-sided (statistical unit = animal)

Table 3: Hematology data of selected parameters of control and high dose group on day 36 and 85, respectively

Day 36:	WBC (giga/l)	RBC (tera/l)	HGB (mmol/l)	HCT (l/l)	MCV (fl)	MCH (fmol)	MCHC (mmol/l)	PLT )giga/l)
Males (n = 10)
0 ppm	7.06 ± 0.63	8.19 ± 0.36	9.47 ± 0.49	0.416 ± 0.027	50.72 ± 1.21	1.16 ± 0.02	22.77 ± 0.62	1000 ± 96
250 ppm	8.18 ± 1.81	7.14 ± 0.51 **	8.72 ± 0.44 **	0.397 ± 0.024	55.56 ± 2.14 **	1.22 ± 0.05 **	22.00 ± 0.57 *	1017 ± 110
Females (n = 10)
0 ppm	4.28 ± 0.72	7.88 ± 0.47	9.16 ± 0.38	0.396 ± 0.021	50.18 ± 1.16	1.16 ± 0.03	23.14 ± 0.58	1136 ± 98
250 ppm	5.88 ± 1.30	6.61 ± 0.44 **	8.40 ± 0.38 **	0.373 ± 0.018	56.50 ± 2.53 **	1.27 ± 0.05 **	22.52 ± 0.53	1093 ± 160
Day 85:
Males (n = 10)
0 ppm	5.87 ± 0.95	8.47 ± 0.54	9.26 ± 0.51	0.414 ± 0.028	48.84 ± 1.03	1.09 ± 0.03	22.38 ± 0.57	1058 ± 111
250 ppm	7.31 ± 1.20	7.44 ± 0.44 **	8.69 ± 0.39 *	0.400 ± 0.020	53.66 ± 1.97 **	1.17 ± 0.05 **	21.74 ± 0.35 **	1101 ± 171
Females (n = 10)
0 ppm	3.90 ± 1.39	8.39 ± 0.42	9.29 ± 0.51	0.41 ± 0.021	48.83 ± 1.06	1.11 ± 0.03	22.65 ± 0.52	1114 ± 73
250 ppm	4.30 ± 0.87	6.49 ± 0.48 **	8.08 ± 0.29 **	0.364 ± 0.016 **	56.13 ± 2.00 **	1.25 ± 0.06 **	22.22 ± 0.44	992 ± 159

Statistics: Dunnet´s test: * = P<0.05, ** = P<0.01, two-sided (statistical unit = animal)