2-Pyridinesulfonamide, N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(2,2,2-trifluoroethoxy)-, sodium salt (1:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4 November 1996 to 20 December 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to GLP and in line with standardised guidelines OECD 407 and EU method B.7 with no deviations thought to impact on the reliability of the presented results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

sodium 4,6-dimethoxy-N-({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)pyrimidin-2-aminide

EC Number:

688-332-8

Cas Number:

199119-58-9

Molecular formula:

C14H13F3N5O6SNa

IUPAC Name:

sodium 4,6-dimethoxy-N-({[3-(2,2,2-trifluoroethoxy)pyridin-2-yl]sulfonyl}carbamoyl)pyrimidin-2-aminide

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: solid
- Storage condition of test material: room temperature
- Stability under test conditions: stable

Test animals

Species:

rat

Strain:

other: Tif:RAIf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 5 weeks
- Weight at study initiation: 157.3 - 190.5 g (males); 115.4 - 156.0 g (females)
- Housing: individually
- Diet: pelleted, certified standard diet available ad libitum
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 16 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From 9 November 1996 to 20 December 1996

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
-Test material was weighed and mixed at an appropriate concentration with pulverised standard diet. About 25 % water was added to the mix before pelleting to ensure the necessary pellet quality. The pellets were subsequently airdried.
- Diet storage: in stainless steel containers at room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration, stability and homogeneity of the test material in the diet was analysed by HPLC. Analysis of homogeneity was performed by analysing samples of each dose group from three different segments (beginning, middle, end) of the pelleting process. Evaluation of stability was performed by analysing samples of each dose group being taken right after preparation and after being stored for 35 and 49 days at room temperature.

- Analysis of samples: 10 g of ground diet pellets was slurried with 20mL 0.2% phosphoric acid. 80mL acetonitrile was added and the mixture shaken, on a lab shaker, for one hour. After sedimentation, an aliquot was withdrawn from the supernatant and diluted with acetonitrile/0.2 % phosphoric acid (40:60 v/v).defined aliquots were quantified by HPLC.
The HPLC was calibrated with standard solutions which were made up at a range of concentrations of test material in acetonitrile/ 0.2 % phosphoric acid mixtures.

- HPLC operating conditions:
Column: C18 5 µm; 125 mm x 4 mm
Temperature: room temperature
Eluent: acetonitrile/ 0.2% phosphoric acid (60:40 v/v)
Flow rate: 1.0 mL/min
Wavelength: 240 nm
Injection volume: 10 µL

- Analytical results: The overall mean concentrations of the homogeneity samples taken during administration were found to be 95.6 %, 94.3 %, 108.6 % and 100.4 % of the nominal concentrations for dose groups of 1000, 4000, 12000 and 20000 ppm, respectively. Therefore, the test material was found to be homogeneously distributed in the diet. In addition, the test material was found to be stable at room temperature over a period of 35 days and 49 days.

Duration of treatment / exposure:

Animals were dosed for 28 days

Frequency of treatment:

Animals were dosed daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1000, 4000, 12000, 20000 mg/kg food
Basis:
nominal in diet

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: dose levels were based on results from a previous 28-day toxicology study in which male rats were dosed test material orally by gavage. Findings from the study indicated that the test material was well tolerated by the rats up to the limit dose level of 1000 mg/kg bw and neither their appearance nor their behaviour were influenced. Minimal to slight effects were noted at 300 and 1000 mg/kg dose levels.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: all animals were checked daily (morning and afternoon) for mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily checks to detect changes in state of health or behaviour, or any reaction to treatment were conducted. Observations were recorded at least weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: during acclimatisation and then weekly for the remainder of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE: yes
- Time schedule: food consumption was recorded weekly and was calculated for periods of one week. The calculation was based on the weight of the offered diet at the beginning of a weighing period and its difference to the re-weighed amount after several days.
- Test material intake was calculated as follows: (food consumption ratio x nominal concentration (ppm)) / 1000

FOOD EFFICIENCY:
The food consumption ratio was calcualted as mean of individual ratios according to the following:
(weekly food consumption (g)/midweek bodyweight (g)) x (1000/7) = g food/kg bodyweight/day

WATER CONSUMPTION: Yes
- Time schedule for examinations: water consumption was recorded weekly and was calculated for periods of one week. The calculation was based on the weight of the water offered at the beginning of a weighing period and its difference to the re-weighed amount after one day.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight prior to blood removal)
- How many animals: all surviving animals
- Parameters checked: red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, total white blood cell count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unsustained cells, differential count, platelet count and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes (overnight prior to blood removal)
- How many animals: all surviving animals
- Parameters checked: urea, creatinine, glucose, albumin, globulin, albumin:globulin ratio, total protein, cholesterol, sodium, potassium, chloride, calcium, total bilirubin, inorganic phosphorous, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase.

URINALYSIS: Yes
- Time schedule for collection of urine: at end of treatment period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: Colour, volume, relative density, glucose, bilirubin, pH, protein, ketone, urobilinogen, erythrocytes and leukocytes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were exanguinated under ether anesthesia and subjected to detailed necropsy.
- Organ weights: At scheduled termination, the body (exanguinated) adrenals, heart, kidneys, liver, spleen, ovaries/testes, thymus and thyroid were removed from all animals and weighed.

HISTOPATHOLOGY: Yes. skin, mammary area, spleen, mesenteric lymph node, axiliary lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary glands, liver, pancreas, oesophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), kidneys, urinary bladder, prostate, seminal vesicle, testes, epididymis, uterus, vagina, ovaries, pituitary gland, adrenal glands, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve, orbital glands, extraorbital lacrimal glands, Zybmal glands, muzzle, tongue and any tissue with gross lesions.
- The tissues were preserved in neutral buffered 4% formalin.

MICROSCOPIC EXAMINATIONS: After fixation, a 3-5 µm section of the liver was taken, embedded, stained and subject to microscopic examination.
The thymus was examined in addition due to the decrease in weights recorded at necropsy.

Any histopathological lesions observed were graded as to degree of severity from grade 1 (minimal/ very few) to grade 5 (massive/ extensive number).

Statistics:

For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods were applied to allow for non normal as well as normal data distribution.

Each treated group was compared to the control group by Wilcoxon's two-sample test and tested for increasing or decreasing trends by Jonckheere's test for ordered alternatives.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of males dosed at 12000 and 20000 ppm were decreased compared to that of the control group (6 and 7 % lower, respectively) throughout the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant, lower mean food consumption values were measured in the female group dosed at 20000 ppm during the treatment period. The overall food consumption was 11 % below the control groups value.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Slightly lower haemoglobin concentrations, associated with a tendency to lower values for heamatocrit (males and females), and MCV (females) were recorded in animals treated at 20000 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment resulted in lower plasma bilirubin levels in males and females at ≥ 12000 ppm, higher cholesterol levels in males at ≥ 12000 ppm and in females at 20000 ppm, and lower phosphate levels in males treated at 20000 ppm and females at ≥ 12000 ppm.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased mean liver weights/mean liver:body weight ratios in males at 4000, 12000 and 20000 ppm and 20000 ppm females. Decreased mean thymus weights/mean thymus:bodyweight ratios in 12000 and 20000 ppm males, also mean thymus weight in 20000 ppm females.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hepatocellular hypertrophy of centrilobular distribution was observed in animals dosed at 4000, 12000 and 20000 ppm of either sex.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities.
No changes in behaviour or relevant clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights of males dosed at 12000 and 20000 ppm were decreased compared to that of the control group (6 and 7 % lower, respectively) throughout the treatment period. The mean body weight gains were 15 and 16 % below that of the control group. In females, the body weight development of the group dosed at 20000 ppm was also slightly impaired; the mean body weight gain during the study was 20 % below that of the control group.

FOOD CONSUMPTION AND TEST ARTICLE INTAKE
Statistically significant, lower mean food consumption values were measured in the female group dosed at 20000 ppm during the treatment period. The overall food consumption was 11 % below the control groups value.
The mean test material intake was 86.79, 342.5, 1038 and 1833 mg/kg bw/day in males and 92.39, 384.2, 1050 and 180 mg/kg bw/day for females. Corrected for the purity of the test material, the mean daily intakes were 83.0, 323, 1127 and 1840 mg/kg bw/day in males and 88.3, 362, 1140 and 1808 mg/kg bw/day in females.

FOOD EFFICIENCY
Compared to the control group, slighlty higher ratios were calcualted for 20000 ppm males at weeks 2, 3 and 5 and for 12000 ppm males at week 4.

WATER CONSUMPTION
There was no influence of treatment on water consumption.

HAEMATOLOGY
Slightly lower haemoglobin concentrations, associated with a tendency to lower values for heamatocrit (males and females), and MCV (females) were recorded in animals treated at 20000 ppm. In addition, males and females of the high dose group had mild eosinopenia, and minimally lower blood clotting activities were noted in males treated at ≥ 4000 ppm. Statistically significant differences from control also occurred for platelett counts of groups treated with doses of ≥ 4000 ppm.These differences resulted from an unusually low mean value in the control group caused by an animal with thrombopenia, and were therefore not judged to be treatment related. Other minor changed were not considered to be treatment related.

CLINICAL CHEMISTRY
The treatment resulted in lower plasma bilirubin levels in males and females at ≥ 12000 ppm, higher cholesterol levels in males at ≥ 12000 ppm and in females at 20000 ppm, and lower phosphate levels in males treated at 20000 ppm and females at ≥ 12000 ppm. Furthermore, slightly higher plasma protein levels (albumin and globulin), and increased plasma potassium levels were observed in males of the top dose group, and one female each of groups dosed at 12000 and 20000 ppm had an increased plasma chloride level. There were a few other statistically significant changes which were considered unrelated to the treatment. HIgher plasma protein, albumin and globulin levels in males of the group dosed at 1000 ppm occurred without any relation to the dose administered. The minor changes of globulin (males, 12000 ppm) and albumin to globumin ratio (males 12000 pm and females 20000 ppm) were of insufficient magnitude to be toxicologically relevant.

URINALYSIS
No treatment related, statistically significant effects were noted.

ORGAN WEIGHTS
At necropsy, the mean carcass weights of males treated at 12000 and 20000 ppm were 6 and 9 % below that of the control group. At the same feeding levels, females had 6 % lower carcass weights each. Mean liver weights/mean liver to body weight ratios were increased in males dosed at dose levels of 4000, 12000 and 20000 ppm by 17/18 %, 13-20 % and 30/43 %, respectively, and in females dosed at 20000 ppm by 16/23 %. The mean thymus weights/mean thymus to bodyweight ratios were decreased in males dosed at 12000 and 20000 ppm by 19/15 % and 15/7 %, respectively. A statistically significant decrease in mean thymus weight of the female group dosed at 20000 ppm was also noted.

GROSS PATHOLOGY
No treatment related macroscopical findings were observed at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
Hepatocellular hypertrophy of centrilobular distribution was observed in animals dosed at 4000, 12000 and 20000 ppm of either sex. In males, cytoplasmic vacuolation occurred in the group dosed at 20000 ppm. Furthermore, slight necrosis and minimally higher numbers of monocellular necrosis of the liver were seen in males dosed at 20000 ppm. One animal simultaneously presented both types of necrosis. Additionally, a variety of other changes, such as lymphohistiocytic infiltration in the liver and phagocytic cells in the thymus was found during the study. They are common occurrences in rats used at the facility and were not considered to be treatment related.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

83 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: body weight; food consumption; haematology; clinical chemistry; organ weights; histopathology

2

Dose descriptor:

NOAEL

Effect level:

88 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: body weight; food consumption; haematology; clinical chemistry; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Bodyweight (means) in g/animal

Week	Dose (ppm)
	0	1000	4000	12000	20000
Males
-1	171.9	179.8	176.1	175.4	173.3
1	234.5	249.5	241.7	230.2	226.8
2	279.9	297.1	279.2	266.1	260.2
3	307.8	334.2	307.0	293.2	284.4
4	333.7	364.5	333.9	313.2	309.3
Females
-1	134.3	133.7	134.9	134.9	140.6
1	162.5	160.2	162.2	161.7	163.1
2	182.3	176.7	178.4	177.0	176.6
3	196.0	188.9	190.1	189.6	186.5
4	205.1	204.0	199.4	201.0	197.4

 

Table 2: Food consumption (means) in g/animal

week	Dose (ppm)
	0	1000	4000	12000	20000
Males
-1	151.5	161.4	159.7	155.3	156.1
1	156.5	170.4	163.8	143.6	143.1
2	180.6	193.9	177.8	176.3	184.1
3	170.2	189.0*	169.5	162.2	169.9
4	175.8	193.1	177.1	182.2	192.4
Females
-1	121.6	127.3	120.7	107.9	124.1
1	118.0	110.8	121.7	102.0*	111.8
2	135.1	120.7	124.5	115.6*-	116.6*-
3	127.4	118.1	118.7	111.5*	107.7*-
4	129.7	120.3	122.3	115.6*	116.5*

* P < 0.05 (Wilcoxon)

+- P < 0.01 (Jonkheere)

 

Table 3: Haematology (means)

	Dose (ppm)
	0	1000	4000	12000	20000
Males
RBC (T/L)	8.034	8.019	8.040	8.130	7.764
Hb (mmol/L)	9.301	9.410	9.460	9.360	8.880*
Hct (L)	0.460	0.463	0.465	0.470	0.444
MCV (fL)	57.29	57.78	57.98	57.80	57.22
RDW (L)	0.117	0.110	0.114	0.106*	0.113
MCH (fmol)	1.163	1.172	1.178	1.154	1.142
MCHC (mmol/L)	20.27	20.29	20.29	19.94	20.00
HDW (mmol/L)	1.756	1.651	1.794	1.518	1.744
WBC (G/L)	10.46	10.11	9.784	10.20	10.30
Neut (L)	0.086	0.111	0.122*	0.093	0.093
Eos (L)	0.008	0.012	0.009	0.008	0.005
Baso (L)	0.003	0.003	0.003	0.003	0.003
Lympho (L)	0.833	0.803	0.801	0.832	0.838
Mono (L)	0.038	0.039	0.038	0.039	0.032
Luc (L)	0.033	0.034	0.027	0.025	0.029
Neut (G/L)	0.885	1.114	1.196	0.908	0.958
Eos (G/L)	0.085	0.116	0.090	0.080	0.058*
Baso (G/L)	0.036	0.026	0.030	0.028	0.030
Lympho (G/L)	8.731	8.118	7.834	8.542	8.616
Mono (G/L)	0.392	0.396	0.374	0.410	0.330
Luc (G/L)	0.325	0.340	0.268	0.258	0.310
Plt (G/L)	851.1	1107	1145*+	1154*+	1167+
PT (rel. L)	0.812	0.835	0.742*	0.707*-	0.699*-
Females
RBC (t/L)	7.950	7.898	7.940	7.662	7.772
Hb (mmol/L)	9.240	9.310	9.040	8.930	8.800*
Hct (L)	0.457	0.449	0.446	0.441	0.430*-
MCV  (fL)	57.50	56.80	56.22	57.55	55.30*
RDW (L)	0.121	0.116	0.119	0.126	0.119
MCH (fmol)	1.168	1.180	1.142	1.169	1.134
MCHC (mmol/L)	20.27	20.78*	20.28	20.30	20.53
HDW (mmol/L)	1.430	1.366	1.554	1.544	1.466
WBC (G/L)	6.446	5.837	6.588	5.562	6.454
Neut (L)	0.091	0.153	0.103	0.172	0.117
Eos (L)	0.013	0.011	0.010	0.016	0.007*
Baso (L)	0.002	0.002	0.003	0.002	0.002
Lympho (L)	0.828	0.783	0.826	0.756	0.823
Mono (L)	0.035	0.026	0.033	0.031	0.031
Luc (L)	0.030	0.026	0.025	0.024	0.019*
Neut (G/L)	0.548	0.867	0.600	0.907	0.724
Eos (G/L)	0.080	0.065	0.060	0.085	0.046*
Baso (G/L)	0.016	0.011	0.022	0.012	0.016
Lympho (G/L)	5.356	4.598	5.526	4.255	5.350
Mono (G/L)	0.252	0.150	0.210	0.176	0.196
Luc (G/L)	0.196	0.146	0.170	0.127	0.124
Plt (G/L)	1016	969.4	1051	1095	1146*
PT (rel. L)	0.914	0.939	0.935	0.929	0.965

* P < 0.05 (Wilcoxon)

+- P < 0.01 (Jonkheere)

 

Table 4: Blood chemistry (means)

	Dose (ppm)
	0	1000	4000	12000	20000
Males
Gluc (mmol/L)	8.008	7.940	7.324	8.020	9.155
Urea (mmol/L)	5.796	6.194	6.066	6.810	7.396
Creatine (µmol/L)	17.16	17.91	16.18	17.92	19.26
Bili-tot (µmol/L)	1.950	1.916	1.546	1.128*-	0.868*-
Prot (g/L)	64.10	67.69*+	65.60	66.69	70.74*
Alb (g/L)	34.20	35.45*	34.71	34.86	36.43*
Glob (g/L)	29.90	32.24*	30.89	31.84*	34.31*
A/G (L)	1.146	1.100	1.128	1.094*	1.068
Chol (mmol/L)	1.742	1.930	1.922	2.226*	2.872*+
Na+ (mmol/L)	142.2	142.0	143.5*	143.3*+	143.4+
K+ (mmol/L)	4.070	3.716	3.749	4.043	4.492
Ca2+ (mmol/L)	2.654	2.732	2.652	2.678	2.682
Cl- (mmol/L)	99.74	98.24	101.2	100.5	100.8
PO4-in. (mmol/L)	2.120	2.180	2.060	1.974	1.864*-
ASAT (GPT) (U/L)	86.21	72.30	85.78	68.54	68.76*
ALAT (GPT (U/L)	32.10	26.88	35.26	31.61	38.02
Alp (U/L)	179.2	144.1	165.1	134.3	176.5
Females
Gluc (mmol/L)	7.102	6.548	6.694	7.417	7.738
Urea (mmol/L)	7.528	6.906	7.566	7.553	7.428
Creatine (µmol/L)	21.40	21.76	21.74	21.71	21.06
Bili-tot (µmol/L)	1.970	1.862	1.754	1.242*	0.847*-
Prot (g/L)	64.85	64.55	64.86	66.35	66.85
Alb (g/L)	35.71	35.23	35.54	36.16	36.11
Glob (g/L)	29.14	29.32	29.31	30.19	30.73
A/G (L)	1.226	1.206	1.212	1.200	1.176*
Chol (mmol/L)	2.028	2.332	2.006	2.402	2.741*
Na+ (mmol/L)	142.7	142.7	143.9	143.8	143.9
K+ (mmol/L)	3.548	3.316	3.184	3.498	3.530
Ca2+ (mmol/L)	2.604	2.604	2.594	2.614	2.590
Cl- (mmol/L)	101.6	101.5	102.6	104.2	104.1
PO4-in. (mmol/L)	1.766	1.816	1.788	1.586	1.586*
ASAT (GPT) (U/L)	81.57	80.70	77.66	70.96	62.22*-
ALAT (GPT (U/L)	26.82	24.86	24.16	24.44	24.92
Alp (U/L)	123.9	120.9	121.6	112.6	111.8

* P < 0.05 (Wilcoxon)

+- P < 0.01 (Jonkheere)

 

Table 5: Organ weights (means)

	Dose (ppm)
	0	1000	4000	12000	20000
Males
Body (g)	314.4	345.5	312.9	296.1	286.5
Heart (g)	1.117	1.191	1.095	1.067	1.223
Liver (g)	14.98	17.61	17.51	16.96	19.43*+
Kidneys (g)	2.354	2.644	2.593	2.266	2.307
Adrenals (mg)	77.42	83.46	76.58	73.10	70.74
Thymus (mg)	734.5	803.4	661.8	592.8	627.4
Testes (g)	3.472	3.647	3.249	3.249	3.437
Spleen (g)	0.634	0.598	0.586	0.586	0.577
Thyroid gland (mg)	25.26	26.68	25.80	25.80	26.58
Females
Body (g)	199.6	195.8	191.2	187.1	187.4
Heart (g)	0.810	0.746	0.764	0.732	0.759
Liver (g)	9.214	9.100	9.236	9.062	10.68
Kidneys (g)	1.719	1.660	1.781	1.651	1.755
Adrenals (mg)	83.00	88.66	101.20	79.66	86.34
Thymus (mg)	547.6	503.6	478.7	470.2	426.3*
Ovaries (g)	168.6	155.2	195.3	181.1	146.5
Spleen (g)	0.426	0.453	0.469	0.423	0.432
Thyroid gland (mg)	21.28	21.82	22.94	27.88	20.24

  

Table 6: Organ to bodyweight ratios (means)

	Dose (ppm)
	0	1000	4000	12000	20000
Males
Heart (x 10^-3)	3.547	3.465	3.499	3.604	4.324
Liver (x 10^-3)	47.57	50.93	55.90*+	57.25	67.97*+
Kidneys (x 10^-3)	7.486	7.699	8.257	7.653	8.086
Adrenals (x 10^-3)	0.246	0.243	0.248	0.247	0.249
Thymus (x 10^-3)	2.334	2.328	2.121	1.996	2.178
Testes (x 10^-3)	11.03	10.63	11.42	10.97	12.07
Spleen (x 10^-3)	2.008	1.735	1.978	1.981	2.024
Thyroid gland (x 10^-3)	0.082	0.078	0.083	0.087	0.094
Females
Heart (x 10^-3)	7.055	3.814	3.994	3.92	4.068
Liver (x 10^-3)	46.16	46.44	48.21	48.43	56.96*+
Kidneys (x 10^-3)	8.626	8.534	9.307	8.853	9.370
Adrenals (x 10^-3)	0.418	0.458	0.529	0.427	0.461
Thymus (x 10^-3)	2.750	2.546	2.527	2.501	2.288
Ovaries (x 10^-3)	0.848	0.812	1.030	0.967	0.786
Spleen (x 10^-3)	2.130	2.316	2.458	2.258	2.317
Thyroid gland (x 10^-3)	0.107	0.110	0.120	0.148	0.109

* P < 0.05 (Wilcoxon)

+- P < 0.01 (Jonkheere)

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, dietary administration of the test material to rats at concentrations of 0, 1000, 4000, 12000 and 20000 ppm for 4 weeks resulted in: decreased bodyweights at 2000 ppm (both sexes) and 12000 ppm (males); decreased food consumption of females at 20000 ppm; lower haemoglobin concentrations and haematocrit (both sexes) and lower MCV (females) at 20000 ppm; eosinopenia at 20000 ppm; lower clotting activities (males) at dose levels ≥ 4000 ppm; lower plasma bilirubin levels at 20000 and 12000 ppm; higher cholesterol levels at 20000 ppm (both sexes) and at 12000 ppm (males); lower phosphate levels at 20000 ppm (both sexes) and at 12000 ppm (females); higher albumin and globulin levels and increased potassium levels at 20000 ppm(males); one female each treated at 20000 and 12000 ppm with an increased plasma chloride level; increased liver weights and liver to body ratios at 20000 ppm and at 12000 and 4000 ppm (males); decreased thymus weights and thymus to body weight ratios at 20000 ppm (both sexes) and 12000 ppm (males); hepatocyte hypertrophy at 20000, 12000 and 4000 ppm (both sexes); cytoplasmic vacuolation and hepatocyte necrosis at 20000 ppm (males).

The no observed effect level for males and females was at 1000 ppm, corresponding to 83 and 88 mg/kg bw/day, respectively. The available information was considered to be complete and suitable for inclusion in risk assessments.

Executive summary:

The oral repeat dose toxicity of the test material was determined in accordance with the standardised guidelines OECD 407 and EU Method B.7. Groups of five male and five female rats were administered test material in the diet at concentrations of 0 (control), 1000, 4000, 12000 and 20000 ppm (corresponding to 0, 83.0, 323, 1127 and 1840 mg/kg bw in males, and 0, 88.3, 362, 1140 and 1808 mg/kg bw in females) for 28 consecutive days. Clinical observations, bodyweights and food consumption were measured throughout the study. In addition, clinical observations were performed. At the end of the scheduled period, the animals were killed and subjected to an examination post mortem. Cardiac blood samples were taken for clinical pathology, selected organs were weighed and specified tissues were taken for subsequent histopathology examination.

Dosing preparations were analysed for achieved concentration, homogeneity and stability and were considered to be satisfactory. Under the conditions of the study, treatment with test material was tolerated without occurrence of overt toxicity. Reduced bodyweight gains were measured in both sexes treated at 20000 ppm. At 20000 ppm, the laboratory examinations revealed decreased haemoglobin concentrations and a mild eosinopenia. At dose levels above 4000 ppm lower blood clotting activities were detected. Changes to some blood chemistry parameters indicated an effect on the liver. At necropsy, liver weights were increased in groups dosed

at or above 4000 ppm in males and at 20000 ppm in females. Histopathology of the livers showed hepatocellular hypertrophy in both sexes at dose levels of 4000 ppm and above, and slight cytoplasmic vacuolation and necrosis in males treated at 20000 ppm. In addition, thymus weights were decreased at 12000 ppm (males) and at 20000 ppm (both sexes). It can be inferred from the observations made during the study, that the no observed effect level for the test material, when offered to rats continuously in their food over a period of four weeks, was 1000 ppm, corresponding to a mean daily intake of 83 mg/kg bw in males and 88 mg/kg bw in females.