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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1981
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1988
Qualifier:
according to
Guideline:
other: EPA-FIFRA, Subdivision F, § 82-1
Version / remarks:
1984
Qualifier:
according to
Guideline:
other: MAFF, Japan
Version / remarks:
1985
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Tif:RAIf (SPF), hybrids of RII/1 x RII/2 (Sprague-Dawley derived)
Details on species / strain selection:
The rat was selected as the test species as it is recognized by international guidelines as a preferred test species. The number of animals used was considered to be the minimum required to meet the scientific objectives of the study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 5 weeks
- Weight at study initiation: 123.3 to 179.5 g (males), 114.7 to 155.1 g (females)
- Housing: Individually in Macrolon cages type 3
- Diet: Pelleted, certified standard diet (Nafag No. 8900 for GLP) ad libitum (except overnight prior to blood collection)
- Water: Drinking water ad libitum (except overnight during urine collection)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55 ± 10%
- Air changes: 16-20 per hour
- Photoperiod: 12 hours light, 12 hours dark

IN-LIFE DATES: Start: 27 Dec 1994, End: 30 Mar 1995

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
Oral treatment in the diet of the animals
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION :
Fresh diets were prepared monthly and stored in stainless steel containers at room temperature. The correct amount of the test substance was added to pulverised diet and homogeneously mixed together. Approximately 25% water was added before pelleting and the pellets subsequently air-dried.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Prepared pelleted samples of diet were analysed from diet used for treatment days 1-36 and 64-90 for achieved concentration and day 1-36 samples were also analysed for homogeneity and stability by an HPLC method. Analysis of homogeneity was performed by analysing samples of each diet from three different segments.
- Concentration analysis results: The mean test substance concentrations were 104.6%, 104.5%, 100.8%, 95.8% and 94.2% of nominal for 25, 250, 1250, 2500 and 5000 ppm, respectively, in the day 1-36 samples and 98.1%, 96.2%, 96.9%, 99.8% and 97.8% of nominal for 25, 250, 1250, 2500 and 5000 ppm, respectively, in the day 64-90 samples.
- Homogeneity results: Homogeneity varied between -6% to +9% of the mean concentrations.
- Stability results: The test substance was stable in rodent diet at room temperature (22 ± 2°C) over a period of 35 days.
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuously
Doses / concentrationsopen allclose all
Dose / conc.:
25 ppm
Remarks:
Dietary concentration; equivalent to 1.74 and 1.88 mg/kg bw/day for males and females, respectively
Dose / conc.:
250 ppm
Remarks:
Dietary concentration; equivalent to 17.6 and 19.2 mg/kg bw/day for males and females, respectively
Dose / conc.:
1 250 ppm
Remarks:
Dietary concentration; equivalent to 84.9 and 92.5 mg/kg bw/day for males and females, respectively
Dose / conc.:
2 500 ppm
Remarks:
Dietary concentration; equivalent to 168 and 182 mg/kg bw/day for males and females, respectively
Dose / conc.:
5 000 ppm
Remarks:
Dietary concentration; equivalent to 329 and 359 mg/kg bw/day for males and females, respectively
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on the results of a 28 day range finding study in rats (dietary).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
All animals were checked daily for mortality (am and pm weekdays, am only weekends and holidays).

DETAILED CLINICAL OBSERVATIONS
All animals were checked daily for signs of toxicity and findings were recorded at least once per week.

BODY WEIGHT
The body weight of each animal was recorded predose and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption for each animal was recorded weekly. Food consumption ratios were calculated as g food/kg body weight/day. Test substance intake was calculated per group.

WATER CONSUMPTION
Water consumption for each animal was recorded weekly.

OPHTHALMOSCOPY EXAMINATION
All animals were examined prior to dosing (week -1) and rats from the control and 5000 ppm groups were examined in week 13. Examination included observation of eye appearance and the periocular region and detection of pupillary reflex using an ophthalmoscope.

HAEMATOLOGY
- At the end of the treatment period, following overnight starvation, blood samples were collected under ether anaesthesia via the orbital sinus, from all surviving animals.
- The following parameters were measured: haemoglobin, haemoglobin concentration distribution width, haematocrit, total white cell count, mean cell volume, differential white cell count, red blood cell count, thrombocyte count, red cell volume distribution width, prothrombin time, mean cell haemoglobin, methaemoglobin, mean cell haemoglobin concentration

CLINICAL CHEMISTRY
- At the end of the treatment period, following overnight starvation, blood samples were collected under ether anaesthesia via the orbital sinus, from all surviving animals.
- The following parameters were measured: urea, alkaline phosphatase activity, creatinine, aspartate aminotransferase activity, glucose, alanine aminotransferase activity, albumin, gamma-glutamyl transpeptidase activity, globulin, calcium, A/G ratio, chloride, total protein, sodium, cholesterol, potassium, total bilirubin, inorganic phosphorus, triglycerides

URINALYSIS
- At the end of the treatment period, urine was collected overnight. Individual animals were held in metabolism cages and food and water were withheld.
- The following parameters were measured: volume, glucose, colour, ketones, relative density, protein, pH, bilirubin, urobilinogen, blood
Sacrifice and pathology:
MACROSCOPIC EXAMINATION
At the end of the study, all control and treated animals were exsanguinated under ether anaesthesia and examined post mortem. This involved an external observation and an internal examination of all organs and structures. A complete necropsy with tissue preservation was performed on one 1250 ppm female which died on day 57.

ORGAN WEIGHTS
At necropsy, the following organs were removed, trimmed free of extraneous tissue and weighed: adrenal glands, ovaries, brain, spleen, heart, thyroid, kidneys, testes, liver, thymus

TISSUE SUBMISSION
The following tissues were removed and examined and fixed in an appropriate fixative: gross lesions including masses, ovary, adrenal gland, pancreas, aorta, peripheral nerve (sciatic), brain (including medulla, pons, cerebral cortex, cerebellar cortex), pituitary gland, caecum, prostate gland, colon, rectum, duodenum, salivary gland (submandibular), epididymis, seminal vesicle, eyes (with optic nerve), skeletal muscle, extra-orbital lachrymal gland, spinal cord, femur (including joint), skin, heart, spleen, ileum, sternum with bone marrow, jejunum, stomach, kidney, testis, liver, thymus, lung, thyroid gland with parathyroid, lymph node - axillary, tongue, lymph node - mesenteric, trachea, mammary area, urinary bladder, muzzle, uterus, oesophagus, vagina, orbital gland, Zymbal gland

MICROSCOPIC EXAMINATION:
The following tissues, from all animals, were sectioned, stained and examined by light microscopy: gross lesions including masses, ovary, adrenal gland, pancreas, aorta, peripheral nerve (sciatic), brain (including medulla, pons, cerebral cortex, cerebellar cortex), pituitary gland, caecum, colon, rectum, duodenum, salivary gland (submandibular), epididymis, femur (including joint), heart, spleen, ileum, jejunum, stomach, kidney, testis, liver, thymus, lung, thyroid gland with parathyroid, lymph node - axillary , tongue, lymph node - mesenteric, uterus, oesophagus, vagina
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 Lepage’s two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere’s test for ordered alternatives.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no treatment-related mortalities, although one female at 1250 ppm died on day 57 with no histopathological treatment-related findings.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were significantly reduced in male groups at ≥ 1250 ppm. Weight gains of all female groups and the male groups at up to 250 ppm were unaffected by treatment.
See Table 1 in "Any other information on results incl. tables" for more information.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was reduced in male groups at ≥ 1250 ppm. Food consumption of all female groups and the male groups at up to 250 ppm were unaffected by treatment.
See Table 2 and 3 in "Any other information on results incl. tables" for more information.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related findings.

Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The only haematological effect was a minimal increase in the number of circulating platelets in males at 5000 ppm. Minimal differences between control and high dose haematological values that were statistically significant or showed a positive trend are considered to reflect physiological variation since individual values were within reference ranges. Lower lymphocyte counts were noted in female groups from 250 ppm onwards attaining level of statistical significance while the dose-response relationship was equivocal.
See Table 4 in "Any other information on results incl. tables" for more information.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Minor treatment-related effects on clinical chemistry occurred in males at ≥1250 ppm and in females at ≥2500 ppm. The effects were increased urea levels in both sexes, and reduced glucose levels and increased creatinine levels in males only. Cholesterol levels were also raised in both sexes but at 5000 ppm only. Minimal changes in the electrolyte balance and phosphate of both sexes are considered to be of no toxicological consequence.
See Table 5 in "Any other information on results incl. tables" for more information.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no effects on any urinary parameters.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and/or relative weights of the adrenals, liver, kidneys, heart and spleen were increased in males at 5000 ppm. Relative liver and kidney weights were also marginally increased in males at 2500 ppm. There was no histopathological correlation to the increased heart weight of males at 5000 ppm. Treatment-related effects in females were confined to a minimal reduction in thymus weights at 5000 ppm.
See Table 6 in "Any other information on results incl. tables" for more information.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The liver, kidney, spleen and adrenal glands were identified as target organs for the test substance. Changes in liver morphology at 2500 and 5000 ppm were minimal to moderate centrilobular hypertrophy and an increased incidence of lymphohistiocytic infiltration of the parenchyma and in the males only, an increased incidence of cholangiofibrosis of bile ducts. Females at 5000ppm also showed minimal pigmentation of Kupffer cells. Changes in kidney morphology occurred at ≥250 ppm in males and ≥2500 ppm in females. Lesions detected in males were minimal to marked hyaline change in the tubular epithelium, acute and chronic tubular lesions, and an increased incidence of tubular basophilic proliferation. Increased incidence of pelvic dilatation and epithelial hyperplasia, and tubular cast formation were also evident. The pattern of effects in male rat kidneys at ≥250 ppm is consistent with α2µ-globulin nephropathy. Immunohistochemical studies have demonstrated the occurrence of α2µ-globulin nephropathy following the substance treatment. α2µ-globulin nephropathy is generally accepted to be unique to male rats and not relevant to human risk assessment. Renal lesions in females were confined to an increased incidence of chronic tubular lesions and an increase in the severity of nephrocalcinosis. There was an increased incidence of fatty change in the adrenal cortex in males at ≥1250 ppm and in females at ≥ 2500ppm. In the spleen, there was an increased incidence and severity of extramedullary haemopoiesis and haemosiderosis in males at 5000ppm and an increase in the severity of haemosiderosis in females at 2500 and 5000ppm
See Table 7 in "Any other information on results incl. tables" for more information.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
17.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
92.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
168 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
17.6 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
182 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
84.9 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
182 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 1: Intergroup comparison of body weights and body weight gain (g) – selected time points

Dose level [ppm]

Dietary concentration of the test substance (ppm)

Males

Females

0

25

250

1250

2500

5000

0

25

250

1250

2500

5000

Body weight [g]:

 

 

 

 

 

 

 

 

 

 

 

 

- week –1

145.4

151.2

147.0

149.8

157.6

151.4

134.7

138.4

135.6

134.9

139.9

135.0

- week 4

348.4

345.1

338.6

316.5$

328.8$

297.8*$

212.6

220.1

217.5

209.2

223.6

209.2

- week 7

439.2

416.2

423.3

382.0$

391.9$

356.6*$

237.2

247.3

241.6

236.2

245.9

232.9

- week 13

528.7

491.0

507.7

448.8-

467.6-

429.5*-

263.6

279.5

269.4

267.9

271.2

256.5

Body weight gain [g]&

383.3

339.8

360.7

299.0

310.0

278.1

128.9

141.1

133.8

133.0

131.3

121.5

% gain (relative to control) &

 

-11.3

-5.9

-22.0

-19.1

-27.4

 

9.5

3.8

3.2

1.9

-5.7

* p0.05 (Wilcoxon); $ negative trend (Jonckheere)

& derived values not analysed statistically

 

Table 2: Intergroup comparison of food consumption (g/animal/week) – selected time points

Dose level [ppm]

Dietary concentration of the test substance (ppm)

Males

Females

0

25

250

1250

2500

5000

0

25

250

1250

2500

5000

Food consumption [g]:

 

 

 

 

 

 

 

 

 

 

 

 

week 1

153.7

156.0

151.7

142.2

142.1$

117.7*$

99.85

110.6

113.6@

103.7

108.5

101.1

week 4

193.3

194.0

193.8

174.6

174.9$

162.8*$

123.7

127.5

130.4

127.0

133.9

122.3

week 7

197.9

182.5

191.5

171.3

179.9

159.0*$

127.2

127.6

124.5

123.3

125.7

120.0

- overall week-1-13&

2551.7

2446.7

2515.9

2280.9

2375.0

2183.4

1641.3

1679.9

1691.7

1636.4

1695.5

1626.5

% (relative to control)&

 

-4.1

-1.4

-10.6

-6.9

-14.4

 

2.4

3.1

-0.3

3.3

-0.9

* p0.05 (Wilcoxon); $ negative trend (Jonckheere); @ positive trend (Jonckheere)

& derived values not analysed statistically

 

Table 3: Mean Dose Received (mg/kg bw/day, based on analysed concentration*)

Test substance (ppm)

25

250

1250

2500

5000

Males

1.74

17.6

84.9

168

329

Females

1.88

19.2

92.5

182

359

* Dose rates (based on nominal dietary levels of the test substance) were calculated in terms of mg /kg body weight.

 

Table 4: Intergroup comparison of selected haematology parameters

Dose level [ppm]

Dietary concentration of the test substance (ppm)

0

25

250

1250

2500

5000

Males

 

 

 

 

 

 

Plt [x10^9/L]

996.0

987.5

957.8

953.0

989.4

1117

Females

 

 

 

 

 

 

Plt [x10^9/L]

956.9

936.2

971.8

1006

1001

999.4

no statistically significant differences

  

Table 5: Intergroup comparison of selected clinical chemistry parameters

Dose level [ppm]

Dietary concentration of the test substance (ppm)

0

25

250

1250

2500

5000

Males

 

 

 

 

 

 

Glucose [mmol/L]

8.244

8.787

7.949

7.274*-

7.530-

7.205-

Urea [mmol/L]

5.422

5.515

5.301

6.141

6.346

6.726+

Creatinine [mmol/L]

52.88

55.77

54.98

60.65+

59.03+

64.28*+

Cholesterol [mmol/L]

2.094

2.096

2.092

2.005

2.375

2.549+

Na+ [mmol/L]

142.8

142.2

142.4

142.0

141.4*-

142.1-

Cl-[mmol/L]

100.6

101.1

100.4

98.96-

98.24-

98.73-

PO4inorganic[mmol/L]

1.656

1.535

1.579

1.733

1.747+

1.810+

Females

 

 

 

 

 

 

Glucose          [mmol/L]

7.320

7.582

7.554

7.154

7.246

7.152

Urea [mmol/L]

7.319

6.828

7.500

6.814

8.340

7.943

Creatinine [mmol/L]

56.23

53.37

59.63

56.19

59.81

57.16

Cholesterol [mmol/L]

2.027

2.249

2.062

2.026

2.256

2.513

Na+ [mmol/L]

142.7

141.9

141.9

140.9

140.8-

141.1-

Cl-[mmol/L]

103.0

102.5

101.9

101.7

101.7-

100.6-

PO4inorganic [mmol/L]

1.171

1.143

1.200

1.233

1.161

1.372

* p 0.05 by Dunnett's "t" Test (two-tailed); - / + negative / positive trend (Jonckheere)

  

Table 6: Intergroup comparison of treatment-related organ weight changes

Organ

Dose level [ppm]

Male

Female

 

 

Absolute weight

Relative weighta

Absolute weight

Relative weighta

Liver [g]

0

20.60

40.82

9.615

38.29

 

25

19.30

40.85

9.792

36.71

 

250

20.16

41.45

9.683

37.70

 

1250

17.58

40.62

9.344

36.83

 

2500

19.39

43.36

9.426

36.63

 

5000

20.52

49.32*+

9.932

40.34

Kidney [g]

0

3.224

6.434

1.947

7.769

 

25

3.133

6.625

2.060

7.719

 

250

3.171

6.534

2.070

8.057

 

1250

2.921

6.764

1.963

7.708

 

2500

3.182

7.115

1.942

7.555

 

5000

3.260

7.857*+

2.005

8.128

Heart [g]

0

1.427

2.837

0.919

3.661

 

25

1.363

2.883

0.940

3.526

 

250

1.356

2.794

0.902

3.516

 

1250

1.373

3.156

0.923

3.634

 

2500

1.343

3.023+

0.914

3.565

 

5000

1.355

3.261*+

0.845-

3.475

Adrenals (both) [mg]

0

73.60

0.148

84.93

0.336

 

25

65.81

0.139

88.15

0.330

 

250

71.54

0.149

99.87

0.392

 

1250

64.15

0.149

84.04

0.330

 

2500

65.65

0.148

82.88

0.325

 

5000

88.68

0.213*+

88.14

0.359

Spleen [g]

0

0.764

1.509

0.466

1.863

 

25

0.756

1.601

0.513

1.927

 

250

0.778

1.600

0.429

1.671

 

1250

0.761

1.752

0.508

2.002

 

2500

0.753

1.688

0.519

2.019

 

5000

0.799

1.916*+

0.504

2.047

Thymus [mg]

0

598.2

1.184

341.6

1.368

 

25

575.2

1.217

338.8

1.266

 

250

596.6

1.232

306.4

1.190

 

1250

487.4

1.127

301.3

1.193

 

2500

527.8

1.183

315.9

1.226

 

5000

529.0

1.272

282.7

1.145

a: % of body weight, *: p0.05 by Dunnett's "t" Test (two-tailed); - / + :negative / positive trend (Jonckheere)

 

Table 7: Incidence of treatment-related histopathological findings

Dose level [ppm]

Dietary concentration of the test substance (ppm)

0

25

250

1250

2500

5000

MALES No. of animals

10

10

10

10

10

10

Liver

 

 

 

 

 

 

 - Hepatocellular hypertrophy   i

0

0

0

0

6

10

 - Lymphohistiocytic infiltration

3

4

6

5

4

9

 - Cholangiofibrosis

2

4

2

4

5

6

 - Pigmentation Kupffer cells

0

0

0

0

0

0

Kidneys

 

 

 

 

 

 

 - Hyaline change

1 (1.0)

0 (0)

4 (1.3)

8 (1.5)

10 (1.7)

10 (2.3)

 - Chronic tubular lesion

0

1

3

6

10

9

 - Acute tubular lesion

0

0

0

3

8

9

 - Lymphohistiocytic infiltration

2

2

1

3

6

3

 - Basophilic proliferation

2

1

2

4

6

10

 - Dilatation renal pelvis

1

0

2

1

5

1

 - Hyperplasia pelvic epithelium.

0

0

0

1

3

0

 - Cast formation

1

3

2

3

3

5

 - Calcification renal cortex

0

0

0

0

0

1

 - Nephrocalcinosis.

0

0

0

0

0

0

Spleen

 

 

 

 

 

 

 - Haemosiderosis

7 (1.0)

8 (1.9)

9 (1.4)

7 (1.6)

9 (1.7)

10 (2.0)

 - Extramedullary haematopoiesis.

3 (1.0)

2 (1.0)

3 (1.0)

5 (1.0)

2 (1.0)

5 (1.8)

Adrenal gland (cortex)

 

 

 

 

 

 

 - Fatty change

4

5

5

7

7

8

FEMALES No. of animals

10

10

10

10

10

10

Liver

 

 

 

 

 

 

 - Hepatocellular hypertrophy   

0

0

0

0

0

8

 - Lymphohistiocytic infiltration

4

5

6

7

9

10

 - Cholangiofibrosis

2

1

2

1

3

1

 - Pigmentation Kupffer cells

0

0

0

0

0

6

Kidney

 

 

 

 

 

 

 - Hyaline change         incidence

0

0

0

0

0

0

 - Chronic tubular lesion

4

5

7

7

9

10

 - Acute tubular lesion

0

0

0

0

0

0

 - Lymphohistiocytic infiltration

0

1

0

0

0

0

 - Basophilic proliferation

2

1

1

2

0

1

 - Dilatation renal pelvis

2

0

1

1

0

0

 - Hyperplasia pelvic epithelium

0

0

0

0

0

0

 - Cast formation

0

1

0

0

2

0

 - Calcification renal cortex

0

0

0

0

0

1

 - Nephrocalcinosis.

10 (1.6)

10 (1.8)

10 (1.9)

10 (1.7)

10 (2.6)

10 (2.7)

Spleen

 

 

 

 

 

 

 - Haemosiderosis

9 (1.7)

10 (2.3)

10 (2.1)

10 (1.9)

10 (2.6)

10 (2.7)

 - Extramedullary haematopoiesis.

4

6

6

7

2

3

Adrenal gland (cortex)

 

 

 

 

 

 

 - Fatty change

0

0

2

1

4

6

 

 

 

Applicant's summary and conclusion

Conclusions:
The NOAEL in this OECD 408 study was 250 ppm (males) and 1250 ppm (females), equivalent to dose levels of 17.6 mg/kg bw/day (males) and 92.5 mg/kg bw/day (females), based on reduced body weight gain and histological findings in the adrenals in males at 1250 ppm and histological changes in the kidneys, adrenals and spleen in females only at 2500 ppm. The absolute NOEL established for males, 25 ppm, is not relevant to human risk assessment since it is based on the occurrence of alpha-2µ-globulin nephropathy, a pathological condition unique to the male rat.
Executive summary:

In a GLP compliant study, performed according to OECD TG 408, groups of 10 male and 10 female Sprague-Dawley-derived rats (Tif:RAIf strain) were treated orally for 90 days with the test substance in the diet at concentrations of 0, 25, 250, 1250, 2500 and 5000 ppm. Clinical signs, body weight, food and water consumption were monitored throughout the study. Ophthalmoscopic examinations were performed before dosing commenced and towards the end of treatment. Haematology, clinical chemistry and urinalysis were performed at the end of the study. All animals were killed, subjected to necropsy and post mortem examination, major organs were weighed and a full range of tissues were examined microscopically. Mean achieved daily dose levels (corrected for the analytical purity of the test substance) were 0, 1.74, 17.6, 84.9, 168 and 329 mg/kg bw/day (males) and 0, 1.88, 19.2, 92.5, 182 and 359 mg/kg bw/day (females), respectively. There were no treatment-related deaths or clinical signs of an adverse reaction to treatment. Ophthalmoscopic examinations revealed no evidence of ocular toxicity. Body weight gains were significantly retarded and food consumption was reduced in male groups at ≥ 1250 ppm. Weight gains and food consumption of all female groups and the male groups at up to 250 ppm were unaffected by treatment. Water consumption in both sexes at 5000ppm was slightly increased. There was a minimal increase in the number of circulating platelets in males at 5000ppm. Lower lymphocyte counts were noted in female groups from 250 ppm onwards attaining level of statistical significance while the dose-response relationship was equivocal. Minor treatment-related effects on clinical chemistry occurred in males at ≥ 1250 ppm and in females at ≥ 2500 ppm. The effects were increased urea levels in both sexes, and reduced glucose levels and increased creatinine levels in males only. Cholesterol levels were also raised in both sexes but at 5000 ppm only. There were no treatment-related effects on urine parameters. There were no treatment-related macroscopic effects on any tissue or organ examined. The absolute and/or relative weights of the adrenals, liver, kidneys, heart and spleen were increased in males at 5000 ppm. Relative liver and kidney weights were also marginally increased in males at 2500 ppm. There was no histopathological correlation to the increased heart weight of males at 5000 ppm. Treatment-related effects in females were confined to a minimal reduction in thymus weights at 5000ppm. Changes in liver morphology at 2500 and 5000 ppm were minimal to moderate centrilobular hypertrophy and an increased incidence of lymphohistiocytic infiltration of the parenchyma and in the males only, an increased incidence of cholangiofibrosis of bile ducts. Females at 5000 ppm also showed minimal pigmentation of Kupffer cells. Changes in kidney morphology occurred at ≥250 ppm in males and ≥2500 ppm in females. Lesions detected in males were minimal to marked hyaline change in the tubular epithelium, acute and chronic tubular lesions, and an increased incidence of tubular basophilic proliferation. Increased incidence of pelvic dilatation and epithelial hyperplasia, and tubular cast formation were also evident. Renal lesions in females were confined to an increased incidence of chronic tubular lesions and an increase in the severity of nephrocalcinosis. There was an increased incidence of fatty change in the adrenal cortex in males at ≥1250 ppm and in females ≥2500 ppm. In the spleen, there was an increased incidence and severity of extramedullary haemopoiesis and haemosiderosis in males at 5000 ppm and an increase in the severity of haemosiderosis in females at 2500 and 5000 ppm. The NOAEL in this study was 250 ppm (males) and 1250 ppm (females), equivalent to dose levels of 17.6 mg/kg bw/day (males) and 92.5 mg/kg bw/day (females), based on reduced body weight gain and histological findings in the adrenals in males at 1250 ppm and histological changes in the kidneys, adrenals and spleen in females at 2500 ppm. The absolute NOEL established for males, 25 ppm, is not relevant to human risk assessment since it is based on the occurrence of alpha-2µ-globulin nephropathy, a pathological condition unique to the male rat.