Registration Dossier

Administrative data

Description of key information

Oral, sub-chronic (OECD 408, rats): NOAEL = 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)

Oral, chronic (OECD 452, dogs): NOAEL = 150 ppm (males and females), equivalent to dose levels of 4.05 mg/kg bw/day (males) and 4.49 mg/kg bw/day (females)

Dermal, sub-chronic (OECD 410, rats): NOAEL = 250 mg/kg bw/day (males) and 60 mg/kg bw/day (females), no local effects were observed

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
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
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
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
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).
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.
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
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
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

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

 

 

 

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
17.6 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP compliant, OECD 408 study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06 Feb 1996 to 07 Mar 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Tif: RAIf, SPF strain
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no

Achieved doses

Concentration analysis results: The mean test substance concentrations were 101.4%, 102.3%, 105.9% and 102.1% of nominal for formulations prepared at concentrations of 1, 10, 100 and 500 mg/mL, respectively predose. During administration concentrations were 116.0%, 115.4%, 113.3% and 109.9% of nominal for dose formulations prepared 10, 30, 125 and 500 mg/mL, respectively.

Homogeneity results: The test substance was homogeneously distributed in the vehicle and varied between ‑2% to +3% of the mean concentrations.

Stability results: The test substance was stable in the vehicle at room temperature over a period of 7 hours, covering the period of dosing.

 

Mortality: There were no mortalities.

 

Clinical observations: There were no treatment-related clinical signs and no local irritation at the application sites.

 

Body weight and weight gain: Weight gain of males at 1000 mg/kg was retarded during the first 2 weeks of treatment but was unaffected by treatment at other dose levels and in females at 1000 mg/kg.

 

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

 

Dose level of the test substance (mg/kg bw/day)

Males

Females

0

20

60

250

1000

0

20

60

250

1000

week 1

277.8

290.3

294.9*+

292.1

277.4

231.8

233.9

234.0

231.6

228.9

week 2

292.6

309.6*

311.4*

307.1

288.8

234.9

232.0

236.0

225.3

237.1

week 4

338.8

353.1

353.1

351.8

324.7

250.9

248.9

250.5

244.0

248.0

* p =< 0.05 (Wilcoxon), +/- p =< 0.01

 

Food consumption and compound intake: There were no treatment related effects on food consumption.

 

Haematology: There were no treatment-related effects on haematological profiles. All statistically significant values either showed no dose-relationship or recorded values were within historical ranges.

 

Clinical chemistry: Females at 250 and 1000 mg/kg had slightly raised plasma glucose levels and minimally raised alkaline phosphatase activities. Triglyceride levels were also slightly raised in females at 1000 mg/kg. Significantly elevated plasma K+ levels in all male groups are considered not to have been influenced by the test substance since control values were unusually low. Other differences from the controls in blood chemistry are considered not to be treatment related because they were too small to be of toxicological relevance (inorganic phosphate levels in males at 1000 mg/kg) or their occurrence showed no relationship to dose level.

 

Table 2: Intergroup comparison of treatment related clinical chemistry parameters

Parameter

Dose level of the test substance (mg/kg bw/day)

Male

Female

0

20

60

250

1000

0

20

60

250

1000

Glucose (mmol/L)

7.528

7.786

7.886

7.592

6.622

6.390

6.126

6.159

9.592*

9.739*+

Alk. phosphatase (u/L)

171.5

140.0

144.7

155.2

135.6

108.3

95.42

95.62

174.5*

139.8+

Triglycerides (mmol/L)

0.501

0.623

0.688

0.708

0.516

0.515

0.506

0.438

0.700

1.091*+

*p < 0.05 (Wilcoxon);+positive trend (p < 0.01 Jonckheere)

 

Sacrifice and pathology

Macroscopic findings: There were no treatment related macroscopic findings..

 

Organ weights: There were no treatment related effects on organ weights or ratios.

 

Microscopic findings: Morphological changes in the skin of the application site occurred at low incidence in both treated and control animals and are considered to have been induced by the treatment procedure rather than by the administration of the test substance. The liver, kidneys and adrenal glands were identified as target organs on the basis of histological findings. An increased incidence of minimal inflammatory cell infiltration and necrosis of single hepatocytes occurred in the liver of females at 250 and 1000 mg/kg. The elevated incidence of this lesion at 60 mg/kg is considered to fall within the limits of normal variation for this strain of rat. Effects on liver morphology were absent in the males. In the kidneys, minimal tubular hyaline change in males and minimal chronic tubular lesions in females were apparent at 1000 mg/kg. A single female at this dose level also showed minimal basophilic cell infiltration in the renal tubules. Lower dose levels were unaffected by these renal changes. In the adrenal glands, minimal inflammatory cell infiltration was evident in the cortex of females at 1000 mg/kg. No other groups were affected.

 

Table 3: Incidence of treatment-related histopathological findings

Organ/finding

Dose level of the test substance (mg/kg bw/day)

Male

Female

0

20

60

250

1000

0

20

60

250

1000

Adrenal glands:

cortical fatty change

inflammatory cell infiltration

 

4

0

 

4

0

 

3

0

 

3

0

 

4

0

 

1

0

 

0

1

 

1

0

 

1

1

 

1

3

Kidneys:

 

 

 

 

 

 

 

 

 

 

chronic progressive nephropathy

0

0

0

0

1

0

1

0

0

0

glomerulosclerosis

0

1

0

0

0

0

0

0

0

0

chronic tubular lesion

1

0

1

1

1

1

2

2

1

4

nephrocalcinosis

1

0

0

1

0

3

5

5

5

5

renal tubular cast

0

0

1

0

1

0

1

0

1

0

hyaline change

1

2

1

1

3

0

0

0

0

0

calcification

0

1

0

0

0

0

0

0

0

0

basophilic proliferation

2

2

0

2

1

0

0

0

0

1

Liver:

 

 

 

 

 

 

 

 

 

 

inflammatory cell infiltration

3

4

4

3

2

2

2

4

5

5

intrahepatic bile duct cholang/fibrosis

1

0

0

1

1

0

0

0

0

0

extramedullary hematopoiesis

0

0

0

0

0

1

0

0

0

1

Kupffer cell hyperplasia

0

0

0

0

0

1

0

1

0

0

hepatocyte necrosis

0

0

0

0

0

2

0

2

3

4

 

 

 

 

 

 

 

 

 

 

 

 

Conclusions:
The no-observed-effect-level (NOEL) was 250 mg/kg (males) and 60 mg/kg (females) based on the occurrence of renal tubular hyaline change and minimal growth retardation at 1000 mg/kg (males) and histological changes in the liver at 250 mg/kg (females). In view of the low order of toxicity by the percutaneous route, a 90-day dermal study is not required.
Executive summary:

In this repeated dose toxicity test, performed according to OECD 410 under GLP, groups of 5 male and 5 female Sprague-Dawley-derived rats (Tif: RAIf, SPF strain) were exposed dermally to the test material at dose levels of 0, 20, 60, 250 and 1000 mg/kg bw/day, by topical application under occlusive dressing for 6 hours/day, 5 days/week, for 4 weeks. The test substance was formulated as a suspension in aqueous 0.5% carboxymethylcellulose/0.1% Tween 80. Mortality and clinical signs were checked daily, individual body weights and food and water consumption were recorded weekly. Haematological and clinical chemistry investigations were performed on all animals towards the end of the treatment period. All animals were subjected to necropsy and post mortem examination, major organs were weighed and selected tissues examined histopathologically.

There were no mortalities, no clinical signs of an adverse reaction to treatment and no local irritation at the application site at any dose level. Weight gain of males at 1000 mg/kg was retarded during the first 2 weeks of treatment but was unaffected by treatment at other dose levels and in females at 1000 mg/kg. Food consumption was unaffected by treatment at all dose levels. There were no treatment related effects on haematology profiles. Females at 250 and 1000 mg/kg had slightly raised plasma glucose levels and minimally raised alkaline phosphatase activities. Triglyceride levels were also slightly raised in females at 1000 mg/kg. No macroscopic anomalies were detected at necropsy and all organ weights and ratios were unaffected by treatment. A treatment related increase in the incidence of minimal inflammatory cell infiltration and necrosis of single hepatocytes occurred in the liver of females at 250 and 1000 mg/kg. In the kidneys, minimal tubular hyaline change in males and minimal chronic tubular lesions in females were apparent at 1000 mg/kg. A single female at this dose level also showed minimal basophilic cell infiltration in the renal tubules. In the adrenal glands, minimal inflammatory cell infiltration was evident in the cortex of females at 1000 mg/kg.

The no-observed-effect-level (NOEL) was 250mg/kg (males) and 60mg/kg (females) based on the occurrence of renal tubular hyaline change and minimal growth retardation at 1000 mg/kg (males) and histological changes in the liver at 250 mg/kg (females). In view of the low order of toxicity by the percutaneous route, a 90-day dermal study is not required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP compliant, OECD 410 study

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP compliant, OECD 410 study

Additional information

Repeated dose toxicity: oral

Sub-acute

The test substance was administered orally for 28 days to groups of 5 male and 5 female Tif: RAif (SPF) rats, by admixture in the diet, at concentrations of 0, 100, 1000, 2500 and 10000 ppm (Bachmann, 1995). Mortality and clinical signs were checked daily, individual body weights and food and water consumption were recorded weekly. Haematological, clinical chemistry and urinalysis investigations were performed on all animals towards the end of the treatment period. All animals were subjected to necropsy and post mortem examination, major organs were weighed and selected tissues examined histopathologically. Mean achieved daily dose levels (corrected for the analytical purity of the test substance) were 0, 8.0, 81.7, 198.6 and 710.6 mg/kg bw/day in males and 0, 8.7, 89.3, 210.6 and 762.6 mg/kg bw/day in females at dose levels of 0, 100, 1000, 2500 and 10000 ppm, respectively. No deaths occurred and no clinical signs of a reaction to treatment were evident during the administration period. Body weight gain was reduced by 29% in males at 10000 ppm but females at this level were not affected. Minimal retardation of weight gain occurred in males at 2500 ppm. Food consumption was markedly reduced at 10000 ppm in males throughout the treatment period but in females was only slightly reduced during the first week. There were no treatment-related effects on haematological profiles at dose levels up to and including 10000 ppm. Cholesterol levels showed a dose-related increase in male groups treated at 1000 ppm, and in females treated at 10000 ppm. Other treatment-related effects were confined to 10000 ppm and are considered to be minor sequelae to renal and hepatic morphological changes. Slight changes occurred in plasma urea levels (increased) and Na+ concentrations (reduced) in both sexes, and in males ASAT activity was elevated and total protein and albumin levels were slightly decreased. All other clinical chemistry findings showed no relationship to dose level and are considered incidental to treatment with the test substance. There were no effects on any urine parameters. Treatment-related effects on organ weights occurred in both males and females at ≥1000 ppm. Liver weights were increased in both sexes at 10000 ppm. Relative kidney weights were increased in males at 2500 ppm, and in females at 1000 and 10000 ppm. At necropsy, dilatation of the renal pelvis occurred in 2 treated males, but no other treatment-related gross lesions were evident. Treatment-related microscopic changes were detected in males treated at ≥1000 ppm and females at ≥2500 ppm. The organs affected were liver, kidneys, adrenal glands and thyroid glands. Minimal to marked hypertrophy of centrilobular hepatocytes occurred in both sexes at 2500 and 10000 ppm. In some animals at 10000 ppm this was accompanied by depletion of glycogen deposits and/or cholangiofibrosis of bile ducts. In the kidneys, minimal to moderate hyaline change to the tubular epithelium occurred in males at 1000 or 2500 ppm and hyperplasia of the pelvic epithelium occurred in one animal of each sex at 10000 ppm. Many of the affected male animals also showed one or more additional kidney lesions. In the thyroid gland, hypertrophy of the follicular epithelium occurred in some males and females, but the severity was greater in males. No other treatment-related microscopic lesions occurred. The observed kidney toxicity in male rats is due to α2μ-globulin nephropathy which is considered not relevant for humans. The test substance at 10000 ppm in the diet provides daily dose levels that exceed the MTD, and the results are a suitable basis on which to establish dose levels for a 90-day study. The no-observed-adverse-effect-level (NOAEL) was 100 ppm in both sexes, equivalent to 8.0 mg/kg bw/day in males and 8.7 mg/kg bw/day in females, based on the findings of increased kidney weights in both sexes and hyaline changes to the renal tubular epithelium in males. A higher NOAEL of 1000 ppm in both sexes, equivalent to dose levels of 81.7 mg/kg bw/day (males) and 89.3 mg/kg bw/day (females), was considered for this study in the CLP Report prepared by the French competent authorities (European Commission 2018).

Sub-chronic

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 (Bachmann 1996). 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 5000 ppm was slightly increased. There was a minimal increase in the number of circulating platelets in males at 5000 ppm. 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 5000 ppm. 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 at ≥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.

Chronic (rat)

In a GLP compliant study (Bachmann, 1998), performed in accordance with OECD TG 453, groups of 70 male and 70 female Sprague-Dawley rats (strain Tif: RAIf, SPF) were administered with the test substance orally for 104 weeks, by admixture in the diet, at concentrations of 0, 10, 30, 500 or 1500 ppm for males (0, 0.41, 1.29, 21.0 and 63.0 mg/kg bw/day) and 0, 10, 30, 1000 or 3000 ppm for females (0, 0.48, 1.56, 50.3 and 155 mg/kg bw/day). Twenty animals/sex/group were designated for clinical laboratory investigations and 50 animals/sex/group for the oncogenicity study. Additional groups of 10 animals/sex/group were similarly treated with the test substance for 52 weeks and sacrificed for interim evaluation. Clinical observations were made daily, body weights and food consumption recorded weekly for 13 weeks and monthly thereafter, and water consumption recorded monthly. The eyes of all animals in the carcinogenicity subgroup were examined pre-test and at 104 weeks. Control and high-dose animals were also examined at weeks 26, 52 and 77. Laboratory investigations were performed at weeks 13, 27, 53, 78 and prior to termination on 20 animals/sex/group for haematology and on 10 animals/sex/group for clinical chemistry and urinalysis. All animals were subjected to detailed necropsy and post mortem examination. Organ weights of all animals which survived until terminal sacrifice were recorded. Tissue/organ samples from all animals were preserved. Microscopic examination of tissues was performed on all animals, of all treatment and control groups, with the exception of the 20 animals/sex/group designated for laboratory investigations. Survival incidence was not affected by treatment, there were no clinical signs of an adverse effect of treatment and the incidences of single and multiple palpable masses were unaffected by treatment at all dose levels. Body weight development in all male groups was unaffected by treatment, but females at 3000 ppm had slightly depressed weight gain from week 3 until termination, at which time cumulative weight gain was depressed by 12.6%. Food consumption was unaffected by treatment in both sexes at all dose levels, but water consumption of males at 1500 ppm only was raised by 13%. Ophthalmological examinations revealed no evidence of ocular toxicity at any time point. There were no treatment-related changes in haematology, blood chemistry and urinalysis parameters. A myeloid leukaemia was identified in one male at 1500 ppm in week 53, but this occurs spontaneously at low incidence in rats of this source and strain, and was considered unrelated to treatment. There were no treatment-related changes in haematology, blood chemistry and urinalysis parameters. There were no treatment-related effects on organ weights. Post mortem examination of animals sacrificed after 52 and 104 weeks, and animals that died spontaneously between 52 and 104 weeks, revealed no treatment-related lesions. No treatment-related neoplastic lesions occurred in animals sacrificed at 52 or 104 weeks or that died during the study. Non-neoplastic, treatment-related lesions at 52 weeks were increased incidences of renal tubular regenerative changes, chronic tubular lesion and tubular basophilic proliferation, in males at 500 and 1500 ppm. Minimally increased incidences of renal tubular and pelvic lymphocytic infiltration also occurred at 1500 ppm, but without tubular hyaline change. These alterations were considered to represent the sequelae of alpha-2µ-globulin mediated nephropathy. The kidneys of females at all dose levels were unaffected. There was a minimal increase in the severity of splenic haemosiderosis in females at 3000 ppm. In the liver, there were increased incidences of cholangiofibrosis in males at 10 ppm (5/10), 500 ppm (6/10) and 1500 ppm (4/10) compared to the controls (2/10), and increased incidence of inflammatory cell infiltration in males at 10 ppm (4/10), 30 ppm (3/10) and 500 ppm (5/10) relative to controls (1/10). These findings, which were not apparent after 104 weeks treatment, are considered incidental to treatment because of the lack of a dose relationship. Treatment-related, non-neoplastic changes in the kidneys and liver occurred in animals sacrificed at 104 weeks or that died between weeks 53 and 104. In the kidneys, a slightly increased incidence of slight/moderate chronic nephropathy occurred in males at 1500 ppm accompanied by a minimal increase in incidence of lymphocytic infiltration of the renal cortex at 1500 ppm. Two animals also showed tubular hyaline change. In the liver, a treatment-related increase occurred in the incidence of slight/moderate focal cellular alteration, generally of the clear cell subtype, in females at 3000 ppm. Based on no excess tumours occurring at any dose level, there was no indication of a carcinogenic potential. The no-observed-effect-levels (NOEL) for toxicity were 30 ppm (males) and 1000 ppm (females), equivalent to mean dose levels of 1.3 mg/kg bw/day (males) and 50.3 mg/kg bw/day (females), based on increased incidence of renal chronic tubular lesions and basophilic proliferation in males at ≥500 ppm, and foci of cellular alteration in the liver, and increased severity of splenic haemosiderosis, in females at 3000 ppm. Since the renal alterations observed in males at 500 and 1500 ppm are considered to be mediated by alpha-2µ-globulin, a mechanism specific to male rats, the observed renal toxicity has no relevance to human risk assessment. Therefore, it is concluded that a no-observed-adverse-effect-level (NOAEL) of >1500 ppm, equivalent to a dose level of >63.0 mg/kg bw/day, is applicable for males. Therefore, for both sexes, the overall NOAEL of relevance to human risk assessment is based on the female NOAEL of 50.3 mg/kg bw/day.

Chronic (dog)

In a chronic toxicity study (Altmann 1998) CGA293343 tech. was administered in the diet at concentrations of 0, 25, 150, 750 and 1500 ppm (=mg/kg diet) (groups 1, 2, 3, 4 and 5, respectively) for a period of at least 52 weeks to a total of 40 pedigreed Beagle dogs (4 males and 4 females per dose group). Mortality, clinical signs, body weight, and food consumption were monitored throughout the study for all animals. Eye examinations were conducted at pre-test, weeks 13, 26, and 52. At necropsy, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected (standard list), prepared for histological evaluation and examined microscopically.

All animals survived to scheduled necropsy and no treatment related changes were observed for clinical signs, eye examination, urine analysis and macroscopical examination. Minimal and transient loss in body weight was noted in group 5 females (1500 ppm) at treatment start and mean food consumption was slightly reduced in females of groups 4 and 5 (750 and 1500 ppm) at the beginning of treatment. Lower thrombin activities were recorded throughout the treatment period for males of group 5 treated at 1500 ppm. Males and females of group 4 and 5 (750 and 1500 ppm) showed a dose-related increase in plasma urea levels throughout the treatment period. In addition lower activities were recorded throughout the treatment period for alanine aminotransferase in males of groups 4 and 5 (750 and 1500 ppm), and females of group 5 (1500 ppm) had minimally lower albumin levels associated with lower albumin to globulin ratios. A minimal decrease in mean testes weight was noted in group 5 males (1500 ppm) due to 2 animals.

Microscopically, tubular atrophy of the testes observed in slightly increased incidence and severity in male animals of groups 4 and 5 (750 and 1500 ppm) was considered to be related to the treatment with the test article. In conclusion, administration of CGA293343 tech. admixed to the diet produced toxic effects at 750 and 1500 ppm. These changes included initial and transient loss in body weight and/or transient reduction in food consumption in females. In addition, histopathological examination of testes indicated a slight increase in the incidence and severity of tubular atrophy. Functional changes observed in the liver and kidneys were without morphological correlates. The no-observed-effect level (NOEL) and no-observed-adverse-effect level (NOAEL) are considered to be 150 ppm in males and females. This concentration in the diet corresponds to a daily intake of CGA293343 tech. of 4.05 mg/kg in males and 4.49 mg/kg in females.

Mode of Action and Human Relevance Assessment of Kidney Toxicity in Male Rats

In subacute (Bachmann, 1995) and subchronic (Bachmann, 1996) rat studies, the main treatment related findings in kidney were renal tubular hyaline change, tubular basophilia, acute tubular lesion (granular casts), chronic tubular lesion and casts formation. Basophilic proliferation or tubular basophilia, chronic tubular lesion and casts (hyaline) were considered parts or individual components of chronic progressive nephropathy and were observed in males only at doses ≥ 250 ppm. In the combined chronic/carcinogenicity study (Bachmann, 1998) an increase in the incidence and severity of tubular basophilia (regenerative changes in renal tubules) (12- month interim sacrifice) and chronic nephropathy (24-month terminal sacrifice) were the main findings at high doses (≥ 500 ppm) in male animals only.

Retrospective investigations (up to 104 weeks of oral exposure) have been performed on male and female rats to assess if these kidney related effects were alpha-2µ-globulin nephropathy driven (Weber 2000 a, b, c, d). These studies are summarised in section 5.10.1.4. Alpha-2µ-Globulin nephropathy is a well-described and well-characterised syndrome in male rats which has been extensively studied and previously demonstrated to be of no human relevance (IARC, 1999; EPA, 1991).

For all retrospective investigations, an increased staining intensity in the renal cortex (proximal tubular segments P1and P2) and other sections of the kidney were found in male rats treated with the test substance. In contrast to male rats, neither control nor treated female rats showed any specific staining in alpha-2µ-globulin immunohistochemistry. The immunohistochemical staining pattern, with respect to distribution and extent, as well as the sex-specificity correspond to effects of known alpha-2µ-globulin nephropathy-inducing compounds. Therefore, the results confirm that the hyaline change seen in renal tubules of males represent alpha-2µ-globulin nephropathy.

Criteria for the establishment of alpha-2µ-nephropathy have been defined by regulatory and authoritative bodies (IARC, 1999; EPA, 1991.) The essential evidence required for establishing a role for alpha-2µ-globulin nephropathy in rat leading to carcinogenicity (Lock and Hard, 2010) are:

1. The renal tumours occur only in male rats.

2. Acute exposure to the chemical causes hyaline droplet accumulation in proximal convoluted tubules.

3. The protein accumulating in hyaline droplets should be identified as alpha-2µ-globulin.

4. Hallmark histopathologic lesions, including granular casts at subchronic time-points and linear papillary mineralization at chronic stages, should be observed.

5. There should be an absence of hyaline droplets and other typical histopathological changes in female rats and mice of both sexes.

6. The chemical should be negative in short - term tests for genotoxicity.

Whilst no renal tumours were observed in any toxicity study with the test substance criteria 1 is fulfilled as the observed hyaline droplets in the renal proximal tubules, tubular basophilia and granular casts were confined to male rats only. Criteria 2 is fulfilled as the accumulation of hyaline droplets in proximal convoluted tubules was observed following short term (28 days) dosing with the test substance. Immunohistochemical staining of kidney tissues demonstrated that the observed hyaline droplets were composed of alpha-2µ-globulin, fulfilling criteria 3. Criteria 4 is fulfilled as the hallmark histopathological findings of alpha-2µ-globulin nephropathy were observed in male rats dosed with test substance for durations of 28 days to 2 years. No hyaline droplets, or any other histopathological changes in the kidney, were observed in female rats or mice of either sex, fulfilling criteria 5. Criteria 6 may be considered not to be applicable to non-cancerous alpha-2µ-globulin nephropathies, however the criteria is fulfilled as no evidence of genotoxicity has been observed in any in vitro or in vivo genotoxicity study with the test substance.

In conclusion, the observed kidney toxicity in male rats treated with the test substance is the result of alpha-2µ-globulin nephropathy and these findings are not relevant for human health risk or hazard assessment.

Mode of action of liver effects in repeat dose toxicity studies in the mouse

Special studies were performed to investigate the aetiology of adenoma and adenocarcinoma formation in mouse liver during an 18 month oncogenicity study (Bachmann 1998a). Upon dietary administration of the substance to Tif:MAGf (SPF) mice for 18 months, foci of cellular change as well as hepatocellular adenomas and adenocarcinomas were observed in the study. The substance was not genotoxic in a battery of in vitro and in vivo mutagenicity studies. Therefore, liver tumour formation in mice was mediated by a non-genotoxic mechanism. An extensive programme was conducted to explore and explain this mechanism. A number of studies performed in the course of the programme are summarised as supporting studies in the repeated dose toxicity section (Persohn 1995, Trendelenburg 1998, Weber 1998, Weber 2003, Noakes 2003a, Noakes 2003b). The mode of action for the development of these tumours, and the lack of relevance to man has been demonstrated. Further information is given in the section on carcinogenicity.

Justification for classification or non-classification

Based on the available information, the substance should be not classified as STOT RE in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.