Registration Dossier

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:
Aug - Oct 1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted in 1998
Deviations:
yes
Remarks:
no ophtalmological examination, functional observations, epididymis/uterus/ovaries/brain weights
GLP compliance:
yes
Remarks:
The study was conducted in compliance with GLP but no check of study conduct by quality assurance unit was performed.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch No.: 9519

Test animals

Species:
rat
Strain:
other: Wistar, TNO W. 74 (Bor: WISW (SPF Cpb))
Details on species / strain selection:
The species is recommended in test guidelines for subchronic toxicological studies. Additionally, animals of this strain have been employed for many years for toxicological studies at the testing laboratory. Historical data on their physiology and spontaneous alterations is available.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 5 weeks
- Weight at study initiation: 65 - 87 g (males), 63 - 82 g (females)
- Housing: individual in Makrolon® cages type II on low-dust wood granules (during study period), several animals separated per sex in Makrolon® cages type III
- Diet: fixed-formula standard diet: Altromin® 1321 meal (during study period), Altromin® 1324 pellets (during acclimatisation period; Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: duration not specified

DETAILS OF FOOD AND WATER QUALITY: The tap water was of drinking quality. The nutritive composition and contaminant content of the standard diet were routinely spot-checked and analysed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): fixed-formula standard diet: Altromin® 1321 meal
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity, stability and content of the test compound in the diet were examined using GC analysis. Before start of study three different samples of the mid dose (500 ppm; start, middle, end) were collected for the homogeneity analysis as well as stability analysis after storage at the refrigerator over a period of 14 days. The formulations were stable and showed homogeneous distribution (99 – 105% of nominal). Additionally, analysis of the high dose (15000 ppm) after 7 days storage under cage conditions proved stability of the formulation (97% of nominal). The test compound content in the diet established by spot-checks during the study corresponded to the specified nominal contents (86 – 131% of nominal). Slight variations in these figures do not affect the assessment of the results of the study.
Duration of treatment / exposure:
at least 3 months
Frequency of treatment:
continously (via diet)
Doses / concentrationsopen allclose all
Dose / conc.:
150 ppm
Remarks:
equivalent to approximately 10 and 20 mg/kg bw/day in males and females, respectively
Dose / conc.:
500 ppm
Remarks:
equivalent to approximately 40 and 50 mg/kg bw/day in males and females, respectively
Dose / conc.:
1 500 ppm
Remarks:
equivalent to approximately 120 and 170 mg/kg bw/day in males and females, respectively
No. of animals per sex per dose:
20
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and holidays)
- Cage side observations included clinical sysmptoms, abnormalities, mortalities

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: before treatment, then once weekly and prior to necropsy

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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 month after start of study and at study termination
- Anaesthetic used for blood collection: Yes (ether; at study termination); no (after 1 month)
- Animals fasted: Not specified
- How many animals: 5 animals/sex/group
- Parameters examined: differential blood count, erythrocyte count, haemoglobin concentration, haematocrit, leukocyte count, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular cell volume (MCV), thromboplastin time, thrombocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1 month after start of study and at study termination (blood collection for the examination of the glucose concentration in the deproteinised whole blood without anaesthetic)
- Animals fasted: Not specified
- How many animals: 5 animals/sex/group
- Parameters examined: glucose, cholesterol, urea, total bilirubin, creatinine, total protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP)

URINALYSIS: Yes
- Time schedule for collection of urine: 1 month after start of study and at study termination (over approx. 16 h periods (overnight) a few days before blood sampling)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: semi-quantitative: ketone body, pH value, blood, glucose, bilirubin, urobilinogen, sediment (leucocytes, erythrocytes, epithelia, cylinders and other), quantitative: total protein

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals which died or were moribund and sacrificed during the study period were dissected at the earliest possible time and organs/tissues were subjected to thorough gross pathological examination. All surviving animals were sacrificed at study termination and a gross pathological examination was performed.

HISTOPATHOLOGY: Yes
The following organs of ten males and ten females of the control and highest dose group were histologically examined: adrenals, aorta, femur with bone marrow, brain, colon, duodenum, epididymis, eyes, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, mamma, oesophagus, ovaries, pancreas, pituitary, prostate, salivary glands, seminal vesicles, skeletal musculature with nervus ischiadicus, spleen, sternum with bone marrow, testicles, thymus, thyroid gland, trachea, urinary bladder, uterus, and all tissues with grossly apparent lesions.
Statistics:
Arithmetic group means, standard deviations and in the case of organ weights the upper and lower confidence limits on the confidence level of 1-alpha = 95% and 1-alpha = 99%. The values for the test collective were compared with the control collective by significance test (U-test) using H.B. Mann and D.R. Whitney´s method, or by Wilcoxon´s method on the significance level alpha = 5% and alpha = 1% (two-tailed).
Differences with p-values ≤ 0.01 and ≤ 0.05 were considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
150 ppm: 1/20 female died in connection with blood sampling in the 4th study week
500 ppm: 1/20 female died in connection with blood sampling in the 4th study week
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
500 ppm: statistically significantly reduced body weight gain in males
1500 ppm: statistically significantly reduced body weight gain in males
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
500 ppm: statistically significantly increased mean corpuscular haemoglobin concentration (MCHC) after 1 month
1500 ppm: statistically significantly increased mean corpuscular haemoglobin concentration (MCHC) after 1 month; statistically significant decreased erythrocyte count at study termination

FEMALES
150 ppm: statistically significantly increased leukocyte count at study termination; statistically significantly increased polymorphonuclear neutrophils at study termination; statistically significantly decreased lymphocyte count at study termination
500 ppm: statistically significantly increased leukocyte count at study termination; statistically significantly increased polymorphonuclear neutrophils at study termination; statistically significantly decreased lymphocyte count at study termination
1500 ppm: statistically significantly increased leukocyte count at study termination
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
150 ppm: statistically significantly decreased protein content at study termination (non-treatment-related); statistically significantly increased glucose content at study termination (non-treatment-related)
500 ppm: statistically significantly decreased alkaline phosphatase after 1 month
1500 ppm: statistically significantly decreased alkaline phosphatase after 1 month; statistically significantly decreased creatinine after 1 month

FEMALES
500 ppm: statistically significantly decreased urea content at study termination (non-treatment-related); statistically significantly decreased bilirubin content at study termination (non-treatment-related)
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
500 ppm: statistically significantly increased protein content at study termination

FEMALES
150 ppm: statistically significantly increased protein content at study termination
500 ppm: statistically significantly increased protein content at study termination
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
150 ppm: statistically significantly decreased lung weight relative to body weight
1500 ppm: statistically significantly decreased absolute liver weight; statistically significantly increased lung weight relative to body weight; statistically significantly increased absolute heart weight relative to body weight

FEMALES
500 ppm: statistically significantly decreased liver weight relative to body weight
1500 ppm: statistically significantly decreased liver weight relative to body weight; statistically significantly decreased kidney weight relative to body weight
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
0 and 1500 ppm: very slight to moderate isolated non-specific cellular or inflammatory cellular infiltrations in various organs, and vacuole formation in the cortical area of the adrenals
1500 ppm: pericarditis in 1/20 animal
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
MORTALITY
The autopsy of the mid- and high dose females which died in connection with blood sampling provided no indication of treatment-related effects. The probable cause of death is an overdose of diethyl ether and/or hypovolaemia.

BODY WEIGHT AND BODY WEIGHT GAIN
Due to the slight differences of body weights between mid- and high-dose males compared to control animals as well as due to the lacking dose-response-relationship, retarded body weight gain, observed in mid- and high-dose males, is considered to be not an adverse effect.

HAEMATOLOGICAL FINDINGS
Statistically significantly reduced erythrocyte count in males at 1500 ppm is within the normal range, and wrongly implies a treatment effect, due to the too high control value. The significantly increased leucocyte counts of the treated females from 150 ppm up to 1500 ppm do not correlate with dose, and represent values which are within the normal range. The isolated instances of significant variations in the females' differential blood counts are not of toxicological relevance, since the control values are relatively too high or too low in comparison to the corresponding figures after one study month. Thus, all haematological findings were not considered adverse or treatment-related.

CLINICAL BIOCHEMISTRY FINDINGS
No dose-response-relationship was observed for decreased protein and increased glucose content in low dose males as well as for decreased bilirubin and urea content in mid dose females. Thus, these effects were not attributed to the test substance.

URINALYSIS
Increased urinary protein content in males at 500 ppm and in females at 150 and 500 ppm were not correlated to the administered dose. Additionally, histological examination did not detect any indications of substance-induced kidney damage in the high dose males and females. Thus, this effect is considered non-adverse and not treatment-related.

ORGAN WEIGHT FINDINGS
Decreased absolute liver weights in males at 1500 ppm as well as increased relative liver weights in females at 500 and 1500 ppm are not associated with any indications of an effect on the liver or its functioning of the clinical chemistry, gross pathological and histopathological examinations. Additionally, variations of the liver weights were of slight nature. Thus, differences in liver weights were not considered adverse. The significantly higher relative weights of heart and lungs in the males, and the significantly lower relative weights of the females' kidneys, in each case after 1500 ppm, are most likely to be random findings without toxicological relevance.

HISTOPATHOLOGICAL FINDINGS
Since the effects noted in males of the control and high dose group were isolated and/or individual findings they are not attributed to the test substance.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 500 ppm
Based on:
test mat.
Remarks:
equivalent to 120 and 170 mg/kg bw/day in males and females, respectively
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Mortality

 

Control

150 ppm

500 ppm

1500 ppm

 

MALES

Number of animals examined

20

20

20

20

Mortality

0/20

0/20

0/20

0/20

 

FEMALES

Number of animals examined

20

20

20

20

Mortality

0/20

1/20

1/20

0/20

 

Table 2: Body weights (g)

Dose (ppm)

 

Week

0

1

2

3

4

5

6

7

8

9

10

11

12

13

MALES

0

Mean

76

114

150

187

213

241

261

280

295

306

319

327

338

341

SD

5

6

10

12

15

19

20

21

22

25

27

26

26

26

150

Mean

76

112

152

188

216

243

264

679

293

303

315

326

333

340

SD

5

7

10

12

16

19

19

21

20

19

21

23

24

27

500

Mean

75

110

145

178

203

227*

247*

263**

277**

286**

296**

305**

315**

322*

SD

6

9

11

14

15

16

17

18

21

21

23

23

22

23

1500

Mean

76

110

146

179

202*

227*

248*

265*

278**

288**

298**

309*

318**

325*

SD

6

7

11

14

15

16

17

18

17

19

19

20

20

23

 

FEMALES

0

Mean

74

98

113

126

134

146

154

162

167

170

173

179

184

186

SD

5

5

7

8

9

10

11

12

12

13

14

15

14

14

150

Mean

74

98

111

121

132

143

154

161

168

171

174

177

183

186

SD

4

5

7

6

6

7

6

7

7

8

7

9

8

10

500

Mean

73

98

111

124

133

144

153

159

165

167

172

176

180

186

SD

5

8

9

10

12

14

14

14

14

14

15

15

16

17

1500

Mean

75

98

112

123

129

141

152

162

167

170

174

178

183

185

SD

4

4

6

8

16

15

12

12

12

12

12

13

13

13

*p < 0.05; ** p < 0.01

Table 3: Haematological findings after 1 month and at study termination

Dose (ppm)

Mean corpuscular haemoglobin concentration (g/L)

Leucocyte count (giga/L)

Erythrocyte count

(tera/L)

Polymorphonuclear neutrophils (%)

Lymphocytes (%)

 

MALES – 1 month

0

304

9.6

7.51

8.6

90.6

150

312

100.5

7.44

9.8

89.6

500

316*

10.7

7.52

7.2

92.4

1500

313*

10.6

7.37

6.4

93.2

 

FEMALES – 1 month

0

313

6.5

7.57

7.6

91.8

150

315

7.7

7.57

6.2

93.8

500

316

7.7

7.29

9.4

90.2

1500

323

7.4

7.53

8.0

91.4

 

MALES – Study termination

0

311

12.1

9.13

6.6

92.4

150

308

12.7

8.79

9.0

89.4

500

312

11.9

8.80

9.0

89.6

1500

310

13.0

8.72*

7.0

91.4

 

FEMALES – Study termination

0

315

7.75

7.7

4.0

95.8

150

314

7.96

10.8*

7.8**

91.8**

500

314

7.82

10.9*

8.0**

91.8**

1500

316

7.87

10.2*

8.4

91.0

*p < 0.05; ** p < 0.01

Table 4: Clinical biochemistry findings after 1 month and at study termination

Dose (ppm)

Alkaline phosphatase (U/L)

Bilirubin (µmol/L)

Protein

(g/L)

Urea (mmol/L)

Creatinine (µmol/L)

Glucose (mmol/L)

 

MALES – 1 month

0

613

3.3

57.7

7.58

49

6.38

150

583

3.1

56.4

7.63

48

6.49

500

522*

3.5

57.6

7.46

44

6.16

1500

516*

3.0

56.2

7.75

42*

6.19

 

FEMALES – 1 month

0

328

3.0

56.6

6.73

46

5.88

150

318

2.8

54.2

7.25

51

5.47

500

325

2.6*

57.4

7.37

47

5.70

1500

336

2.8

56.7

7.09

48

5.68

 

MALES – Study termination

0

269

3.3

65.4

8.96

53

5.48

150

275

2.8

62.2**

8.53

54

6.11*

500

282

3.2

64.1

8.54

52

5.99

1500

254

2.7

64.2

8.45

56

5.88

 

FEMALES – Study termination

0

225

3.7

58.2

9.97

55

6.07

150

194

3.0

56.4

9.11

60

5.65

500

191

3.0

58.0

7.64**

52

5.65

1500

217

3.1

57.9

8.58

47

5.26

*p < 0.05; ** p < 0.01

Table 5: Urinalysis findings after 1 month and at study termination

Dose

Protein (g/L) after 1 month

Protein (g/L) at study termination

 

MALES

0

0.64

1.06

150

0.86

1.69

500

0.96

3.92**

1500

0.94

1.69

 

FEMALES

0

0.32

0.20

150

0.23

0.60*

500

0.25

1.39*

1500

0.13

0.35

*p < 0.05; ** p < 0.01

Table 6: Absolute and relative organ weights (mg)

Dose

Body weight (g)

Heart

Lung

Liver

Kindey

 

MALES - Absolute

0

341

938

1232

12339

2003

150

340

964

1284

12378

2053

500

322*

928

1212

11864

1910

1500

325*

974

1235

11653*

1928

 

MALES – Relative to body weight

0

n.a.

275

362

3607

588

150

n.a.

284

373*

3644

606

500

n.a.

288

375

3677

593

1500

n.a.

300*

380*

3584

595

 

FEMALES - Absolute

0

186

620

860

6770

1217

150

186

626

881

6575

1172

500

186

644

893

6478

1163

1500

185

631

901

6427

1146

 

FEMALES – Relative to body weight

0

n.a.

334

463

3639

655

150

n.a.

337

475

3540

632

500

n.a.

347

481

3488*

627

1500

n.a.

341

486

3458*

619*

*p < 0.05; ** p < 0.01

Applicant's summary and conclusion

Executive summary:

The potential toxicity of the test substance was assessed in a 90-day sub-chronic toxicity feeding study in the Wistar rat performed according to US EPA Guideline 82-1 and similar to OECD Guideline 408 and in compliance with GLP. 20 male and female Wistar rats per sex and dose group received the test substance at concentrations of 0, 150, 500 and 1500 ppm in the diet for 3 continuous months. Observations for clinical signs and mortality were made twice daily (once daily on weekends and holidays). Individual body weights, food consumption as well as detailed physical examinations for clinical signs of toxicity were determined/ performed once each week on all animals. Blood samples were collected for haematological and clinical chemistry examination from all surviving animals after 4 weeks and at study termination. Urine specimens were collected in 16 h intervals at several timepoints during the study period in each case a few days before blood sampling. All animals found dead or were moribund and sacrificed during the study period were dissected and the organs/tissues were subjected to thorough gross pathological examination. All surviving animals were sacrificed at termination and a gross pathological examination was performed. Organ weights of the heart, testicles, liver, lung, spleen, kidneys, adrenals and thymus were determined. Histopathological examinations were performed with the tissues from 10 males and females of the control and high dose group. Appearance, general behaviour, mortality and food intake were unaffected in any dose group. Two females (1/20 at 150 ppm, 1/20 at 500 ppm) died in connection with blood sampling. Autopsy of these animals did not indicate a relation between the treatment and death. Body weight gain was retarded in mid- and high-dose males while no effect on the body weights of females was noted. Since observations on the male body weight were of slight degree the effect is considered non-adverse. Urinalysis and haematological examinations revealed no treatment-related effects. With respect to clinical biochemistry, at the 1 month blood sampling significantly decreased alkaline phosphatase activity was noted in males at 500 and 1500 ppm. Since this effect was not apparent at study termination and in the absence of corresponding histopathological finding, decreased enzyme activity was not considered adverse. Clinical chemistry examination provided no further treatment-related effects. No treatment-related effects or abnormalities were found during gross pathological and histopathological examinations. Decreased absolute liver weights in males at 1500 ppm as well as increased relative liver weights in females at 500 and 1500 ppm were not associated with any indications of an effect on the liver or its functioning as evident in the clinical chemistry, gross pathological and histopathological examinations. Additionally, variations of the liver weights were of slight nature. Thus, differences in liver weights were not considered adverse. Further isolated findings on kidney, heart or lung weights were most likely to be random findings without toxicological relevance. Thus, under the conditions of the study, the NOAEL over a 3-month period of dietary administration with the test substance to the rat was ≥ 1500 ppm in both sexes (equivalent to 120 and 167 mg/kg bw/day in males and females, respectively).