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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 February - 21 March 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
other: Dose range finding study for short-term study
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16 - 30 January 1996
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Remarks:
Summary dose range finding study (documentation insufficient for assessment)
Reason / purpose for cross-reference:
other: Dose range finding study for short-term study
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Dose range finding study (subacute)
- Short description of test conditions: Female rats were treated with the test substance at a dose of 100, 300 or 1000 mg/kg bw/day for a period of 12 days. The test substance was administered dermally for 6h/day and 5 days per week.
- Parameters analysed / observed: clinical symptoms, skin redness, food consumption, body weights, clinical chemistry analysis (cholinesterase inhibition), organ weights and macroscopic analysis
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
female
Type of coverage:
not specified
Vehicle:
not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
12 days
Frequency of treatment:
5 days per week in the first week, 6h/day
7 days per week in the second week, 6h/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

CLINICAL OBSERVATIONS: Yes

DERMAL IRRITATION: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes (cholinesterase inhibition)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (macroscopic analysis, organ weights)

HISTOPATHOLOGY: No
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: decreased cholinersterase activity ChE activity in erythrocytes (42%), plasma (33%) and brain (9%)
300 mg/kg bw/day: decreased cholinersterase activity ChE activity in erythrocytes (33%), plasma (30%) and brain (2%)
100 mg/kg bw/day: decreased cholinersterase activity ChE activity in erythrocytes (21%), plasma (23%) and brain (2%)

Urinalysis findings:
not examined
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):
1000 mg/kg bw: adrenal weights increased
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Based on the results of the conducted dose range finding study, especially the flat dose-response curve in ChE inhibition, doses of 50 - 250 - 1000 mg/kg bw/day were applied in the short-term repeated dose study (please refer to 1997k).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
Adopted: 12 May 1991
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide
EC Number:
605-140-1
Cas Number:
158237-07-1
Molecular formula:
C16H20ClN5O2
IUPAC Name:
4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HsdCpb:WU
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks (males), 15 weeks (females)
- Weight at study initiation: 243 to 279 g (males), 217 to 239 g (females)
- Housing: 5 animals (<180g body weight) or 3 animals (>180g body weight) separated by sex in polycarbonate cages type III during adaption period, individally in polycarbonate cages type IIA. Low-dust wood shavings type S 8/15 (Ssniff, Spezialdiaten GmbH, Soest/Westphalia, Germany) were used as litter.
- Diet: Altromin® 1321 meal (Altromin GmbH, Lage), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least seven days

DETAILS OF FOOD AND WATER QUALITY:
The tap water complied with drinking water standards in accordance with the Deutsche Trinkwasserverordnung.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55% ± 5%
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Remarks:
moistened with water
Details on exposure:
TEST SITE
- Area of exposure: 5.5 x 5.5 cm = 30.25 cm²
- % coverage: >10% body surface area
- Type of wrap if used: gauze-layer ,,Hansapor steril" patch moistened with water, gauze strip was secured by using ,,Peha-Haft" cohesive stretch tape (8 x 23 cm). Additionally, the mobility of the rats was impaired by a ,,Lomir Biomedical Inc." rat jacket, which was connected in two rats with a safety pin to the stretch tape.
- Time intervals for shavings or clipplings: twice weekly

REMOVAL OF TEST SUBSTANCE
- Washing: cleaned with soap and water
- Time after start of exposure: 6h after exposure

TEST MATERIAL
- Amount(s) applied: 50, 250 and 500 mg/kg bw/day
- Constant volume or concentration used: no
- For solids, paste formed: no, moistened with tap water

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analytical determinations (stability, homogeneity) were not performed, because the test substance was applied undiluted and only moistened with water immediately before application.
Duration of treatment / exposure:
4 weeks exposure + 2 weeks recovery
Frequency of treatment:
5 days per week for the first three weeks, 6h/day
7 days per week in the fourth week, 6h/day
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (main study)
5 (recovery period: control and 1000 mg/kg bw/day)
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: 12 day dose-range finding study (1996d) in female rats. In this study, the doses were 0-100-300-1000 mg/kg. On the application area neither erythema nor oedema of the skin were observed. The administration of 100 and 300 mg/kg showed no toxicologically significant effects. At 1000 mg/kg body weight increased adrenal weights were observed. At all dose levels cholinesterase activity was decreased in erythocytes, plasma and brain.
- Rationale for selecting satellite groups: reversibility of adverse effects
- Post-exposure recovery period in satellite groups: 2 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day, once daily at weekends and public holidays
- Cage side observations checked: body surfaces and orifices, posture, general behavior, breathing and excretory products, including irritation at the dose site, any clinical signs (findings) and abnormalities were recorded

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice a day, once daily at weekends and public holidays

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: before start of the study and during the study each day before treatment

BODY WEIGHT: Yes
- Time schedule for examinations: before the study was initiated and at the beginning of each study week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 (main groups) and Day 43 (recovery groups)
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: leucocytes (leuco), erythrocytes (ery), hemoglobin (HB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelet count (thro), hepato-quick (hquick) differential blood count: lymphocytes (lym), polymorphonuclear granulocytes (segm), eosinophils (eos), monocytes (mono), normal red blood cells (norm RBC)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: glucose test - Day 22/23 (main groups) and Day 41 (recovery groups), Day 28 (main groups) and Day 43 (recovery groups)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: aspartat aminotransferase (ASAT), alkaline phosphatase (APh), alanine aminotransferase (ALAT), , glutamate dehydrogenase activities (GLDH), gamma-glutamyl transpeptidase activity (γGT), cholesterol (Chol), triglycerides (trigl), creatinine (crea), urea, bilirubin total (bible-t), protein total (prot), albumin, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), phosphorus (P), glucose, cholinesterase activity in plasma (CHE), cholinesterase activity in erythrocytes (CHE/E) and cholinesterase activity in brain (CHE/B)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organ weights: adrenal glands, brain, heart, kidneys, liver, lung, spleen, testes, thymus

HISTOPATHOLOGY: Yes (adrenal glands, brain, heart, kidneys, liver, skin (treated and untreated), spleen, testes, thymus, thyroid gland with parathyroid gland, urinary bladder, physical identifier (tattooed ears), one liver lobe and lungs and all organs or tissues with macroscopic findings)
Statistics:
The quantitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by "+" for p<0.05 and "++" for p<0.01.
The Dunnett test:body weight, feed consumption and organ weight data (relative organ weights subsequent to logarithmic transformation).
The clinical findings are presented by individual animal findings with information on the time of occurrence in question.
Calculation of means and variances was based in part on the non-rounded original values.


Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
no effects observed
Dermal irritation:
no effects observed
Description (incidence and severity):
no effects observed
Mortality:
no mortality observed
Description (incidence):
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
For details please refer to Tables 1 and 2 in "any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
For details please refer to Tables 3 and 4 in "any other information on results incl. tables".
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):
250 mg/kg bw/day: decreased MCV in females
1000 mg/kg bw/day: decreased MCV in females; no correlation with the doses administered and with the other sex, and all individual values were within the 2 standard deviation-range of the historical control values

For details please refer to Tables 5 in "any other information on results incl. tables".
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: decreased ChE activity in erythrocytes in both sexes, in brain in females only (reversible), increased protein and albumin levels in males (reversible)

For details please refer to Tables 6 in "any other information on results incl. tables".
Urinalysis findings:
not examined
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):
1000 mg/kg bw/day: increased thymus and testes weights at end of treatment period, but values were comparable to the controls of the recovery group (in the recovery group all organ weights were comparable to controls)

For details please refer to Tables 7 in "any other information on results incl. tables".
Gross pathological findings:
no effects observed
Description (incidence and severity):
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
no effects observed
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to this dose level
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
System:
nervous system
Organ:
other: ChE activity inhibited in erythrocytes and brain (statistically significant inhibition by 20% or more in erythrocytes or brain is considered a clear toxicological effect)
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Body weight (g) - main groups

dose (mg/kg)

day 0

day 7

day 14

day 21

day 28

sex

0

260

 

279

299

311

323

male

50

253

265

283

301

316

male

250

264

284

303

319

335

male

1000

253

278

295

311

321

male

0

227

227

232

234

237

female

50

225

224

230

233

236

female

250

227

224

229

233

232

female

1000

228

226

230

231

235

female

* statistical evaluation not possible

+ = 5 % significance level

 

Table 2: Body weight (g) - recovery groups

dose (mg/kg)

day 0

day 7

day 14

day 21

day 28

day 35

day 42

sex

0

259

271

278

290

295

320

319

male

1000

261

278

294

305

321

342

339

male

0

227

229

233

236

241

247

239

female

1000

227

227

231

233

242

243

237

female

+ = 5 % significance level

 

Table 3: Mean feed intake

Mean feed intake – main groups

 

day 7

day 14

day 21

day 28

day 7

day 14

day 21

day 28

 

dose (mg/kg)

g/animal/day

g/kg bw/day

sex

0

23

25

24

23

83

84

77

70

male

50

22

24*

24

24

84

84*

80

77

male

250

24

26

26

23

83

86

80

70

male

1000

23

25

25

26

83

83

80

81+

male

0

17

19

18

19

73

82

77

79

female

50

17

19

17

18

74

81

75

77

female

250

18

19

19

19

78

85

80

83

female

1000

17

19

18

19

76

84

77

83

female

* statistical evaluation not possible

+ = 5 % significance level

 

Table 4: Mean feed intake – recovery groups

Mean feed intake – recovery groups

 

day 7

day 14

day 21

day 28

day 35

day 42

day 7

day 14

day 21

day 28

day 35

day 42

 

dose (mg/kg)

g/animal/day

g/kg bw/day

sex

0

22

23

23

23

24

23

82

82

79

76

75

73

male

1000

24

25+

25

26

25

23

87+

86+

82

81

74

68

male

0

18

19

19

21

20

17

79

83

82

86

81

72

female

1000

18

20

19

21

20

17

81

85

82

85

82

73

female

+ = 5 % significance level

 

Table 5: Hematology

dose (mg/kg)

leuco (109/L)

ery (1012/L)

HB (g/L)

HCT (L/L)

MCV

(fl)

MCH (pg)

MCHC (g/L ery)

thro (109/L)

hquick sec

sex

0

7.6

8.86

148

0.473

53.3

16.7

314

894

31.8

male (main group)

50

7.3

8.74

150

0.478

54.7

17.2

314

1029

30.6

male (main group)

250

8.8

8.94

151

0.482

54.2

17.0

314

1036

31.3

male (main group)

1000

7.3

8.78

149

0.473

53.8

17.0

316

1070

32.3

male (main group)

0

6.0

8.57

146

0.490

57.1

17.0

298

1015

27.2

female (main group)

50

5.8

8.51

145

0.466

54.8

17.0

310++

842+

27.1

female (main group)

250

5.9

8.54

139

0.454+

53.2++

16.3

306+

892

26.8

female (main group)

1000

5.4

8.44

138

0.455

53.9+

16.3

303

952

26.4

female (main group)

0

8.3

9.14

148

0.481

52.6

16.2

308

1100

26.2

male (recovery group)

1000

7.1

8.90

146

0.479

53.8

16.4

305

920

26.1

male (recovery group)

0

4.4

8.18

139

0.448

54.7

17.0

311

901

24.9

female (recovery group)

1000

5.5

8.35

140

0.446

53.4

16.7

314

910

25.2

female (recovery group)

+ = 5 % significance level

++ = 1 % significance level

 

Table 6: Activity cholinesterase

dose (mg/kg)

CHE plasma kU/L

CHE plasma %

CHE erythrocytes kU/L

CHE erythrocytes %

CHE brain kU/L

CHE

brain %

sex

0

0.41

-

0.83

-

12.00

-

male

50

0.41

0

0.74

-11

12.64+

+5

male

250

0.43

+5

0.73

-12

12.36

+3

male

1000

0.46

+12

0.62+

-25+

1165

-3

male

0

1.95

-

0.63

-

12.09

-

female

50

1.75

-10

0.65

+3

11.75

-3

female

250

1.92

-2

0.62

-2

11.42

-6

female

1000

1.55

-21

0.41++

-35++

10.88+

-10

female

0

0.55

-

1.39

-

12.15

-

male (recovery group)

1000

0.50

-10

1.02

-27

11.92

-2

male (recovery group)

0

1.98

-

1.04

-

11.49

-

female (recovery group)

1000

2.13

+8

1.10

+6

11.17

-3

female (recovery group)

+ = 5% significance level

++ = 1 % significance level

Table 7: Absolute and relative organ weights

Absolute organ weights (mg)

dose (mg/kg)

body weight (g)

brain

adrenals

heart

lung

liver

spleen

thymus

kidneys

testes

sex

0

323

1843

43

1062

1665

12956

602

367

2136

3062

male (main group)

50

316

1699

43

1042

1542

12481

637

401

1920

3075

male (main group)

250

335

1706

42

1035

1730

12907

661

437

2069

3208

male (main group)

1000

321

1769

47

1069

1713

13668

633

487

2191

3468+

male (main group)

0

237

1672

64

870

1399

9160

521

309

1543

-

female (main group)

50

236

1727

64

848

1293

8685

504

304

1558

-

female (main group)

250

232

1779

67

832

1225

9312

449

253

1494

-

female (main group)

1000

235

1753

59

865

1268

9245

500

305

1526

-

female (main group)

0

320

1788

37

972

1613

11918

608

407

2092

3346

male (recovery group)

1000

342

1833

41

1107

1586

13340

623

407

2291

3315

male (recovery group)

0

242

1738

59

941

1275

8791

472

334

1608

-

female (recovery group)

1000

237

1639

57

877

1264

8543

546

356

1451

-

female (recovery group)

Relative organ weights (mg)

dose (mg/kg)

body weight (g)

brain

adrenals

heart

lung

liver

spleen

thymus

kidneys

testes

sex

0

323

579

13

330

519

4013

188

112

663

960

male (main group)

50

316

538

14

329

486

3944

202

127

607

972

male (main group)

250

335

512

13

310

517

3858

199

131

621

961

male (main group)

1000

321

556

14

332

531

4250

197

152+

683

1085

male (main group)

0

237

708

27

368

591

3871

220

131

653

-

female (main group)

50

236

731

27

359

547

3676

213

129

660

-

female (main group)

250

232

767

29

358

527

4005

193

109

644

-

female (main group)

1000

235

745

25

367

539

3933

213

130

649

-

female (main group)

0

320

560

12

303

503

3726

189

126

655

1046

male (recovery group)

1000

242

536

12

323

465

3894

183

229

670

971

male (recovery group)

0

242

720

24

389

528

3636

195

138

666

-

female (recovery group)

1000

237

693

24

369

533

3594

229

150

612

-

female (recovery group)

+ = 5 % significance level

 

Applicant's summary and conclusion