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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21. Mar. 2005 - 18. Apr. 2005 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
460-100-9
EC Name:
-
Cas Number:
342573-75-5
Molecular formula:
Hill formula: C8H16N2O4S CAS formula: C6H11N2.C2H5O4S
IUPAC Name:
3-ethyl-1-methyl-1H-imidazol-3-ium ethyl sulfate

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System : Rat: Wi star Crl:(WI) BR (outbred, SPF-Quality). Recognised by international guidelines as the recommended test system (e.g. EPA,
FDA, OECD, EC).
Source : Charles River Deutschland, Sulzfeld, Germany.
Age at Start of Treatment : Approximately 6 weeks.
Number of animals : 20 males, 20 females (females were nulliparous and non-pregnant).
Randomisation : Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within 20% of the sex mean.
Identification : Earmark and tattoo.
Health inspection : A health inspection was performed prior to commencement of treatment to ensure that the animals are in a good state of health.
Animal Husbandry
Conditions : Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 20.0-23.2°C), a relative humidity of 30-70% (actual range: 25 - 73%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation : Group housing of 5 animals per sex in Macrolon plastic cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in Mill type; height 15 cm.) with sterilised sawdust as bedding material (Woody-Clean type 3/4, Tecnilab-BMI BV, Someren, The Netherlands) and paper as cage-enrichment (Enviro-dri, BMI, Helmond, The Netherlands). No cage-enrichment was provided during overnight activity monitoring. Certificates of analysis are examined and then retained in the NOTOX archives. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet : Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Water : Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Results of analysis for ingredients and/or contaminants of bedding, diet and water were assessed and did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Method : Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing (except on days 5 and 6).
Frequency : Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of 5 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.
Dose volume : 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared for use on day 3 were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 417285).
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days a week
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150, 1000 mg/kg bw.
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of a 5-day dose range finding study (NOTOX Project 431268).
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: no
- Section schedule rationale (if not random): random

Examinations

Observations and examinations performed and frequency:
Mortality / Viability : At least twice daily.
Clinical signs : At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded: Maximum grade 1: grade 0 = absent, grade 1 = present Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe
Functional Observations : During week 4 of treatment, the following tests were performed on all animals (abbreviations mentioned in the respective tables indicated between brackets): hearing ability (HEARING), pupillary reflex (PUPIL LlR), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent). motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Body weights : On days 1, 8, 15, 22 and 28.
Food consumption : Weekly.
Water consumption : Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Sacrifice and pathology:
All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution: (see table)
Other examinations:
Clinical Laboratory Investigations : Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (1.0 ml) and Liheparin treated tubes for clinical biochemistry parameters (0.5 ml). Parameters from haematology, clotting potential and clinical biochemistry were determined:

Results and discussion

Results of examinations

Clinical signs:
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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
(increased alanine aminotransferase activity, cholesterol, potassium and albumin levels in highest dose group)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY : No mortality occurred during the study period. There were no clinical signs of toxicity. Incidental findings that were noted in males included alopecia on head and/or neck, scabs, a broken tail apex and broken lower incisors. These findings are occasionally noted in rats of this age and strain, which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were observed in females.

BODY WEIGHT AND WEIGHT GAIN : Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period

FOOD EFFICIENCY : Food consumption before or after allowance for body weight was similar between treated and control animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)


HAEMATOLOGY : No toxicologically relevant changes occurred in haematological parameters of treated rats. The minor statistically significant decrease of prothrombin time in both sexes and lower partial thromboplastin time and relative neutrophil granulocyte counts in males at 150 mg/kg/day occurred in the absence of a dose-related distribution. Therefore, no toxicological significance was ascribed to these changes.

CLINICAL CHEMISTRY : The following statistically significant differences distinguished animals treated at 1000 mg/kg/day from control animals:
Increased alanine aminotransferase activity levels (males); Increased cholesterol levels (males); Increased potassium levels (males and females); Increased albumin levels (females). Changes achieving a level of statistical significance in females at 50 or 150 mg/kg/day (lower bilirubin and urea levels and higher inorganic phosphate levels) occurred in the absence of a treatment-related distribution. Therefore, these alterations in clinical biochemistry parameters were considered to be of no toxicological relevance.

NEUROBEHAVIOUR : Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.

ORGAN WEIGHTS : Organ weights and organ to body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY : Necropsy did not reveal any toxicologically relevant alterations. Incidental findings included enlarged and/or discoloured mandibular lymph nodes, fracture of the tip of the tail and fluid in uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance. No macroscopic findings were observed in control males, in males at 50 mg/kg/day and in females at 1000 mg/kg/day.

HISTOPATHOLOGY : There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Body weight

 

Treatment

Days

1

8

15

22

28

Days

1

8

15

22

28

Weeks

1

2

3

4

4

Weeks

1

2

3

4

4

Animal

males

Animal

females

GROUP 1 (CONTROL)

GROUP 1 (CONTROL)

1

155

196

236

258

274

21

152

187

213

233

244

2

183

244

301

339

358

22

151

175

191

219

229

3

191

255

304

340

368

23

157

183

208

224

236

4

178

242

296

329

353

24

140

167

188

200

219

5

176

241

298

341

365

25

153

182

214

236

249

GROUP 2 (50 MG/KG)

GROUP 2 (50 MG/KG)

6

163

218

267

307

332

26

151

181

202

222

228

7

174

228

275

302

332

27

148

167

179

202

218

8

180

222

256

275

287

28

154

185

204

232

253

9

181

238

297

339

364

29

147

178

202

223

235

10

187

249

309

353

374

30

151

182

204

211

231

GROUP 3 (150 MG/KG)

GROUP 3 (150 MG/KG)

11

168

220

266

292

312

31

155

179

204

217

243

12

171

218

263

295

323

32

155

190

208

235

252

13

187

246

310

356

378

33

155

177

187

210

223

14

177

230

280

315

336

34

146

175

197

221

235

15

180

235

295

331

358

35

152

178

202

208

239

GROUP 4 (1000 MG/KG)

GROUP 4 (1000 MG/KG)

16

187

245

305

354

381

36

147

182

197

224

231

17

172

233

285

314

336

37

144

175

196

218

228

18

175

236

296

339

367

38

152

176

197

225

236

19

177

239

303

347

374

39

151

178

215

240

249

20

177

230

265

328

344

40

145

181

209

227

237

Applicant's summary and conclusion

Conclusions:
From the results presented in this report and based on the EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), a definitive No Observed Adverse Effect Level (NOAEL) for 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE of 1000 mg/kg/day was established.
Executive summary:

SUMMARY: Repeated dose 28-Day oral toxicity study with 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE by daily gavage in the rat.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day. The study was based on the following guidelines: EC Directive 96/54/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996. OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995 OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000. The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS: Accuracy and homogeneity of formulations of test substance in water (Milli-U) were demonstrated by analyses. Findings that distinguished treated from control animals included: 50 mg/kg/day: None. 150 mg/kg/day: None. 1000 mg/kg/day: Clinical biochemistry deviations: Increased alanine aminotransferase activity and cholesterol levels (males); Increased potassium levels (males/females); Increased albumin levels (females).

CONCLUSION: Changes in clinical biochemistry parameters at 1000 mg/kg/day occurred in the absence of morphological evidence of organ dysfunction. Therefore, although a relation to treatment with the test substance was considered likely, these changes were considered not to be adverse in toxicological terms. From the results presented in this report and based on the EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), a definitive No Observed Adverse Effect Level (NOAEL) for 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSUL.FATE of 1000 mg/kg/day was established.