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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Shell Toxicology Laboratory
- Age at study initiation: 5 weeks
- Mean body weight at study initiation: 138 - 143 g (males), 130 - 132 g (females)
- Housing: animals were individually housed in suspended polypropylene cages which had stainless steel grid floors and cellulose-lined trays underneath (North Kent Plastic Cages Ltd., Dartford, Kent, UK)
- Diet: expanded powdered diet (Spratts Laboratory diet No. 2, LAD 2, Spratts Patent Ltd., Barking, Essex, UK), formulated with the appropriate amount of test material, ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):18.3 - 24.4
- Humidity (%): 45 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with LAD 2: Diet containing 10000 ppm of the test material was prepared by mixing the melted material with acetone in the ratio of 2:1 w/v to make a uniform mix with LAD 2 powdered diet. The 3000 ppm and 1000 ppm concentrations were prepared by mixing the 10000 ppm diet with the appropriate amount of untreated diet and the 300 ppm concentration was prepared by mixing the 3000 ppm diet with the appropriate amount of untreated diet. The plain diet for the control group was prepared with acetone.
- Storage temperature of food: ambient temperature
- Rate of preparation of diet (frequency): The diets were prepared in three batches.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method used is based upon the method of Tobin et. al. (1976) The test material is extracted, degraded and derivatised with hydrogen bromide in acetic acid into 1,2-dibromoethane, 1-bromotetradecane and 1-bromopentadecane. These derivatives are analysed and quantified by gas-liquid chromatography as a measure of the test material content.
The recovery based on the derivate 1,2-dibromoethane was 103, 114 and 146% at 10000, 3000 and 1000 ppm; 106, 97 and 128% at 10000, 3000 and 1000 ppm and 104% at 10.000 ppm for the first, second and third batch of diet, respectively.
The recovery based on the derivate 1-bromotetradecane was 103, 102 and 90% at 10000, 3000 and 1000 ppm; 96, 75 and 106% at 10000, 3000 and 1000 ppm and 105 and 102% at 10.000 ppm for the first, second and third batch of diet, respectively. Due to interference with dietary peaks the recovery was repeated using a modified protocol. The recovery based on the derivate 1-bromotetradecane was 114, 102, 104 and 90% at 10000, 3000, 1000 and 300 ppm; 75, 84 and 96% at 3000, 1000 and 300 ppm for the first and second batch of diet, respectively.
The recovery based on the derivate 1-bromopentadecane was 108, 111 and 79% at 10000, 3000 and 1000 ppm; 91, 69 and 105% at 10000, 3000 and 1000 ppm and 134 and 87% at 10.000 and 300 ppm for the first, second and third batch of diet, respectively.
Re-analysis of the 300 ppm diet of batch 3 proved the stability of the test material in the diet (102 vs 93% based on the derivate 1-bromotetradecane and 87 vs. 93% based on the derivate 1-bromopentadecane.
Although not all results were within the protocol specification of ± 10% the analytical verification proved the nominal dose level.
Reference: Tobin et al. 1976, Water Research, 10 (6), 529-535
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
300 ppm
Remarks:
nominal in diet
Dose / conc.:
1 000 ppm
Remarks:
nominal in diet
Dose / conc.:
3 000 ppm
Remarks:
nominal in diet
Dose / conc.:
10 000 ppm
Remarks:
nominal in diet
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 (control), 12 (dose groups)
Control animals:
other: plain diet mixed with acetone
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: All animals were daily examined for overt signs of toxicity, ill-health, mortality or behavioural change.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Time schedule for examinations: Individual body weights were recorded on Day 0 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION: YES
- Food consumption was recorded at weekly intervals.

WATER CONSUMPTION: No Data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological investigations were performed on all animals from each test and control group at the end of the study (13 weeks). Terminal blood samples were taken by cardiac puncture. Moreover, blood for the estimation of glucose concentration was obtained from the tail vain of all rats during the eleventh week.
- Animals fasted: No
- How many animals: all animals
- Parameters examined: haemoglobin (Hb), haematocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), erythrocyte count (RB), total leukocyte count (WBC) mean cell volume (MCV), prothrombin time (PT), kaolin cephalin clotting time (KCCT), differential leucocyte count, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During the eleventh week blood for the estimation of glucose concentration was obtained from the tail vain of all rats. Clinical chemistry investigations were performed on all animals from each test and control group at the end of the study (13 weeks). Terminal blood samples were taken by cardiac puncture.
- Animals fasted: No
- How many animals: all animals
- Parameters examined: urea nitrogen, total protein, alkaline phosphatase, chloride (Cl-), bilirubin, calcium, sodium (Na+), potassium (K+), cholesterol, glucose, alanine amino transferase (ALT), aspartate amino transferase (AST)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from 10 male and 10 female rats from each of the control and 10000 ppm dose groups during the eleventh week.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined: volume, pH, nitrite, protein, glucose, ketone, urobilinogen, bilirubin, blood, epithelial cells, tubule cells, leucocytes, erythrocytes, sperm, casts, bacteria, triple phosphate

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- full histopathology was performed with tissues from control, 3000 and 10000 ppm dose groups including: mammary gland, mesentheric and submandibular lymph nodes, pancreas, stomach, intestine at 6 levels, spleen, liver, adrenals, kidneys, ovaries, testes, uterus, prostate, seminal vesicles, urinary bladder, thyroids with oesophagus and trachea, heart, lungs, thymus, eyes and lachrymal glands, salivary gland, brain, spinal cord, pituitary, tongue, sciatic nerves, muscle, knee joint and femur
Other examinations:
ORGAN WEIGHT:
- The following organs, removed from animals that were killed at study termination (13 weeks) were weighed: brain, heart, liver, spleen, kidneys, testes, ovaries,
Statistics:
Body and organ weights were analysed by covariance analysis using initial bodyweight as the covariate. Reported means were adjusted for initial bodyweight if a significant covariance relationship existed; where no significant covariance relationship was found, unadjusted means were reported. Organ weights were further examined by covariance analysis using the terminal bodyweight as the covariate. The organ weight means are reported as adjusted for terminal bodyweight if a significant covariance relationship existed; the absence of a significant covariance relationship is indicated where appropriate. Food intakes, clinical chemical and haematological variates were examined using a two-way analysis of variance. The significance of any difference between treated and control group means was tested using the Williams “t” test. On occasions where a monotonic dose response was not seen Dunett´s test was used.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
significantly decreased body weight in the 10000 ppm group (both sexes) and in the 3000 ppm group (females) (non-adverse)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
significantly decreased mean food intake in the 10000 ppm group (both sexes) (non-adverse)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
significantly increased WBC, absolute lymphocytes in the 3000 ppm and 10000 ppm group (males); significantly increased WBC, lymphocytes and significantly decreased mean cell volume, mean cell haemoglobin in the 10000 ppm group (females) (non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
significantly increased urea, calcium, potassium in the 10000 ppm group (males); significantly increased urea, chloride, calcium, cholesterol in the 10000 ppm group (females) (non-adverse)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
significantly increased liver weights in the 1000 ppm (females), 3000 and 10000 ppm (males) group; significantly increased spleen weight in the 10000 ppm group (males); significantly increased kidney weights in the 1000 ppm group (females) (non-adverse)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no treatment-related clinical signs in either sex throughout the study. No mortality occurred during the entire study period.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights of males fed 10000 ppm and of females fed 10000 ppm and 3000 ppm of the test material were significantly lower throughout the study period when compared to the control group. It seems clear that this was directly attributable to the reduced intake of an unpalatable diet. Further evidence of this was the increased food spillage in the same treatment groups (refer to table 1).

FOOD CONSUMPTION AND COMPOUND INTAKE
The food intakes of males and females fed 10000 ppmof the test material were significantly lower throughout the study when compared to the control group. Males and females in the 3000 ppm treatment group had lower food intakes, but these were only statistically significantly lower than control values at week 3 (males) and at weeks 5, 8, 10, 12 and 13 (females). The food spillage by both males and females fed 10000 ppm of the test material was higher than in other treatment groups (refer to table 2).

HAEMATOLOGY
In one or both sexes fed 10000 ppm of the test material, values of total leukocytes and absolute lymphocytes were significantly increased and values of absolute neutrophils, percentage neutrophils, mean cell volume, mean cell haemoglobin, and prothrombin time were significantly decreased when compared with controls. In the 3000 ppm group only the male values of total leukocytes, absolute lymphocytes and prothrombin time differed significantly from those of male controls (refer to table 3).

CLINICAL CHEMISTRY
In rats fed 10000 ppm of the test material, plasma concentrations of urea, potassium, chloride, calcium and cholesterol and the plasma activity of alkaline phosphatase were significantly increased in one or both sexes, when compared with the control group. At lower dietary concentrations, the only difference from control was the significantly elevated plasma concentration of urea in 1000 ppm and 3000 ppm females (refer to table 6).

ORGAN WEIGHTS
When compared to control, heart and kidney weights of both sexes, brain weights of males and spleen weights of females all had significantly lower mean values in the 10000 ppm group when unadjusted or adjusted for initial body weight but not after adjustment for terminal body weight. Liver weights of males in the 3000 ppm and 10000 ppm groups were significantly increased (when unadjusted and after adjustment for terminal body weight). Liver weights of females in the 1000 ppm and higher treatment groups showed a significant increase after adjustment for terminal body weight; adjustment for initial body weight showed a significant difference only at the 1000 ppm level. Adjustment for terminal body weight showed a significant increase in the spleen weight of males in the 10000 ppm group when compared with the controls. Females in the 1000 ppm group showed significantly increased kidney weights when adjusted for initial body weight and when adjusted for terminal body weight (refer to tables 5 and 6).

GROSS PATHOLOGY
No treatment-related macroscopic abnormalities were identified at necropsy.

HISTOPATHOLOGY
Histological examination revealed no specific effects attributable to exposure to the test material; neither the incidence nor the severity of spontaneous background lesions was significantly influenced by treatment.

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean body weights for rats for 13 weeks

Dietary concentration [ppm]

Number of animals

Mean body weight [g] at week

0

1

2

3

4

5

6

7

8

9

10

11

12

13

Males

0

300

1000

3000

10000

 

24

12

12

12

12

 

+ a

+

+

+ a

+

+

+ a

+

+

+

+

+

+

143

203

244

294

337

361

384

403

421

433

442

448

460

468

143

202

243

292

332

358

379

397

413

427

440

449

462

466

142

204

246

295

336

361

380

402

416

432

442

453

467

471

141

201

242

289

333

356

377

399

416

430

440

451

459

466

138

178**

215**

258**

292**

316**

331**

350**

364**

375**

386**

388**

398**

409

SD of a single observation

 

9.4

4.7

6.3

9.4

11.6

12.8

14.3

14.4

16.1

17.6

18.6

19.7

21.6

22.2

 

Females

0

300

1000

3000

10000

 

24

12

12

12

12

 

+

+

+

+

+

+

+

+

+

+

+

+

+

130

161

183

207

224

239

247

259

270

276

283

285

291

294

131

162

183

205

223

235

244

256

266

274

281

286

291

293

132

162

185

207

226

241

151

262

270

279

286

287

294

296

132

158*

178*

199*

214**

226**

233**

246**

253**

262**

267**

269**

274**

276**

130

141**

160**

180**

193**

201**

211**

223**

226**

234**

238**

237**

241**

242**

SD of a single observation

 

7.6

3.4

5.8

8.6

10.4

11.4

12.5

12.9

12.7

13.0

13.0

13.2

14.3

14.3

+: adjusted for initial body weight

*: p 0.05

**: p 0.01

a: body weight measured 1 day late

SD: standard deviation

Table 2: Mean food intake for rats for 13 weeks

Dietary concentration [ppm]

Number of animals

Mean food intake [g] at week

1

2

3

4

5

6

7

8

9

10

11

12

13

Males

0

300

1000

3000

10000

 

24

12

12

12

12

a

b

 

a

b

 

a

b

 

 

 

 

 

182

152

187

217

160

178

213

162

181

179

171

176

174

183

150

185

216

163

170

206

157

179

181

177

181

171

181

150

186

217

161

171

207

154

178

182

180

183

171

180

150

178*

216

155

172

211

158

179

179

176

173

167

146**

130**

161**

192**

140**

148**

180**

133**

150**

162**

144**

154**

151**

SD of a single

observation

 

9.2

8.1

10.3

10.0

10.3

14.5

11.1

10.2

9.7

13.3

10.6

13.9

12.6

 

 

Females

0

300

1000

3000

10000

 

24

12

12

12

12

 

 

 

 

 

 

 

 

 

 

 

 

 

133

135

142

145

138

138

150

150

145

150

138

146

139

136

136

139

141

138

126

145

144

144

147

141

139

134

134

137

139

145

145

141

152

144

150

149

145

148

140

127

130

138

137

129*

125

145

137**

136

141**

136

136*

129**

97**

121**

125**

124**

117**

114**

135**

118**

126**

120**

115**

122**

108**

SD of a single

observation

 

8.5

10.2

12.5

13.3

10.2

17.9

13.9

9.7

15.4

8.7

10.2

10.6

9.9

+: adjusted for initial body weight

*: p0.05

**: p0.01

a: food intake measured over an 8 day period

b: food intake measured over a 6 day period

SD: standard deviation

Table 3: Mean haematological values for rats for 13 weeks

Dietary concentration [ppm]

Number of animals

Haematology parameters

WBC x10^3/cmm

RBC x10^6/cmm

Hb g/100 mL

Hct %

Mean cell volume µ3

Mean cell haemoglobin pg

Platelets

Mean cell haemoglobin concentration g/100 mL

P.T. sec

KCCT sec

Males

0

300

1000

3000

10000

 

24

12

11

11

12

 

 

 

 

 

 

 

 

 

 

3.91

7.93

15.3

42.0

54

19.5

543.6

36.5

15.4

30.0

4.18

7.90

15.3

42.0

54

19.5

560.8

36.5

15.5

31.7

3.99

7.94

15.3

42.0

54

19.4

544.1 A

36.6

15.2 A

28.7 A

4.50*

8.12

15.4

42.3

53

19.1

558.8

36.5

14.4*

27.0

4.64**

7.96

15.4

42.7

54

19.5

571.4

36.2

14.4*

28.2

SD of a single observation

 

0.71

0.287

0.43

1.27

1.2

0.45

74.51

0.48

1.35

5.47

 

 

Females

0

300

1000

3000

10000

 

22

12

12

11

12

 

 

 

 

 

 

 

 

 

 

2.63

7.41

15.1

41.4

56

20.5

531.0

36.5

12.8

21.9

2.66

7.49

15.2

41.3

55

20.4

516.7

36.9

12.6

21.9

2.83

7.44

15.1

41.6

56

20.5

522.1

36.5

13.0

22.3

2.70

7.61

15.4

42.2

55

20.4

546.9

36.6

12.7

21.5

3.02*

7.53

14.9

41.0

54**

19.9**

586.7

36.4

13.0

21.5

SD of a single observation

 

0.504

0.324

0.55

1.48

1.1

0.43

85.02

0.42

0.60

2.07

*: p0.05

**: p0.01

SD: standard deviation

A: based on 12 observations

Applicant's summary and conclusion