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Toxicological information

Carcinogenicity

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Description of key information

2 Year rat study: No carcinogenic effects observed in male and female rats. Alden (1994).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
22 December 1986 - 21 December 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Principles of method if other than guideline:
The test material was administered to animals in feed for a period of 104 weeks. Animals were observed for mortality and clinical signs; body weights and fed consumption were monitored. At the end of the study, animals were subjected to necropsy. Haematology, clinical chemistry, urinalysis and histopathologic evaluations were carried out. Furthermore, neurotoxicity evaluations were conducted.
GLP compliance:
yes
Test material information:
Composition 1
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks, average age 43 days.
- Housing: Animals were housed in groups of 5 in polycarbonate cages changed twice weekly. Hardwood chips changed twice weekly were used as bedding.
- Diet: ad libitum NIH-07 open formula meal diet, changed twice weekly.
- Water: tap water available ad libitum (municipal supply) via automatic watering system.
- Acclimation period: 11 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Average temperature 22.5 °C
- Humidity (%): 40 - 56 %
- Air changes (per hr): minimum of 10 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light per day.

IN-LIFE DATES: From: 22 December 1986 To: 21 December 1988
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): The dose formulations were prepared weekly by mixing the test material with feed to give the required concentrations. Formulations were discarded 2 weeks after the date of preparation.
- Mixing appropriate amounts with (Type of food): NIH-07 open formula meal diet.
- Storage temperature of food: The dose formulations were stored in plastic buckets lined with plastic bags, sealed with lids, and protected from light at -20 °C.
- Preparation: A premix of feed and the test material was prepared, then layered into the remaining feed and blended in a Patterson-Kelley twin-shell blender with the intensifier bar on for 5 minutes and off for 10 minutes.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS
Homogeneity and stability studies of the dose formulations were performed. The dose formulations were analysed every 6 - 10 weeks.

Homogeneity studies were carried out at 100 and 10 000 ppm concentrations; aliquots were extracted with methanol and centrifuged. An aliquot of each extract was mixed with methanol and hexanophenone in methanol as an internal standard and diluted with a water:methanol solution (25:75). HPLC was performed with a Brownlee RP-18 column. The mobile phase was a mixture of water:methanol at a ratio of 25:75 and a flow rate of 1 mL/minute.
Homogeneity was confirmed.

Stability studies were carried out at 250 and 25 000 ppm concentrations; aliquots were extracted with methanol and centrifuged. An aliquot of each extract was mixed with 100 ppm of nonanophenone in methanol as an internal standard and diluted with methanol. HPLC was performed with a Brownlee RP-18 column. The mobile phase was a mixture of water:methanol at a ratio of 23:77 and a flow rate of 1mL/minute.
The stability of the dose formulations was confirmed for at least 3 weeks at -20 °C when stored in the dark, as well as for at least 3 days when exposed to air and light.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
Daily in feed
Remarks:
Doses / Concentrations:
0, 500, 1000 and 2500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 20, 40, 100 mg/kg bw/day (equivalent in males)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 20, 45 an d 120 mg/kg bw/day (equivalent in females)
Basis:
actual ingested
No. of animals per sex per dose:
115 males and 75 females per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were based on the finding of a 90 day oral toxicity study (reported in the same paper), where reduced body weight and liver and kidney toxicity was observed at 5000 ppm in males and females.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical observations were made twice daily. They were recorded initially, weekly for 13 weeks, monthly thereafter and at the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed initially, weekly for 13 weeks, monthly thereafter and at the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Feed consumption was recorded monthly by cage.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Fifteen male and 15 female rats from each group were evaluated at 3, 9 and 15 months for alterations and then discarded. An additional 10 male and 10 female rats from each group were also evaluated at 15 months for alterations and were then subjected to complete necropsy and histopathologic evaluations. Blood was collected from the orbital sinus.
- Parameters examined: haematocrit, haemoglobin, erythrocytes, mean erythrocyte volume, mean erythrocyte haemoglobin, mean erythrocyte haemoglobin concentration, platelets, reticulocytes, leukocyte differentials and nucleated erythrocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Fifteen male and 15 female rats from each group were evaluated at 3, 9 and 15 months for alterations and then discarded. An additional 10 male and 10 female rats from each group were also evaluated at 15 months for alterations and were then subjected to complete necropsy and histopathologic evaluations. Blood was collected from the orbital sinus.
- Parameters examined: urea nitrogen, creatinine, sodium, potassium, chloride, calcium direct bilirubin (15 month rats), total bilirubin, alkaline phosphatase, alanine aminotransferase, sorbitol dehydrogenase and bile salts.

URINALYSIS: Yes
- Time schedule for collection of urine: Fifteen male and 15 female rats from each group were evaluated at 3, 9 and 15 months for alterations and then discarded.
- Parameters examined: Creatinine, alkaline phosphatase, lactate dehydrogenase, N-acetyl-β-D-glucosaminidase, volume and β-galactosidase.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Forty of the 115 male rats in each exposure group were designated for neurotoxicity evaluation at 3 and 6 months.
- Dose groups that were examined: all
- Battery of functions tested: At 3 months, startle reflex and fore-and hindlimb grip strength were measured in all 40 animals. Ten males per group were killed and received electrophysiologic evaluations, including measurements of sciatic nerve conduction time following various frequencies of electrical stimulation and contractile tension of the gastrocnemius muscle following various frequencies of electrical stimulation or following graded electrical stimulation.
An additional 10 males per group received whole body perfusion for histopathologic examination of the left quadriceps muscle and left sciatic nerve and of teased nerve preparations of the sciatic nerve. The remaining 20 male rats in each group were fed the control diet for 13 additional weeks to determine the reversibility of test material-induced changes. At 6 months, grip strength tests were repeated in all 20 rats per group. These 20 rats were then split into two groups of 10 and given electrophysiologic and neuropathologic evaluations as described above.
Sacrifice and pathology:
PATHOLOGY
- A necropsy was performed on all animals except those designated for haematology, clinical chemistry and urinalysis evaluations at 3, 9 and 15 months and the 40 male rats per group designated for neurotoxicity and neuropathologic evaluations.
- Method of Sacrifice: Carbon dioxide asphyxiation or pentobarbital anaesthesia with exsanguination and transcardial perfusion (neurotoxicity evaluation rats).
- The brain, gastrointestinal tract, right kidney, liver and spleen were weighed.
- Tissues for microscopic examination were fixed and preserved in 10 % neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 - 6 µm and stained with haematoxylin and eosin.

HISTOPATHOLOGY
- Histopathologic examinations were performed on all animals with the exception of the clinical pathology group and the neurotoxicity group male rats.
- In addition to gross lesions and tissue masses, the tissues examined included: adrenal gland, bone (including marrow), brain, clitoral gland, heart, kidney, large intestine (cecum, colon, rectum), liver, lung, mammary gland with surface skin, mandibular or mesenteric lymph node, nose, oesophagus, ovary, pancreas, parathyroid gland, pharynx, pituitary gland, preputial gland, prostate gland, salivary gland, skeletal muscle, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thyroid gland, thymus, trachea, urinary bladder, and uterus.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY AND CLINICAL FINDINGS
Estimates of survival probabilities are presented in Table 1. Survival rates of exposed rats were similar to those of the controls.
The behaviour and general health and appearance of exposed male and female rats were similar to those of controls.

BODY WEIGHTS AND FEED CONSUMPTION
Throughout most of the study, the mean body weights of 2500 ppm male rats were approximately 3 % lower than those of the controls and the final mean body weight was 5 % lower than that of the controls. Mean body weights of 500 and 1000 ppm males were similar to those of the controls during the study, but the final mean body weights of these groups were 5 % and 6 % lower than that of the controls, respectively. The mean body weights of 2500 ppm females began to decrease 12 weeks into the study and at week 65 was 14 % lower than that of the controls. The final mean body weight, however, was 6 % lower than that of the controls (Tables 2 and 3). Exposure levels of 500, 1000 or 2500 ppm of the test material resulted in a daily ingestion of 20, 40 or 100 mg/kg body weight for males or 20, 45 or 120 mg/kg body weight for females.
Feed consumption by male and female rats was similar to that by controls.

HAEMATOLOGY
Slight but significant decreases in haematocrit levels, haemoglobin concentrations and erythrocyte counts were observed in one set of 1000 and 2500 ppm males at 15 months, but not in the other set. These differences were not observed in males at 3 or 9 months. Similar significant decreases in haematocrit level and haemoglobin concentration occurred in 2500 ppm females at 9 months; haemoglobin concentrations of 2500 ppm females were significantly decreased in both sets evaluated at 15 months, but haematocrit levels were similar to those of the controls. Mean erythrocyte haemoglobin counts and concentration in the 2500 female group were significantly lower than those of the controls at 9 months and in both sets of animals evaluated at 15 months. Platelet counts in 2500 ppm males and females were slightly but significantly higher than those of the controls at 3 and 9 months, as were the platelet counts of 2500 ppm males in one set of animals evaluated at 15 months and of 2500 ppm females in the other set.
While the results of the haematology evaluations were somewhat variable, they do suggest a slight chemical-related effect. It is not clear, however, if these differences indicate a direct effect on stem cells in the bone marrow or on circulating erythrocytes, or if they are secondary to other physiological alterations caused by the test material.

CLINICAL CHEMISTRY
Clinical chemistry results for rats evaluated at 3 and 9 months and for the two sets of rats evaluated at 15 months were generally similar. Serum activities of alkaline phosphatase, alanine aminotransferase and sorbitol dehydrogenase in 2500 ppm males were significantly greater than those of the controls at each evaluation.
Alkaline phosphatase activities in both sets of 1000 ppm males evaluated at 15 months were also significantly greater than those of controls. Serum activities of alanine aminotransferase and sorbitol dehydrogenase in 2500 ppm females were also significantly greater than those in the controls at each evaluation. These results are consistent with hepatocellular damage caused by the test material.

URINALYSIS
Urine volumes of all exposed groups of males and females were significantly lower than those of the controls at 3 months, but not at later evaluations. This is consistent with decreased water or feed intake in the exposed groups, but it is not considered a direct chemical effect. Elevated urine creatinine concentrations at the 3 month evaluation, particularly in exposed groups of male rats, indicate that the urine constituents were more highly concentrated in these groups and are consistent with the volume measurements. Urine specific gravity was not measured, however. The urinary activity of N-acetyl-β-D-glucosaminidase was mildly increased at all evaluations in 2500 ppm females in comparison to controls. Differences in other urine enzyme activities between exposed and control rats were variable and not considered to be treatment related.

NEUROTOXICITY EVALUATION
At 3 months, there was no difference in startle reflex between exposed and control male groups and there were no differences in forelimb or hindlimb grip strength between exposed and control groups in the first three trials. The standard methodology for measuring grip strength consists of three trials. Eight trials were used in this study, and the grip strength of control groups decreased with subsequent trials, apparently due to fatigue or habituation. Although the grip strength of exposed groups also decreased with repeated trials, the decrement was less than that of the controls. Thus, grip strength in later trials (particularly that of the forelimbs) of each exposed group was significantly greater than controls. The electrophysiologic evaluation revealed no significant inhibitory effects of the test material on motor nerve excitability or conduction, neuromuscular transmission or muscle contractility. Further, there were no microscopic lesions that could be attributed to the test material observed in the sciatic nerve, quadriceps muscle or teased nerve preparations of the sciatic nerve.
In the reversibility study, the effects on grip strength observed at 3 months were no longer evident at the 6 month evaluation. The results of the remaining neurotoxicity studies at 6 months were similar to those at 3 months, and there were no significant effects of the test material on motor nerve excitability or conduction, neuromuscular transmission, muscle contractility or pathology.

PATHOLOGY AND STATISTICAL EVALUATION
This section describes the statistically significant or biologically noteworthy changes in the incidences of neoplasms and non-neoplastic lesions in the liver, kidney, thyroid gland, uterus, and mammary gland.

- Liver
At the 15-month interim evaluation, both the absolute and relative liver weights of 2500 ppm females were significantly greater than those of the controls. Relative liver weights of 2500 ppm males and 1000 ppm females were also significantly greater than those of the controls.
The incidence of Kupffer cell hypertrophy was significantly increased in 2500 ppm males and females at the 15 month interim evaluation and at the end of the 2 year study (Table 4). At 15 months, the incidence of cytoplasmic vacuolisation was significantly increased in all exposed groups of males and in 2500 ppm females. At 2 years, the incidence of cytoplasmic vacuolisation was slightly increased in 1000 and 2500 ppm males and significantly increased in 1000 and 2500 ppm females. Also at 2 years, the incidence of fatty change was significantly increased in 2500 ppm females. Cytoplasmic vacuolisation was characterised by the presence of multiple, small vacuoles, whereas fatty change was indicated by the presence of single, large cytoplasmic vacuoles. In both instances, these changes are presumably the result of lipid accumulation.
At 15 months, the incidence of basophilic foci was significantly increased in 2500 ppm males and these foci were present in all females; the incidences in exposed males and females at terminal sacrifice were similar to those in the controls. Incidences of mixed cell foci were significantly increased in 2500 ppm males and females at 15 months and in 1000 and 2500 ppm males and females at the end of the study; at each time point, the incidence of mixed cell foci in 2500 ppm females was twice that in 2500 ppm males. Hepatocyte foci were characterised as basophilic, eosinophilic, clear or mixed based on cytoplasmic staining properties. These differences in staining properties are generally attributed to variations in the amounts of rough or smooth endoplasmic reticulum, glycogen or fat. Thus, basophilic foci consist predominantly of cells with greater amounts of rough endoplasmic reticulum, while eosinophilic foci consist of cells with more of the smooth endoplasmic reticulum. Clear cell foci consist of cells with vacuolated cytoplasm caused by the accumulation of lipid or with clear cytoplasm caused by the accumulation of glycogen. The mixed cell foci consist of cells with either basophilic or eosinophilic cytoplasm and cells with vacuolated or clear cytoplasm.
The incidences of hepatocellular adenoma or carcinoma (combined) in exposed male rats were not significantly greater than that in the control group (Table 4).

- Kidney
Nephropathy is a common occurrence in aging F344/N rats and was observed in nearly all males and the majority of females in this study. In comparison to the control group, the severity of nephropathy was significantly increased in 2500 ppm females both at 15 months and 2 years (Table 5). The number of females with a moderate severity of nephropathy was much higher in the 2500 ppm group than in the control group, whereas the reverse was true for minimal nephropathy. The severity of nephropathy was similar among all groups of male rats.

- Thyroid gland
The incidence of C-cell adenoma or carcinoma (combined) occurred with a significant positive trend in female rats and was slightly, but not significantly, increased in the 1000 and 2500 ppm groups at the end of the 2 year study (0 ppm, 3/49; 500ppm, 4/49; 1000 ppm, 8/50; 2500 ppm, 9/50). This positive trend was not considered test material related because the incidence in 2500 ppm females was only slightly above the historical average of 15 % and well within the range of 6 to 31 %for historical controls. Further, C-cell hyperplasia was decreased in females (0 ppm, 28/49; 500 ppm, 24/49; 1000 ppm, 27/50; 2500 ppm, 18/50), although the decrease in 2500 ppm females was not statistically significant by pairwise comparison.

- Uterus
Stromal polyps occurred with a significant positive trend (0 ppm, 2/50; 500 ppm, 5/50; 1000 ppm, 9/50; 2500 ppm, 9/50). Increased incidences of stromal polyps in females exposed to 1000 or 2500 ppm were significant; however, the incidences are only slightly above the historical control average of 16 % and are well within the historical control range of 2 to 30 %. The incidence in controls is unusually low compared to that in historical controls. Stromal sarcoma was also present in one 500 ppm and one 2500 ppm female.

- Mammary gland
The incidence of fibroadenoma occurred with a statistically significant negative trend in female rats (0 ppm, 29/50; 500 ppm, 24/50; 1000 ppm, 11/50; 2500 ppm, 16/50), and the decreases were significant in the 1000 and 2500 ppm groups. There was also a significant negative trend in the incidence of mammary gland fibroadenoma, adenoma, or carcinoma (combined) in females (0 ppm, 32/50 500 ppm, 24/50; 1000 ppm, 11/50; 2500 ppm, 16/50).

CONCLUSIONS
There was no evidence of carcinogenic activity of the test material in male or female F344/N rats administered 500, 1000 or 2500 ppm. Non-neoplastic lesions associated with exposure to the test material included: Kupffer cell hypertrophy, cytoplasmic vacuolisation and mixed cell foci in the liver of male and female rats, fatty change in the liver of female rats and an increase in the severity of nephropathy in the kidney of female rats. In addition, decreased incidences of fibroadenoma, adenoma, or carcinoma (combined) were observed in the mammary gland of female rats.
Relevance of carcinogenic effects / potential:
There was no evidence of carcinogenic activity of the test material in male or female F344/N rats administered 500, 1000 or 2500 ppm of the test material.
Dose descriptor:
NOAEL
Effect level:
500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on enhancement of seemingly age-related effects in the liver at 1000 ppm.
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on enhancement of seemingly age-related effects in the liver at 1000 ppm.
Remarks on result:
other: Effect type: toxicity (migrated information)

Table 1 Survival of the Rats

Male

Dose (ppm)

0

500

1000

2500

Animals Initially in the Study

60

60

60

60

15 Month Interim Evaluation

Natural Deaths

Moribund

Animals Surviving to Study Termination

% Probability of Survival at End of Study*

Mean Survival (days)**

10

9

23

18

36

614

10

8

14

28

56

637

10

6

22

22

42

633

10

9

23

18

36

620

Survival Analysis***

P = 0.506

P = 0.049N

P = 0.540N

P = 1.000N

Female

Dose (ppm)

0

500

1000

2500

Animals Initially in the Study

60

60

60

60

15 Month Interim Evaluation

Natural Deaths

Moribund

Animals Surviving to Study Termination

% Probability of Survival at End of Study

Mean Survival (days)

10

5

11

34

68

663

10

5

14

31

62

651

10

2

16

32

64

645

10

6

16

28

56

644

Survival Analysis

P = 0.202

P = 0.559

P = 0.711

P = 0.195

Animals in the 15 month interim evaluation were censored from the survival analyses.

*Kaplan-Meier determinations based on the number of animals alive on the first day of terminal sacrifice.

**Mean of all deaths (uncensored, censored and terminal sacrifice).

***The result of the life table trend test (Tarone, 1975) is in the control column and the results of the life table pairwise comparisons (Cox, 1972) with the controls are in the exposed columns. A lower mortality in an exposure group is indicated by N.

‡Three males rats exposed to 1000 ppm were killed moribund prior to the 15 month interim evaluation.

†Includes one animal that died in the last week of the study.

 

Table 2 Mean Body Weights and Survival of Male Rats

Weeks on Study

Dose (ppm)

0

500

1000

2500

Average Weight (g)

No. of Survivors

Average Weight (g)

Weight (% of Control)

No. of Survivors

Average Weight (g)

Weight (% of Control)

No. of Survivors

Average Weight (g)

Weight (% of Control)

No. of Survivors

1

2

3

4

5

6

7

8

9

10

11

12

13

17

22

25

29

33

37

41

45

49

53

57

61

65

69

73

77

81

85

89

93

97

101

104

134

188

215

245

261

281

292

310

323

329

342

350

355

383

397

412

419

431

438

441

444

451

453

461

464

454

462

459

455

455

448

442

445

437

446

441

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

59

59

59

59

58

47

46

46

43

41

39

35

28

24

21

20

135

190

218

246

264

280

295

307

321

332

341

349

358

377

389

405

412

425

434

442

444

451

455

462

462

453

458

458

457

451

453

447

435

432

428

417

100

101

101

100

101

100

101

99

100

101

100

100

101

99

98

98

98

99

99

100

100

100

101

100

100

100

99

100

100

99

101

101

98

99

96

95

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

59

59

58

47

47

44

44

44

43

42

40

33

31

29

134

189

218

246

266

282

298

312

325

335

343

351

360

383

395

406

417

428

437

440

440

449

453

456

455

452

455

453

453

445

442

441

430

427

410

413

100

100

101

100

102

100

102

101

101

102

100

100

102

100

99

99

100

99

100

100

99

100

100

99

98

100

98

99

100

98

99

100

97

98

92

94

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

56

48

46

46

46

44

43

40

37

33

27

23

132

186

216

246

265

280

294

305

315

325

332

338

346

365

382

392

405

413

425

428

431

436

440

447

445

445

446

444

437

436

431

429

416

413

418

417

99

99

100

100

102

100

101

98

98

99

97

97

97

95

95

95

97

96

97

97

97

97

97

97

96

98

96

97

96

96

96

97

94

95

94

95

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

59

48

48

48

46

42

39

35

32

25

20

18

Mean for Weeks:

1 - 13

14 - 52

53 - 104

279

427

451

 

280

424

452

100

99

99

 

281

422

442

101

100

98

 

275

409

433

99

96

96

 

Interim evaluation occurred.

 

Table 3 Mean Body Weights and Survival of Female Rats

Weeks on Study

Dose (ppm)

0

500

1000

2500

Average Weight (g)

No. of Survivors

Average Weight (g)

Weight (% of Control)

No. of Survivors

Average Weight (g)

Weight (% of Control)

No. of Survivors

Average Weight (g)

Weight (% of Control)

No. of Survivors

1

2

3

4

5

6

7

8

9

10

11

12

13

17

22

25

29

33

41

45

49

53

57

61

65

69

73

77

81

85

89

93

97

101

104

107

134

146

156

165

173

178

182

186

192

192

196

199

209

216

219

225

235

246

252

266

277

293

304

312

322

326

330

334

336

335

339

341

338

333

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

49

49

49

47

47

47

47

46

40

40

37

107

137

148

159

169

176

182

186

192

196

197

200

203

211

219

223

228

236

249

253

263

277

287

297

303

316

324

327

335

335

331

327

327

336

326

100

102

102

102

102

102

103

102

103

103

103

102

102

101

101

102

101

100

101

100

100

100

98

98

97

98

100

99

100

100

99

96

96

100

98

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

59

59

48

48

48

48

48

46

44

42

38

33

32

107

136

147

158

166

174

178

184

188

192

194

196

198

208

215

218

223

232

241

248

261

272

284

294

301

312

319

320

328

326

327

324

328

331

327

100

101

101

101

101

101

101

101

101

100

101

100

100

99

100

100

99

99

98

98

98

98

97

97

97

97

98

97

98

97

98

96

96

98

98

60

60

59

59

59

59

59

59

59

59

59

59

59

59

59

59

59

59

59

59

59

59

59

59

49

48

48

48

46

45

43

41

40

35

32

107

137

149

160

168

175

180

184

189

191

193

193

197

202

209

212

216

221

228

231

240

246

257

262

268

278

286

286

293

295

304

305

305

307

311

100

102

102

102

102

101

101

101

102

100

101

99

99

97

97

97

96

94

93

92

90

89

88

86

86

86

88

87

88

88

91

90

90

91

94

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

60

49

49

48

47

46

43

39

38

35

31

28

Mean for Weeks:

1 - 13

14 - 52

53 - 104

170

234

323

 

173

236

318

12

101

98

 

171

231

314

101

99

97

 

171

220

286

101

94

89

 

Interim evaluation occurred.

The number of animals weighed for this week is less than the number surviving.

 

Table 4 Incidences of Neoplasms and Non-neoplastic Lesions of the Liver

Male

Dose (ppm)

0

500

1000

2500

15 Month Interim Evaluation

Liver

Kupffer Cell Hypertrophy

Cytoplasmic Vacuolisation

Basophilic Focus

Mixed Cell Focus

10

0

1 (1.0)

5

1

10

0

10** (1.1)

2

1

7

0

7** (1.0)

7

1

10

10** (1.2)

10** (1.7)

10*

5

2 Year Study

Liver

Kupffer Cell Hypertrophy

Cytoplasmic Vacuolisation

Basophilic Focus

Mixed Cell Focus

Clear Cell Focus

Eosinophilic Focus

50

2 (1.5)

13 (102)

18

6

2

3

50

3 (1.0)

11 (1.5)

22

14

0

7

50

2 (1.0)

19 (1.4)

23

18*

1

2

49

31** (2.1)

18 (2.0)

22

15*

1

1

Hepatocellular Adenoma

Overall Rates¹

Adjusted Rates²

Terminal Rates³

First Incidence (days)

Logistic Regression Test

 

1 / 50 (2 %)

5.6 %

1 / 18 (6 %)

729 (T)

P = 0.091

 

2 / 50 (4 %)

7.1 %

2 / 28 (7 %)

729 (T)

P = 0.653

 

3 / 50 (6 %)

13.6 %

3 / 22 (14 %)

729 (T)

P = 0.377

 

4 / 49 (8 %)

17.0 %

2 / 18 (11 %)

625

P = 0.177

Hepatocellular Carcinoma

0 / 50 (0 %)

1 / 50 (2 %)

0 / 50 (0 %)

1 / 49 (2 %)

Hepatocellular Adenoma or Carcinoma

Overall Rates

Adjusted Rates

Terminal Rates

First Incidence (days)

Logistic Regression Test

 

1 / 50 (2 %)

5.6 %

1 / 18 (6 %)

729 (T)

P = 0.056

 

3 / 50 (6 %)

10.7 %

3 / 28 (11 %)

729 (T)

P = 0.472

 

3 / 50 (6 %)

13.6 %

3 / 22 (14 %)

729 (T)

P = 0.377

 

5 / 49 (10 %)

21.0 %

2 / 18 (11 %)

625

P = 0.100

Female

Dose (ppm)

0

500

1000

2500

15 Month Interim Evaluation

Liver

Kupffer Cell Hypertrophy

Cytoplasmic Vacuolisation

Basophilic Focus

Eosinophilic Focus

Mixed Cell Focus

10

1 (1.0)

0

10

0

0

10

0

1 (1.0)

10

0

1

10

5 (1.0)

1 (1.0)

10

1

0

10

10** (2.7)

8** (1.0)

10

0

10**

2 Year Study

Liver

Kupffer Cell Hypertrophy

Cytoplasmic Vacuolisation

Fatty Change

Basophilic Focus

Mixed Cell Focus

Eosinophilic Focus

Clear Cell Focus

Adenoma

5

11 (1.2)

12 (1.3)

9 (1.4)

37

5

5

0

0

50

10 (1.5)

10 (1.4)

8 (1.5)

34

4

7

1

0

50

0 (1.0)

20* (1.3)

15 (1.3)

38

14*

8

1

0

50

42** (2.7)

34** (2.7)

19* (1.5)

36

34**

4

1

1

Number of animals with organ examined microscopically.

Number of animals with lesion

( ) = Average severity grade of lesions in affected animals. 1 = minimal; 2 = mild; 3 = moderate; 4 = marked.

¹Number of animals with lesion per number of animals examined microscopically.

²Kaplan-Meier estimated neoplasm incidence at the end of the study adjusted for intercurrent mortality.

³Observed incidence at terminal kill.

⁴Beneath the control incidence are the P values associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the control and the exposed group. The logistic regression test regards these neoplasms as non-fatal.

⁵Historical incidence for 2 year feed studies with untreated control groups (mean ± standard deviation): 41 / 1251 (3.3 % ± 3.6 %); range 0 - 10 %.

**Significantly different from the control group (p≤0.05) by the Fischer exact test (15 month interim valuation) or the logistic regression test (terminal sacrifice).

**(p≤0.01)

T = terminal sacrifice

Table 5 Incidences and Severity of Nephropathy in Female Rats

Dose (ppm)

0

500

1000

2500

15 Month Interim Evaluation

Kidney

Nephropathy‡

-Absent (Grade 0)

-Minimal (Grade 1)

-Mild (Grade 2)

-Moderate (Grade 3)

-Marked (Grade 4)

10

9

1

6

3

0

0

10

10

0

8

2

0

0

10

10

0

9

1

0

0

10

10

0

0

8

2

0

Group Average Severity Grade

1.2

1.2

1.1

2.2**

2 Year Study

Kidney

Nephropathy

-Absent (Grade 0)

-Minimal (Grade 1)

-Mild (Grade 2)

-Moderate (Grade 3)

-Marked (Grade 4)

50

44

6

17

26

1

0

50

41

9

14

25

2

0

50

46

4

19

22

5

0

50

48

2

1

29

18

0

Group Average Severity Grade

1.4

1.4

1.6

2.3**

Number of animals with organ examined microscopically.

Number of animals with lesion

**Significantly different from the control group (p≤0.01) by the Mann-Whitney U test.

Conclusions:
Under the conditions of this study, the NOAEL was 500 ppm based on the enhancement of seemingly age-related effects in the liver at 1000 ppm. There was no evidence of carcinogenic activity.
Executive summary:

A 2 year feed study was conducted in order to assess the test material when administered to male and female F344/N rats. The methodology followed is equivalent to the standardised OECD guideline 453.

Groups of 115 male and 75 female rats were fed diets containing 0, 500, 1000 or 2500 ppm of the test material for 104 weeks. Corresponding to ingested dose levels of 0, 40, 100 mg/kg bw/day in males and 0, 20, 45 an d 120 mg/kg bw/day in females.

Two year survival rates and mean body weights of exposed male and female rats were generally similar to those of the controls.

A 15 month interim evaluation was conducted, at which the absolute and relative liver weights of 2500 ppm female rats were significantly greater than those of controls; at 15 months and at the end of the study, the incidences of Kupffer cell hypertrophy, hepatocyte cytoplasmic vacuolisation and mixed cell foci were also significantly increased. At the end of the study, the incidence of hepatocellular fatty change was significantly increased in 2500 ppm females. The incidence of Kupffer cell hypertrophy was significantly increased in 2500 ppm males at 15 months and at 2 years; the incidence of cytoplasmic vacuolisation was significantly increased in all exposed males at 15 months but only moderately increased in 1000 and 2500 ppm males at 2 years; the incidence of basophilic foci was significantly increased in 2500 ppm males at 15 months and the incidence of mixed cell foci was significantly increased in 1000 and 2500 ppm male rats at 2 years. The incidences of hepatocellular adenoma or carcinoma (combined) in exposed male rats were not significantly greater than that in the controls. The severity of nephropathy was significantly increased in 2500 ppm female rats.

 

There was no evidence of carcinogenic activity of the test material in male or female F344/N rats administered 500, 1000 or 2500 ppm of the test material. Non-neoplastic lesions associated with exposure to the test material included: Kupffer cell hypertrophy, cytoplasmic vacuolisation and mixed cell foci in the liver of male and female rats, fatty change in the liver of female rats and an increase in the severity of nephropathy in the kidney of female rats. In addition, decreased incidences of fibroadenoma, adenoma, or carcinoma (combined) were observed in the mammary gland of female rats.

 

Under the conditions of this study, the NOAEL was 500 ppm based on the enhancement of seemingly age-related effects in the liver at 1000 ppm. There was no evidence of carcinogenic activity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Another study on the same analogue has also been provided as supporting data. The study follows the same methodology in mice. The overall quality of the data set is considered sufficient for classification purposes.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The carcinogenic potential of the test material has been assessed using evidence from the structural analogue, 4,4’-thiobis(6-t-butyl-m-cresol), provided on the basis of read-across due to its structural similarities to the registered substance, 2,2'-Methylenebis(6-nonyl-p-cresol).

 

The key study Alden (1994) is a 2 year chronic carcinogenicity and repeat dose toxicity study performed in male and female F344/N rats. Rats were fed diets containing 0, 500, 1000 or 2500 ppm of the test material for 104 weeks. Corresponding to ingested dose levels of 0, 40, 100 mg/kg bw/day in males and 0, 20, 45 and 120 mg/kg bw/day in females.

There was no evidence of carcinogenic activity in male or female rats. Based on enhancement of seemingly age-related effects in the liver at 1000 ppm, the NOAEL was determined to be 500 ppm.

 

The supporting study Alden (1994) is a 2 year chronic carcinogenicity and repeat dose toxicity study performed in male and female B6C3F1 mice. Mice were fed diets containing 0, 250, 500 or 1000 ppm of the test material for 104 weeks. Corresponding to ingested dose levels of 0, 30, 60 and 145 mg/kg bw/day for males and 0, 45, 110 and 225 mg/kg bw/day for females.

In the liver of male mice, negative trends in the incidences of fatty change, clear cell foci and adenoma or carcinoma combined occurred at the end of the study. There were no test material related increased incidences of neoplasms or non-neoplastic lesions. There was no evidence of carcinogenic activity. Based on decreased body weight gain and effects on the liver seen at 1000 ppm, the NOAEL was determined to be 500 ppm.

 

Both studies were performed under GLP conditions and followed a methodology similar to that outlines in OECD 453 (Combined Chronic Toxicity / Carcinogenicity Studies). Both studies were performed to a good standard and reported with sufficient detail to assess the reliability of the submitted data. Accordingly the studies were assigned reliability scores of 2 in line with Klimisch (1997).

 

Based on the read-across data the registered substance is not considered to be carcinogenic. Read-across evidence from an in vitro mutagenicity test further indicates that the substance is not carcinogenic.


Justification for selection of carcinogenicity via oral route endpoint:
A chronic carcinogenicity study in rats has been selected as key, since the study was performed on the preferred species. The study was conducted under GLP conditions and performed in line with sound scientific principles. The study followed a methodology similar to OECD guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies). The test was performed using the structural analogue 4,4’-thiobis(6-t-butyl-m-cresol), which was considered valid based on its structural similarities to the target substance. Accordingly the study was assigned a reliability score of 2 in line with Klimisch (1997).

Justification for classification or non-classification

According to the criteria outlined in Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, this substance does not meet the criteria for classification for carcinogenicity.