Registration Dossier

Administrative data

Description of key information

The registered substance was tested for repeated dose toxicity in a 90-day subchronic toxicity study according to OECD TG 408 in line with the principles of GLP. After daily administration of 0, 100, 300 and 750 mg/kg body weight via gavage for 90 consecutive days to rats, no significant differences in mortality, clinical findings, neurobehaviour, clinical biochemistry, urinalysis parameters,  organ weights, or gross pathology were revealed. Increase in white blood cell counts in high dose animals correlates very well with observed inflammatory changes in the forestomach of high and mid dose animals. These forestomach findings are considered to be secondary local effects ("port-of-entry") but not systemic toxic effects and are not considered relevant for human health hazard / risk assessment. Comparable forestomach effects were not seen in the rats from the low- and mid-dose groups. Since the forestomach changes are considered to be `site of first contact` and thus local effects, the NOAEL with regard to systemic toxicity was established at 750 mg/kg body weight per day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015 - 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
The animal room was air-conditioned with adequate (above 10) air changes per hour. The experimental room was continuously monitored for tem¬pera¬ture and relative humidity. The ranges for room tem¬pera¬ture and relative humidity were 20.9°C to 23.1°C and 59 to 68%, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Once daily by oral gavage for 90 consecutive days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability, homogeneity analysis as well as dose formulation analysis.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
4 dose groups plus a control recovery group and a high dose recovery group
Observations and examinations performed and frequency:
Viability/ Mortality, clinical signs (daily), Ophthalmoscopy (Before start of treatment and towards end of the treatment period), body weights and feed consumption (once weekly) during treatment and recovery period. urinalysis and clinical pathology (all animals from tretament and recovery groups).
Sacrifice and pathology:
Macroscopic and microscopic observations (end of treatment and recovery period) as well as histopathology of all preserved organs (control and high dose groups). Gross lesions (all groups) and stomach from low, intermediate, high and recovery groups.
Statistics:
The following statistical methods were used to analyze the body weight, feed consumption, organ weights as well as clinical pathology data.
•            Data was summarized in tabular form. Statistical analysis was performed using statplus program.
•            All the data was checked for normality with Shapiro-Wilk W test
•            Data for each group of animals was subjected to analysis of variance (ANOVA). Values were given as mean ± standard deviation (SD).
•            t-test was used to compare the difference between treated and control groups. Statistical significances of differences were calculated with one-way analyses of variance. P≤0.05 (5% level of significance) was considered to represent the significance in the respective parameters
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Minor clinical signs of toxicity (dullness) observed in some animals of the mid and high dose group. Reversed during the recovery period.
In intermediate dose group (group 3), all the animals appeared normal up to day 65. Dullness was observed in from day 66 (Animal No. 046 and 056, 1 male and 1 female), from day 68 (Animal No. 059, 1female), from day 69 (Animal No. 053 and 054, 2 males) until day 90 (last day of observation).
In high dose group (group 4), all the animals appeared normal up to day 51. Dullness and Burrowing were observed in from day 52 (Animal No. 069, 070 and 079, 080, 2 males and 2 females), from day 53 (Animal No. 065, 066, 067, 068 and 075, 076, 077, 078, 4 males and 4 females), from day 60 (Animal No. 061, 062, 063, 064 and 071, 072, 073, 074, 4 males and 4 females) until day 90. Dullness alone was observed on day 91 of observation period (before terminal sacrifice) in all the animals.
In high dose recovery group group 4R, all the animals appeared normal up to day 51. Dullness and Burrowing were observed in from day 52 (Animal No. 109, 110 and 119, 120, 2 males and 2 females), from day 53 (Animal No. 101, 102, 103, 104 and 111, 112, 113, 114, 4 male and 4 female), from day 54 (Animal No. 105, 106, 107, 108 and 115, 116, 117, 118, 4 male, 4 female) until day 90. Dullness alone was observed from day 91 to 94. All the animals recovered from clinical signs and appeared normal from day 95 to until day 119 (last day of observation).
Mortality:
no mortality observed
Description (incidence):
Animals treated in study with test item at dose levels of 100, 300 and 750 mg/kg body weight did not reveal any mortality in both males and females throughout the experimental period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effect on body weight and bodyweight gain (%) was detected for treated animals when compared with vehicle control.
The body weight and body weight gain (%) was generally decreased in males at high dose group (G4) compared to control group from day 15 to 90. In females there was no decrease in body weight or body weight gain (%) in any of the treated animals of low, intermediate and high dose group (G2 to G4). However, high dose recovery females as well as males showed decreased body weight and body weight gain (%) when compared with respective vehicle control. Concurrently a significantly reduced food consumption was observed in high dose males and females. Based hereupon, the reduced body weights and body weight gains may be explained by lower food intake due to local irritative effects of the test item at the port of entry as indicated in the forestomach of the test animals. Thus, these findings are not considered to be adverse.
Detailed data see the tables in section "Any other information on results inc. tables"
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean feed consumption was slightly but statistically significantly decreased in males and females at high dose (G4) when compared with vehicle control (G1). This lower food intake is considered due to local irritative effects of the test item at the port of entry as indicated in the forestomach of the test animals. Thus, these findings are not considered to be adverse.
Food efficiency:
not examined
Description (incidence and severity):
not applicable because no drinking water study.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Opthalmological examiniation did not reveal any abnormalities in the vehicle control and high dose treated animals at the end of treatment period.
Haematological findings:
no effects observed
Description (incidence and severity):
Erythrocyte count (RBC), hemoglobin (Hb), hematocrit (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (thrombocyte) count (PLT), total leukocyte count (WBC), differential leukocyte count (DC), prothrombin time (PT) and activated partial thromboplastin time (APTT) did not show any toxicologically relevant finding in both sexes. In recovery group at end of week-17, no significant changes were observed in females whereas, in males, MCHC significantly increased in high dose recovery (G4R) when compared with vehicle control recovery group (G1R).
The changes observed in the hematological parameters are marginal, not clinically significant and could not be attributed to the test item administration as these values were within biological variation.
Detailed data see the tables in section "Any other information on results inc. tables"
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Biochemical parameters like glucose (GLU), urea, creatinine (CREA), cholesterol (CHOL), total triglycerides (TRIGL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, sodium (Na), potassium (K), chloride (Cl), total protein (TPO), albumin (ALB), globulin (GLB) and A/G ratio did not show any toxicologically relevant findings in both the sexes in treatment and recovery group with the exception of statistically significant increase in alanine transaminase (ALT) which was noticed in high dose (G4) male animals when compared to vehicle control group (G1). Total proteins (TPO) in high dose (G4) male was increased when compared to vehicle control group (G1) at end of week 13 only.
In recovery group animals at the end of week-17, significant increase in glucose (GLU), potassium (K) aspartate aminotransferase (AST) in males was observed. In high dose recovery females (G4R) a decrease in aspartate aminotransferase (AST) was observed as compared to vehicle control recovery group (G1R).
Although statistically significant, all observed changes fall within the historical control range, were not clinically significant and did not correlate with any microscopic finding observed in high dose animals. Hence, the test item had no effect on the biochemical parameters of treated animals up to the high dose of 750 mg/kg body weight.
Detailed data see the tables in section "Any other information on results inc. tables"
Urinalysis findings:
no effects observed
Description (incidence and severity):
Treatment had no significant effect on urinary parameters in any of the groups with exception of decreased urinary volume in high dose recovery group (G4R) whern compared with vehicle control (G1R). These changes were not clinically significant and do not correlate with any microscopic finding. Hence, the test item had no effect on the urine parameters of treated animals upto the dose of 750 mg/kg body weight.
Detailed data see the tables in section "Any other information on results inc. tables"
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No functional behavioural effects have been observed during the course of the study.
Immunological findings:
no effects observed
Description (incidence and severity):
Despite inflammatory related findings, no effects in immunological competent organs have been detected.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant test item-related differences in absolute and relative organ weights were observed.
In main study groups, there was no significance observed in absolute organ weights of male animals. Sporadically observed increased in absolute and relative organ weights of female animals showed no dose response relationship and are not considered treatment related.
In recovery groups, absolute organ weights of heart, spleen and thymus of male animals from G4R (750 mg/kg) was significantly lower than that of control recovery group G1R (0 mg/kg) male animals. Relative weights of liver and brain in male animals of group G4R (750 mg/kg) was significantly higher than those of group G1R. Regarding female recovery animals there was no significance noted in absolute organ weight when compared to controls. Relative organ weights of heart, spleen and kidneys of females from 750 mg/kg recovery group G4R was significantly higher than that of control recovery group G1R female animals.
Despite these differences, microscopically no test item-related change was observed in these organs. Hence, the changes observed in organ weight were not considered to be biologically significant.
Detailed data see the tables in section "Any other information on results inc. tables"
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy performed at the end of the treatment period revealed slightly thickened forestomach (9 males, 4 female) and red foci in glandular stomach (7 male, 1 female) in group G4 (750 mg/kg) wherase only slightly thickened forestomach was observed in 3 males and 1 female animal from G3 (300 mg/kg) group. No gross findings were observed on completion of treatment in animals of Group G2 (100 mg/kg) as well as in male and females recovery animals of G4R (750 mg/kg).
These findings are of no relevance for humans as discussed in the executive summary and are considered a response to the observed port of entry effects in the forestomach of test animals. In addition, these effects were reversed after the treatment free recovery period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination revealed forestomach and glandular stomach findings in high dose animals comparable to the observed gross pathological changes. In high dose animals, forestomach submucosal hemorrhages (2 male, 1 female), polymorphonuclear (4 male) and/or mononuclear cells infiltrate (4 male, 3 female), mucosal necrosis (4 male, 1 female), hyperkeratosis (8 male, 4 female) and hyperplasia (9 male, 6 female) of squamous epithelium were observed.
Animals treated with test item at 300 mg/kg (G3) revealed glandular stomach hemorrhage (2 male), forestomach submucosal hemorrhage (2 male), polymorphonuclear and mononuclear cells infiltrate (2 male, 1 female), mucosal necrosis (2 male, 1 female), hyperkeratosis (3 male) and hyperplasia of squamous epithelium (3 female).
These changes are not considered having relevance for humans, were reversible as demonstrated in the high dose recovery animals and are considered as port of entry effects due to the local irritant effect of the test item at mentioned doses and treatment period (see also discussion section in the executive summary).
No abnormality attributable to the test item in group G2 (100 mg/kg) and recovery group G4R (recovery group, 750 mg/kg) was revealed on microscopic examination.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No indications of neoplastic histopathological findings revealed
Other effects:
no effects observed
Details on results:
No other effects of relevance observed.
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
The only significant effects observed were related to the forestomach of rats. This finding is not considered relevant for humans (details see discussion section).
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
other: forestomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Observed forestomach lesions consisted of acanthosis, parakeratosis, inflammation, ulceration and erosions and are considered to be `site of first contact` findings due to local irritative effects. Application of rodent forestomach effect data for predicting risk or even hazard for humans is in general not justified, given that a human counterpart for the rodent forestomach does not exist. This view is also reflected by the endpoint specific guidance given by ECHA (Chapter R.7.2.1). The forestomach changes in rats are thus considered to be of minor or no relevance for human health hazard / risk assessment (for a detailled assessment see discussion section in the executive summary).

Further data in tabular form:

BODY WEIGHTS (G)– SUMMARY

Males

TREATMENT

Group 1

Group 2

Group 3

Group 4

Day 1

Mean

133.0

133.6

134.7

134.3

SD

3.80

3.77

3.00

2.80

Min

128.1

127.7

129.4

130.4

Max

138.6

138.9

138.5

138.2

N

10

10

10

10

Day 8

Mean

181.6

183.1

183.2

183.6

SD

5.01

3.62

4.53

4.28

Min

173.2

176.5

175.9

172.9

Max

187.4

188.2

188.7

187.1

N

10

10

10

10

Day 15

Mean

309.8

239.7

236.5

231.5*

SD

14.06

4.72

10.58

8.46

Min

284.0

229.7

214.0

216.7

Max

328.3

246.6

246.1

243.6

N

10

10

10

10

 

*Significant at p ≤ 0.05 level with group 1

 

 


BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

Males

TREATMENT

Group 1

Group 2

Group 3

Group 4

Day 22

Mean

279.6

273.4

275.7

258.3*!

SD

11.11

8.24

20.37

13.92

Min

255.7

261.5

243.1

233.5

Max

291.7

283.7

314.0

277.3

N

10

10

10

10

Day 29

Mean

309.8

300.6

300.9

286.2*

SD

14.06

15.96

21.31

11.74

Min

284.0

282.6

266.4

266.1

Max

328.3

326.8

343.3

301.3

N

10

10

10

10

Day 36

Mean

334.3

323.5

323.0

303.5*

SD

18.05

20.67

22.96

16.11

Min

302.1

298.8

292.4

284.0

Max

366.7

357.6

368.2

327.8

N

10

10

10

10

Day 43

Mean

349.8

340.2

337.1

319.5*

SD

18.89

22.73

25.35

17.15

Min

321.8

319.1

305.7

294.4

Max

386.0

380.6

394.0

350.1

N

10

10

10

10

*Significant at p ≤ 0.05 level with group 1

*!Significant at p ≤ 0.05 level with group 1, 3

 

 


BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

Males

TREATMENT

Group 1

Group 2

Group 3

Group 4

Day 50

Mean

366.1

353.7

351.4

334.7*

SD

21.14

22.38

31.47

18.38

Min

335.2

333.4

314.6

319.6

Max

403.9

390.1

419.8

371.2

N

10

10

10

10

Day 57

Mean

376.4

365.8

362.3

343.0

SD

24.26

27.05

34.04

22.43

Min

345.3

339.2

326.8

320.2

Max

422.4

416.8

438.7

381.8

N

10

10

10

10

Day 64

Mean

389.9

378.3

374.5

351.8*

SD

25.71

30.97

31.65

22.21

Min

355.6

341.1

338.5

319.8

Max

437.6

427.7

445.9

390.8

N

10

10

10

10

Day 71

Mean

395.1

384.0

382.1

356.3*

SD

27.08

30.62

32.33

24.64

Min

357.4

343.0

339.4

317.1

Max

441.9

430.7

451.4

403.8

N

10

10

10

10

*Significant at p ≤ 0.05 level with group 1


BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

Males

TREATMENT

Group 1

Group 2

Group 3

Group 4

Day 78

Mean

405.4

394.3

392.8

361.4*

SD

28.36

32.56

34.48

26.63

Min

371.4

352.7

348.0

317.8

Max

457.3

449.3

462.2

416.9

N

10

10

10

10

Day 85

Mean

413.7

401.9

401.0

367.2*

SD

27.94

32.39

34.64

27.61

Min

377.8

362.9

361.8

316.8

Max

460.8

456.7

471.2

421.3

N

10

10

10

10

Day 90

Mean

420.6

407.7

407.5

371.4*

SD

28.16

32.08

34.55

29.04

Min

383.2

367.5

370.8

316.2

Max

468.7

459.9

476.8

427.4

N

10

10

10

10

 

*Significant at p ≤ 0.05 level with group 1


BODY WEIGHTS (G)– SUMMARY

 

FEMales

TREATMENT 

Group 1

Group 2

Group 3

Group 4

Day 1

Mean

131.8

132.1

132.4

132.0

SD

4.54

4.79

4.27

4.62

Min

125.3

125.4

126.9

125.4

Max

139.2

139.5

139.4

138.7

N

10

10

10

10

Day 8

Mean

156.9

160.3

158.4

158.9

SD

7.27

8.94

4.55

11.56

Min

144.6

151.2

146.3

132.5

Max

165.7

178.3

162.7

170.1

N

10

10

10

10

Day 15

Mean

177.0

181.4

178.7

179.0

SD

12.37

5.44

5.34

12.82

Min

155.2

170.8

170.6

149.1

Max

189.2

186.7

186.5

193.9

N

10

10

10

10

 

 

BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

FEMales

TREATMENT

Group 1

Group 2

Group 3

Group 4

Day 22

Mean

191.7

188.7

190.7

188.7

SD

13.44

8.57

10.14

14.41

Min

162.5

175.8

178.4

158.0

Max

206.7

201.4

212.6

208.2

N

10

10

10

10

Day 29

Mean

201.7

201.8

199.2

199.8

SD

14.20

9.22

13.16

15.26

Min

173.2

185.9

178.6

171.3

Max

222.6

216.4

228.2

220.8

N

10

10

10

10

Day 36

Mean

207.0

208.8

203.9

207.6

SD

13.88

9.24

14.69

15.96

Min

179.4

194.6

181.3

175.3

Max

227.1

223.4

237.7

227.9

N

10

10

10

10

Day 43

Mean

213.0

214.8

210.0

212.7

SD

14.78

9.74

17.12

15.70

Min

183.4

198.3

186.7

178.6

Max

236.4

227.7

250.6

233.6

N

10

10

10

10

 

 


BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

FEMales

TREATMENT

Group 1

Group 2

Group 3

Group 4

Day 50

Mean

218.3

220.7

215.8

221.4

SD

15.64

10.72

17.54

14.85

Min

187.4

202.4

188.6

190.6

Max

243.7

234.8

256.6

246.4

N

10

10

10

10

Day 57

Mean

220.6

223.1

219.1

223.7

SD

15.79

10.77

17.71

15.09

Min

189.0

203.4

189.8

192.3

Max

245.6

236.0

259.6

248.7

N

10

10

10

10

Day 64

Mean

223.8

226.2

223.0

224.5

SD

15.65

11.30

17.76

15.40

Min

191.2

205.4

193.3

190.4

Max

247.5

240.8

260.8

243.9

N

10

10

10

10

Day 71

Mean

226.3

228.5

223.0

223.0

SD

15.87

12.30

17.76

16.91

Min

193.6

206.7

193.3

189.4

Max

252.0

249.2

260.8

248.3

N

10

10

10

10

 


BODY WEIGHTS (G)– SUMMARY (CONTD.)

 

FEMales

TREATMENT

Group 1

Group 2

Group 3

Group 4

Day 78

Mean

229.3

230.8

226.2

223.0

SD

15.89

12.75

17.41

18.37

Min

194.6

207.2

198.9

187.4

Max

254.4

251.3

263.4

251.5

N

10

10

10

10

Day 85

Mean

232.0

232.6

228.5

222.4

SD

16.40

12.58

17.08

19.69

Min

196.0

210.3

205.6

185.3

Max

258.2

252.7

267.3

254.4

N

10

10

10

10

Day 90

Mean

234.4

235.0

230.7

222.7

SD

16.48

12.83

17.40

21.53

Min

197.8

214.2

208.6

183.8

Max

261.3

255.3

270.2

257.6

N

10

10

10

10

 

 


BODY WEIGHTS (G) – SUMMARY

MALES  

RECOVERY

Group 1R

Group 4R

Day 1

Mean

135.4

135.5

SD

2.21

1.70

Min

131.6

132.7

Max

138.9

137.9

N

10

10

Day 8

Mean

183.8

183.6

SD

4.43

3.44

Min

176.2

179.5

Max

189.9

189.8

N

10

10

Day 15

Mean

239.5

239.4

SD

5.07

5.89

Min

227.5

228.4

Max

246.8

249.5

N

10

10

Day 22

Mean

275.1

272.8

SD

8.44

18.28

Min

261.1

253.1

Max

287.0

318.0

N

10

10

 


BODY WEIGHTS (G) – SUMMARY(CONTD.)

MALES

RECOVERY

Group 1R

Group 4R

Day 29

Mean

300.3

296.8

SD

8.48

24.62

Min

284.5

257.4

Max

310.5

355.5

N

10

10

Day 36

Mean

322.5

314.4

SD

11.56

22.22

Min

304.3

271.6

Max

337.2

360.5

N

10

10

Day 43

Mean

342.2

326.6

SD

12.11

19.17

Min

324.6

290.6

Max

355.7

365.6

N

10

10

Day 50

Mean

356.5

343.1

SD

12.26

19.64

Min

332.5

301.8

Max

370.5

377.6

N

10

10

 


BODY WEIGHTS (G) – SUMMARY(CONTD.)

MALES

RECOVERY

Group 1R

Group 4R

Day 57

Mean

368.0

346.0

SD

14.52

20.68

Min

345.8

306.5

Max

385.6

371.5

N

10

10

Day 64

Mean

382.4

351.9

SD

16.99

22.28

Min

362.0

317.2

Max

411.2

378.3

N

10

10

Day 71

Mean

391.7

355.3

SD

18.44

25.13

Min

371.6

317.5

Max

423.4

385.1

N

10

10

 


BODY WEIGHTS (G) – SUMMARY(CONTD.)

MALES

RECOVERY

Group 1R

Group 4R

Day 78

Mean

402.1

361.0

SD

20.04

28.46

Min

383.3

317.6

Max

440.2

391.3

N

10

10

Day 85

Mean

409.4

364.3

SD

18.78

30.03

Min

390.4

316.7

Max

445.5

395.6

N

10

10

Day 91

Mean

419.9

368.6

SD

21.49

33.51

Min

396.8

315.7

Max

460.3

401.2

N

10

10

Day 98

Mean

425.6

373.3

SD

20.19

34.06

Min

405.7

318.4

Max

463.1

405.5

N

10

10

 

 

 

BODY WEIGHTS (G) – SUMMARY(CONTD.)

MALES

                                                                                                                          

RECOVERY

Group 1R

Group 4R

Day 105

Mean

431.8

380.7

SD

21.12

33.85

 

Min

410.3

322.3

Max

475.2

412.7

N

10

10

Day 112

Mean

437.8

387.9

SD

21.11

33.81

Min

418.3

329.4

Max

481.3

421.3

N

10

10

Day 118

Mean

444.8

395.4

SD

21.44

33.84

Min

424.9

336.4

Max

490.5

429.5

N

10

10

 


BODY WEIGHTS (G) – SUMMARY

FEMALES  

RECOVERY

Group 1R

Group 4R

Day 1

Mean

132.6

132.7

SD

4.30

4.00

Min

126.7

127.9

Max

138.6

137.9

N

10

10

Day 8

Mean

165.6

158.6

SD

5.12

5.52

Min

157.2

148.2

Max

172.4

166.2

N

10

10

Day 15

Mean

183.4

179.7

SD

6.21

7.37

Min

174.9

166.7

Max

192.2

186.2

N

10

10

Day 22

Mean

192.4

185.5

SD

9.55

9.27

Min

180.3

167.7

Max

206.2

200.6

N

10

10

 


BODY WEIGHTS (G) – SUMMARY(CONTD.)

FEMALES

RECOVERY

Group 1R

Group 4R

Day 29

Mean

202.5

196.7

SD

8.22

8.09

Min

191.0

183.3

Max

213.3

208.8

N

10

10

Day 36

Mean

209.8

201.8

SD

9.17

8.53

Min

199.3

185.5

Max

226.6

214.3

N

10

10

Day 43

Mean

214.2

207.0

SD

8.37

9.26

Min

203.4

187.3

Max

229.7

218.3

N

10

10

Day 50

Mean

219.6

212.7

SD

9.09

9.75

Min

207.4

191.2

Max

234.2

224.4

N

10

10

 


BODY WEIGHTS (G) – SUMMARY(CONTD.)

FEMALES

RECOVERY

Group 1R

Group 4R

Day 57

Mean

224.0

213.1

SD

8.39

9.78

Min

209.7

193.0

Max

236.7

226.8

N

10

10

Day 64

Mean

228.0

215.6

SD

9.34

10.74

Min

211.6

201.6

Max

241.5

232.6

N

10

10

Day 71

Mean

230.1

214.7

SD

9.25

11.56

Min

212.6

199.9

Max

243.2

235.8

N

10

10

Day 78

Mean

232.8

213.9

SD

9.52

12.54

Min

214.7

197.5

Max

247.6

238.5

N

10

10

 


BODY WEIGHTS (G) – SUMMARY(CONTD.)

FEMALES

RECOVERY

Group 1R

Group 4R

Day 85

Mean

235.6

213.5

SD

9.28

13.08

Min

216.7

197.3

Max

249.2

239.1

N

10

10

Day 91

Mean

239.1

213.2

SD

9.75

13.87

Min

220.6

196.9

Max

254.2

240.1

N

10

10

Day 98

Mean

241.4

215.5

SD

9.97

13.74

Min

222.4

197.2

Max

255.6

242.5

N

10

10

Day 105

Mean

244.4

218.7

SD

10.18

14.07

Min

225.3

199.2

Max

258.0

245.3

N

10

10

*Significant at p ≤ 0.05 level with group 1R


BODY WEIGHTS (G) – SUMMARY(CONTD.)

FEMALES

RECOVERY

Group 1R

Group 4R

Day 112

Mean

248.0

222.9

SD

9.98

14.28

Min

229.7

202.4

Max

261.4

249.5

N

10

10

Day 118

Mean

252.2

227.0

SD

10.46

14.48

Min

232.4

205.3

Max

266.4

253.4

N

10

10

*Significant at p ≤ 0.05 level with group 1R

 

SPERM COUNT SUMMARY – MALES 

TREATMENT

Total Count

Mean x 20 x 106

Group 1

Mean

194

SD

27.014

Group 2

Mean

194

SD

25.289

Group 3

Mean

193

SD

50.711

Group 4

Mean

197

SD

49.389

 WEEK -17

RECOVERY

Total Count

Mean x 20 x 106

Group 1R

Mean

241

SD

71.237

Group 4R

Mean

213

SD

74.643

 

HEMATOLOGY SUMMARY – MALES

Week -13

 

TREATMENT

RBC

Hb

PCV

MCV

MCH

MCHC

PLT

x106Cells

g/dL

%

fL

pg

g/dL

x103cells

Group 1

Mean

8.53

15.81

44.62

52.41

18.58

35.42

684.60

SD

0.490

0.495

1.652

1.629

1.389

1.650

113.256

Group 2

Mean

8.36

15.37

43.18

51.67

18.52

35.87

688.00

SD

0.769

0.732

3.896

1.191

1.956

3.933

108.056

Group 3

Mean

8.47

14.97

43.76

51.67

17.71

34.25

734.70

SD

0.665

1.142

2.659

1.649

0.555

1.449

88.043

Group 4

Mean

8.18

15.58

44.14

54.05#!

19.10

35.33

712.90

SD

0.720

0.879

3.231

1.635

1.155

1.380

106.371

 

 

TREATMENT

WBC

DC (%)

PT

APTT

x103cells

NEUT

LYMPH

MONO

EOS

BASO

Sec

Sec

Group 1

Mean

5.13

19.85

74.05

3.23

1.81

0.31

15.41

15.54

SD

1.192

4.037

4.641

1.136

0.530

0.074

0.889

1.932

Group 2

Mean

4.59

25.42

67.73

3.69

1.76

0.24

15.89

13.62

SD

1.120

9.648

9.646

1.050

0.341

0.126

0.805

2.604

Group 3

Mean

4.33

21.15

72.37

3.34

1.91

0.27

15.74

14.96

SD

1.058

9.774

9.596

0.854

0.832

0.082

0.752

3.630

Group 4

Mean

5.57

23.09

70.27

3.35

1.69

0.27

15.44

14.57

SD

1.420

7.890

8.140

1.125

0.593

0.095

0.550

1.657

#!Significant at p ≤ 0.05 level with group 2, 3

 

  


HEMATOLOGY SUMMARY – FEMALES

Week -13

 

TREATMENT

RBC

Hb

PCV

MCV

MCH

MCHC

PLT

x106Cells

g/dL

%

fL

pg

g/dL

x103cells

Group 1

Mean

7.88

15.12

42.54

53.97

19.21

35.60

752.70

SD

0.284

0.365

1.746

1.429

0.814

1.306

158.800

Group 2

Mean

7.95

14.88

42.77

53.78

18.71

34.79

758.40

SD

0.299

0.439

1.398

1.111

0.443

0.285

145.489

Group 3

Mean

8.05

15.10

43.57

54.20

18.80

34.73

714.60

SD

0.435

0.665

2.358

2.744

0.800

1.570

100.334

Group 4

Mean

7.93

14.94

43.15

54.45

18.87

34.68

791.70

SD

0.575

0.769

2.466

1.522

0.721

1.258

55.428

 

 

TREATMENT

WBC

DC (%)

PT

APTT

x103cells

NEUT

LYMPH

MONO

EOS

BASO

Sec

Sec

Group 1

Mean

3.13

16.39

78.36

2.53

1.59

0.23

15.46

13.99

SD

0.587

2.800

3.351

0.645

0.453

0.125

0.648

1.223

Group 2

Mean

2.98

21.87

72.63

2.65

1.82

0.28

15.21

15.68

SD

0.913

9.926

10.328

0.597

0.637

0.193

0.409

4.099

Group 3

Mean

3.15

17.99

76.14

3.04

1.84

0.27

15.34

14.19

SD

0.808

4.405

5.491

0.828

0.665

0.082

0.738

2.059

Group 4

Mean

3.41

20.03

74.10

2.83

1.70

0.28

15.43

15.55

SD

1.460

6.631

7.158

0.672

0.720

0.140

0.891

3.137

 

 


HEMATOLOGY SUMMARY – MALES

Week -17

 

RECOVERY

RBC

Hb

PCV

MCV

MCH

MCHC

PLT

x106Cells

g/dL

%

fL

pg

g/dL

x103cells

Group 1R

Mean

8.49

15.69

44.91

52.98

18.50

34.95

653.70

SD

0.244

0.314

1.290

1.991

0.516

0.610

54.718

Group 4R

Mean

8.51

15.81

44.63

52.43

18.59

35.43*

625.10

SD

0.378

1.041

3.054

2.439

0.844

0.211

134.622

 

 

 

RECOVERY

WBC

DC (%)

PT

APTT

x103cells

NEUT

LYMPH

MONO

EOS

BASO

Sec

Sec

Group 1R

Mean

5.41

21.68

72.61

2.70

1.75

0.30

15.43

13.25

SD

0.982

6.820

7.035

0.952

0.420

0.067

0.926

1.362

Group 4R

Mean

4.46

18.96

74.86

3.18

1.50

0.37

14.89

13.28

SD

1.063

5.807

6.646

0.981

0.455

0.095

0.601

1.522

 

 

*Significant at p ≤ 0.05 level with group 1R


HEMATOLOGY SUMMARY – FEMALES

Week -17

 

RECOVERY

RBC

Hb

PCV

MCV

MCH

MCHC

PLT

x106Cells

g/dL

%

fL

pg

g/dL

x103cells

Group 1R

Mean

7.81

15.03

42.69

54.72

19.27

35.22

722.70

SD

0.256

0.231

1.000

2.205

0.750

0.571

130.177

Group 4R

Mean

7.77

15.04

42.62

54.87

19.36

35.31

707.50

SD

0.611

1.343

3.646

2.635

0.943

0.448

163.011

 

 

 

 

RECOVERY

WBC

DC (%)

PT

APTT

x103cells

NEUT

LYMPH

MONO

EOS

BASO

Sec

Sec

Group 1R

Mean

2.61

19.35

74.98

2.64

2.11

0.17

15.01

12.51

SD

0.666

3.654

4.363

0.824

0.881

0.067

0.601

1.455

Group 4R

Mean

2.51

15.00

79.44

2.29

2.28

0.23

14.78

13.32

SD

0.457

7.487

9.049

0.557

1.802

0.067

0.880

1.219

*Significant at p ≤ 0.05 level with group 1R

 

CLINICAL BIOCHEMISTRY SUMMARY – MALES

WEEK -13

 

TREATMENT

GLU

UREA

CREA

CHOL

TRIGL

AST

ALT

ALP

mmol/L

mmol /L

µmol /L

mmol/L

mmol /L

U/L

U/L

U/L

Group 1

Mean

8.18

7.04

36.22

2.12

0.57

72.35

33.52

63.06

SD

0.764

0.708

4.062

0.375

0.114

23.667

6.992

13.658

Group 2

Mean

8.29

7.29

38.20

2.13

0.61

62.83

28.26

52.61

SD

0.864

1.402

7.395

0.320

0.121

3.604

4.733#

12.913

Group 3

Mean

8.25

6.82

36.73

2.30

0.56

68.40

39.21

54.57

SD

1.166

1.231

5.849

0.447

0.194

11.151

6.218

17.782

Group 4

Mean

8.02

7.33

38.02

2.11

0.60

70.73

55.26*#!

63.10

SD

1.234

1.243

4.450

0.293

0.105

7.720

8.678

15.241

 

 

 

TREATMENT

BIL

Na

K

Cl

TPO

ALB

GLB

A/G

µmol /L

mmol /L

mmol/L

mmol/L

g/L

g/L

g/L

_

Group 1

Mean

1.49

147.99

3.55

111.40

67.66

43.00

24.66

1.81

SD

0.829

2.976

0.286

3.142

6.763

1.520

6.222

0.293

Group 2

Mean

1.47

146.23

3.51

110.13

66.58

44.17

22.41

1.99

SD

0.634

2.066

0.377

2.307

3.909

4.441

1.629

0.304

Group 3

Mean

1.39

147.28

3.71

111.01

67.29

43.72

23.57

1.87

SD

0.603

3.739

0.339

3.293

5.423

5.085

1.980

0.257

Group 4

Mean

1.18

146.69

3.58

110.08

64.77

43.93

20.84

2.13

SD

0.581

1.227

0.392

1.598

1.470

1.784

1.928

0.271

*Significant at p ≤ 0.05 level with group 1,#Significant at p ≤ 0.05 level with group 2,!Significant at p ≤ 0.05 level with group 13

 


CLINICAL BIOCHEMISTRY SUMMARY – FEMALES  

WEEK -13

TREATMENT

GLU

UREA

CREA

CHOL

TRIGL

AST

ALT

ALP

mmol/L

mmol/L

µmol /L

mmol/L

mmol/L

U/L

U/L

U/L

Group 1

Mean

7.32

7.88

40.25

2.16

0.51

62.95

28.47

26.14

SD

0.721

1.267

3.665

0.270

0.123

19.452

6.219

7.245

Group 2

Mean

7.96

7.65

41.61

2.06

0.51

65.51

27.10

32.64

SD

1.074

1.039

5.728

0.301

0.089

9.856

7.517

12.331

Group 3

Mean

7.61

7.68

38.56

1.86

0.48

62.31

31.02

34.51

SD

1.603

0.872

6.578

0.343

0.143

8.117

7.142

15.659

Group 4

Mean

7.87

7.49

36.87

1.77

0.54

67.95

62.58

42.02

SD

1.268

1.044

4.073

0.402

0.178

20.950

61.769

33.439

 

 

 

 

 

TREATMENT

BLI

Na

K

Cl

TPO

ALB

GLB

A/G

µmol /L

mmol /L

mmol/L

mmol/L

g/L

g/L

g/L

_

Group 1

Mean

1.69

147.27

3.39

111.24

72.83

54.06

18.77

2.91

SD

0.861

1.709

0.359

2.459

1.889

1.942

1.829

0.329

Group 2

Mean

1.27

146.49

3.46

110.38

70.65

51.34

19.31

2.71

SD

0.581

2.402

0.351

2.438

3.750

2.915

2.682

0.444

Group 3

Mean

1.66

147.11

3.42

110.40

69.60

51.57

18.03

2.89

SD

0.674

2.657

0.317

3.552

4.531

5.363

1.518

0.465

Group 4

Mean

1.44

146.83

3.65

110.78

68.21*

51.16

17.05

3.05

SD

0.724

2.314

0.333

2.086

2.546

3.738

2.018

0.519

*Significant at p ≤ 0.05 level with group 1

 

 


CLINICAL BIOCHEMISTRY SUMMARY – MALES 

WEEK -17

RECOVERY

GLU

UREA

CREA

CHOL

TRIGL

AST

ALT

ALP

mmol/L

mmol /L

µmol /L

mmol/L

mmol/L

U/L

U/L

U/L

Group 1R

Mean

8.17

6.67

37.69

1.82

0.58

58.42

27.95

52.80

SD

0.773

0.591

3.793

0.249

0.135

4.709

4.776

8.127

Group 4R

Mean

9.12*

6.99

38.27

2.00

0.68

70.55*

32.02

55.73

SD

0.868

0.720

5.430

0.256

0.185

15.368

5.961

7.811

 

   

 

RECOVERY

 

BLI

Na

K

Cl

TPO

ALB

GLB

A/G

µmol /L

mmol /L

mmol/L

mmol/L

g/L

g/L

g/L

_

Group 1R

Mean

1.15

146.84

3.55

110.34

63.42

41.04

22.39

1.84

SD

0.636

1.055

0.144

0.458

2.189

1.432

1.694

0.160

Group 4R

Mean

0.81

145.84

4.32*

109.58

65.84

42.69

23.15

1.85

SD

0.277

1.283

0.899

1.457

4.003

2.061

2.161

0.115

 

*Significant at p ≤ 0.05 level with group 1R.

  

 

 


CLINICAL BIOCHEMISTRY SUMMARY – FEMALES

WEEK -17

RECOVERY

GLU

UREA

CREA

CHOL

TRIGL

AST

ALT

ALP

mmol/L

mmol/L

µmol /L

mmol/L

mmol/L

U/L

U/L

U/L

Group 1R

Mean

8.21

8.01

38.51

2.03

0.60

105.08

47.37

26.00

SD

0.604

0.993

6.709

0.477

0.217

59.888

43.679

8.220

Group 4R

Mean

8.36

7.94

41.11

2.26

0.51

61.51*

22.69

25.66

SD

1.483

0.881

4.879

0.455

0.069

10.059

6.971

9.801

 

 

 

 

 

RECOVERY

BLI

Na

K

Cl

TPO

ALB

GLB

A/G

µmol /L

mmol/L

mmol/L

mmol/L

g/L

g/L

g/L

_

Group 1R

Mean

0.77

145.12

3.77

109.80

68.50

48.87

19.63

2.49

SD

0.680

1.199

1.080

1.560

3.901

3.413

1.141

0.193

Group 4R

Mean

1.36

145.06

3.71

109.81

69.56

51.09

18.47

2.81

SD

0.753

1.556

0.350

1.754

4.496

4.551

2.154

0.508

 

*Significant at p ≤ 0.05 level with group 1R.

URINE ANALYSIS – SUMMARY - MALES

Week 13

TREATMENT

Volume

SG

pH

Pro

GLU

KET

UBG

BIL

Color

Clarity

mL

-

-

mg/dl

-

mg/dl

mg/dl

mg/dl

-

-

Group 1

Mean

5.00

1.01

8.50

30.00

0.00

5.50

0.00

0.20

NA

NA

SD

0.931

0.003

0.850

15.811

0.000

5.503

0.000

0.422

NA

NA

Group 2

Mean

4.93

1.01

8.60

35.00

0.00

4.50

0.00

0.20

NA

NA

SD

0.452

0.003

0.843

21.082

0.000

1.581

0.000

0.422

NA

NA

Group 3

Mean

4.92

1.01

8.60

30.00

0.00

4.00

0.00

0.00

NA

NA

SD

0.713

0.003

0.699

25.820

0.000

4.743

0.000

0.000

NA

NA

Group 4

Mean

4.58

1.01

8.80

20.00

0.00

1.50

0.00

0.00

NA

NA

SD

0.666

0.003

0.422

30.732

0.000

2.415

0.000

0.000

NA

NA

 

TREATMENT

ERY

LEU

/µL

/µL

Group 1

Mean

2.00

47.50

SD

4.216

36.228

Group 2

Mean

4.50

102.50

SD

8.317

144.554

Group 3

Mean

1.00

62.50

SD

3.162

39.528

Group 4

Mean

0.00

55.00

SD

0.000

38.730

 

Key:NA – Not Applicable
URINE ANALYSIS – SUMMARY – FEMALES

Week 13

TREATMENT

Volume

SG

pH

Pro

GLU

KET

UBG

BIL

Color

Clarity

mL

-

-

mg/dl

-

mg/dl

mg/dl

mg/dl

-

-

Group 1

Mean

4.31

1.01

8.30

22.50

0.00

2.50

0.00

0.00

NA

NA

SD

0.695

0.003

0.949

7.906

0.000

2.635

0.000

0.000

NA

NA

Group 2

Mean

3.95

1.01

9.00

40.00

0.00

3.00

0.00

0.00

NA

NA

SD

0.595

0.002

0.000

31.623

0.000

4.830

0.000

0.000

NA

NA

Group 3

Mean

3.88

1.01

8.90

45.00

0.00

3.00

0.00

0.00

NA

NA

SD

0.403

0.003

0.316

25.820

0.000

2.582

0.000

0.000

NA

NA

Group 4

Mean

4.15

1.02

8.50

25.00

0.00

5.00

0.00

0.20

NA

NA

SD

0.403

0.002

0.850

20.412

0.000

5.774

0.000

0.422

NA

NA

 

TREATMENT

ERY

LEU

/µL

/µL

Group 1

Mean

1.00

47.50

SD

3.162

36.228

Group 2

Mean

2.50

107.50

SD

7.906

143.880

Group 3

Mean

0.00

87.50

SD

0.000

148.254

Group 4

Mean

3.50

82.50

SD

8.182

150.946

 

Key:NA – Not Applicable
URINE ANALYSIS – SUMMARY–MALES

Week 17

RECOVERY

Volume

SG

pH

Pro

GLU

KET

UBG

BIL

Color

Clarity

mL

-

-

mg/dl

-

mg/dl

mg/dl

mg/dl

-

-

Group 1R

Mean

5.71

1.01

8.70

27.50

0.00

4.00

0.00

0.10

NA

NA

SD

0.509

0.002

0.483

18.447

0.000

2.108

0.000

0.316

NA

NA

Group 4R

Mean

5.16*

1.01

9.00

35.00

0.00

2.50

0.00

0.00

NA

NA

SD

0.427

0.002

0.000

45.947

0.000

2.635

0.000

0.000

NA

NA

 

 

 

RECOVERY

ERY

LEU

/µL

/µL

Group

1R

Mean

0.00

70.00

SD

0.000

38.730

Group

4R

Mean

0.00

117.50

SD

0.000

138.969

Key: NA = Not Applicable

* Significant at p ≤ 0.05 with Group 1R

 


URINE ANALYSIS – SUMMARY - FEMALES

Week 17

RECOVERY

Volume

SG

pH

Pro

GLU

KET

UBG

BIL

Color

Clarity

mL

-

-

mg/dl

-

mg/dl

mg/dl

mg/dl

-

-

Group 1R

Mean

4.82

1.01

8.90

27.50

0.00

3.00

0.00

0.10

NA

NA

SD

0.478

0.002

0.316

18.447

0.000

2.582

0.000

0.316

NA

NA

Group 4R

Mean

4.78

1.02

9.00

70.00

0.00

2.50

0.00

0.00

NA

NA

SD

0.399

0.002

0.000

152.662

0.000

2.635

0.000

0.000

NA

NA

 

 

RECOVERY

ERY

LEU

/µL

/µL

Group

1R

Mean

0.00

70.00

SD

0.000

38.730

Group

4R

Mean

1.00

82.50

SD

3.162

37.361

Key: NA = Not Applicable

ORGAN WEIGHTS (GRAM) – SUMMARY - MALES

 

TREATMENT

Group 1

Group 2

Group 3

Group 4

Group 1R

Group 4R

Body Weight

Mean

404.67

394.99

394.06

357.37

424.29

379.75

SD

404.32

30.92

34.88

27.68

25.20

33.61

N

10

10

10

10

10

10

Adrenals

Mean

0.0812

0.0799

0.0744

0.0745

0.0788

0.1467

SD

0.0298

0.0147

0.0076

0.0110

0.0063

0.2240

N

10

10

10

10

10

10

Kidneys

Mean

2.2282

2.1630

2.1143

2.0029

2.2439

2.0790

SD

0.2602

0.1850

0.2035

0.2037

0.1990

0.1653

N

10

10

10

10

10

10

Liver

Mean

11.3176

11.5933

11.1981

10.9633

11.3923

11.0422

SD

1.3017

1.3157

1.4083

1.0043

0.7604

0.8848

N

10

10

10

10

10

10

Heart

Mean

1.2417

1.2417

1.2311

1.1959

1.2562

1.1550*

SD

0.1432

0.0957

0.1214

0.1340

0.0830

0.1072

N

10

10

10

10

10

10

Thymus

Mean

0.5052

0.5677

0.5077

0.4598

0.5765

0.4816*

SD

0.0996

0.0934

0.0637

0.1274

0.0557

0.1030

 

N

10

10

10

10

10

10

 

*Significant at p ≤ 0.05 level with group 1R


ORGAN WEIGHTS (GRAM) – SUMMARY - MALES (CONTD.)

 

Organs

TREATMENT (WEEK- 13)

RECOVERY(WEEK-17)

Group 1

Group 2

Group 3

Group 4

Group 1R

Group 4R

Spleen

Mean

0.6606

0.7199

0.6965

0.7091

0.7916

0.7072*

SD

0.1052

0.0691

0.0770

0.1261

0.0654

0.1026

N

10

10

10

10

10

10

Testes

 

Mean

3.9899

3.8488

4.0070

3.8737

3.9596

3.7811

SD

0.2702

0.2606

0.2590

0.3286

0.3038

0.2269

N

10

10

10

10

10

10

 Right Epididymides

 

Mean

0.7738

0.7674

0.7697

0.7233

0.8974

0.8727

SD

0.1062

0.0478

0.0563

0.1205

0.0995

0.0651

N

10

10

10

10

10

10

Brain

 

Mean

2.1784

2.1707

2.1753

2.0792

2.1835

2.1791*

SD

0.1091

0.0803

0.0698

0.0763

0.0754

0.0879

N

10

10

10

10

10

10

Left Epididymides

Mean

0.7633

0.7156

0.7117

0.6859

0.8071

0.7532

SD

0.1152

0.0625

0.0484

0.0813

0.0962

0.0811

N

10

10

10

10

10

10

Left Cauda

Mean

 

0.2058

0.2018

0.2078

0.1805

0.1920

0.1898

SD

0.0188

0.0229

0.0328

0.0312

0.0266

0.0227

N

10

10

10

10

10

10

 

*Significant at p ≤ 0.05 level with group 1R

 

 

 


ORGAN WEIGHTS (GRAM) – SUMMARY FEMALES

 

Organs

TREATMENT (WEEK- 13)

RECOVERY(WEEK-17)

Group 1

Group 2

Group 3

Group 4

Group 1R

Group 4R

Body Weight

Mean

224.39

222.26

220.24

212.35

237.58

217.68

SD

17.35

12.47

17.54

22.10

9.57

14.02

N

10

10

10

10

10

10

Adrenals

Mean

0.0803

0.0991*

0.0933

0.0838#

0.1916

0.0771

SD

0.0138

0.0090

0.0104

0.0146

0.3362

0.0127

N

10

10

10

10

10

10

Kidneys

Mean

1.4152

1.5144

1.4906

1.4668

1.4768

1.4914

SD

0.1328

0.1503

0.1379

0.1439

0.1120

0.1238

N

10

10

10

10

10

10

Liver

Mean

7.0592

7.3385

7.0130

7.2293

6.8651

6.8447

SD

0.8370

0.9889

0.9861

0.6709

0.9525

0.8560

N

10

10

10

10

10

10

Heart

Mean

0.8300

0.9344

0.9142

0.8206

0.8252

0.8551

SD

0.0943

0.1176

0.0915

0.0748

0.0738

0.0891

N

10

10

10

10

10

10

Thymus

Mean

0.4052

0.4440

0.3833

0.3998

0.4233

0.3761

SD

0.0766

0.0796

0.0843

0.1019

0.0751

0.0767

N

10

10

10

10

10

10

*Significant at p ≤ 0.05 level with group 1 and 1R

#Significant at p ≤ 0.05 level with group 2

 

 


ORGAN WEIGHTS (GRAM) – SUMMARY FEMALES (CONTD.)

 

Organs

TREATMENT (WEEK- 13)

RECOVERY(WEEK-17)

Group 1

Group 2

Group 3

Group 4

Group 1R

Group 4R

Spleen

Mean

0.5175

0.4881

0.4808

0.4883

0.5048

0.5355

SD

0.1033

0.0748

0.1370

0.0861

0.0435

0.0724

N

10

10

10

10

10

10

Ovaries

Mean

0.3317

0.1641

0.2220

0.1497

0.1657

0.1429

SD

0.5704

0.0355

0.1829

0.0377

0.0497

0.0316

N

10

10

10

10

10

10

Uterus

Mean

0.8555

0.9271

0.7668

0.8594

0.8009

0.8334

SD

0.2900

0.4389

0.3018

0.3363

0.2646

0.1427

N

10

10

10

10

10

10

Brain

Mean

2.0013

1.9595

2.0287

1.9679

1.9716

1.9480

SD

0.0987

0.0898

0.0696

0.0639

0.0984

0.1230

N

10

10

10

10

10

10

 

ORGAN WEIGHTS RATIO (%) – SUMMARY- MALES

 

Organs

TREATMENT (WEEK- 13)

RECOVERY(WEEK-17)

Group 1

Group 2

Group 3

Group 4

Group 1R

Group 4R

Body Weight

Mean

404.67

394.99

394.06

357.37

424.29

379.75

SD

27.921

30.92

34.88

27.68

25.20

33.61

N

10

10

10

10

10

10

Adrenals

Mean

0.0201

0.0203

0.0191

0.0209

0.0186

0.0397

SD

0.0074

0.0037

0.0030

0.0029

0.0018

0.0619

N

10

10

10

10

10

10

Kidneys

Mean

0.5500

0.5488

0.5382

0.5615

0.5288

0.5513

SD

0.0432

0.0417

0.0500

0.0506

0.0329

0.0677

N

10

10

10

10

10

10

Liver

Mean

2.7904

2.9397

2.8419

3.0726

2.6885

2.9231

SD

0.1656

0.3013

0.2507

0.2412

0.1646

0.2902

N

10

10

10

10

10

10

Heart

Mean

0.3067

0.3151

0.3142

0.3361

0.2970

0.3063

SD

0.0265

0.0231

0.0380

0.0430

0.0264

0.0391

N

10

10

10

10

10

10


ORGAN WEIGHTS RATIO (%) – SUMMARY MALES (CONTD.)

 

Organs

TREATMENT (WEEK- 13)

RECOVERY (WEEK-17)

Group 1

Group 2

Group 3

Group 4

Group 1R

Group 4R

Thymus

Mean

0.1248

0.1444

0.1297

0.1291

0.1361

0.1276

SD

0.0228

0.0267

0.0199

0.0359

0.0132

0.0291

N

10

10

10

10

10

10

Spleen

Mean

0.1630

0.1444

0.1777

0.1981*

0.1870

0.1881

SD

0.0217

0.0267

0.0222

0.0300

0.0180

0.0342

N

10

10

10

10

10

10

Testes

 

Mean

0.9875

0.9768

1.0220

1.0870*#

0.9356

1.0033

SD

0.0560

0.0607

0.0902

0.0953

0.0843

0.1138

N

10

10

10

10

10

10

 Right Epididymides

Mean

0.1908

0.1954

0.1971

0.2028

0.2119

0.2307

SD

0.0186

0.0208

0.0253

0.0337

0.0245

0.0182

N

10

10

10

10

10

10

Brain

Mean

0.5399

0.5523

0.5555

0.5843

0.5159

0.5786

SD

0.0356

0.0437

0.0471

0.0416

0.0285

0.0639

N

10

10

10

10

10

10

Left Epididymides

Mean

0.1886

0.1821

0.1819

0.1924

0.1900

0.1992

SD

0.0236

0.0208

0.0196

0.0225

0.0164

0.0222

N

10

10

10

10

10

10

Left Cauda

Mean

0.0510

0.0512

0.0533

0.0505

0.0453

0.0504

SD

0.0049

0.0058

0.0105

0.0077

0.0057

0.0078

N

10

10

10

10

10

10

*Significant at p ≤ 0.05 level with group 1,*#Significant at p ≤ 0.05 level with group 1, 2


ORGAN WEIGHTS RATIO (%) – SUMMARY FEMALES

 

Organs

TREATMENT (WEEK- 13)

RECOVERY(WEEK-17)

Group 1

Group 2

Group 3

Group 4

Group 1R

Group 4R

Body Weight

Mean

224.39

222.26

220.24

212.35

237.58

217.68

SD

17.35

12.47

17.54

22.10

9.57

14.02

N

10

10

10

10

10

10

Adrenals

Mean

0.0359

0.0447*

0.0428

0.0400

0.0842

0.0355

SD

0.0064

0.0043

0.0069

0.0087

0.1533

0.0064

N

10

10

10

10

10

10

Kidneys

Mean

0.6324

0.6817

0.6767

0.6967

0.6234

0.6862*

SD

0.0573

0.0573

0.0271

0.0945

0.0640

0.0558

N

10

10

10

10

10

10

Liver

Mean

3.1419

3.3062

3.1786

3.4421

2.8872

3.1418

SD

0.2361

0.4396

0.3089

0.5090

0.3586

0.3101

N

10

10

10

10

10

10

Heart

Mean

0.3695

0.4195*

0.4169

0.3893

0.3477

0.3928*

SD

0.0227

0.0371

0.0476

0.0459

0.0320

0.0322

N

10

10

10

10

10

10

Thymus

Mean

0.1808

0.1999

0.1755

0.1893

0.1778

0.1728

SD

0.0324

0.0357

0.0445

0.0505

0.0284

0.0344

N

10

10

10

10

10

10

*Significant at p ≤ 0.05 level with group 1 & 1R
ORGAN WEIGHTS RATIO (%) – SUMMARY FEMALES (CONTD.)

 

Organs

TREATMENT (WEEK- 13)

RECOVERY(WEEK-17)

Group 1

Group 2

Group 3

Group 4

Group 1R

Group 4R

Spleen

Mean

0.2305

0.2205

0.2192

0.2316

0.2126

0.2462*

SD

0.0408

0.0392

0.0636

0.0441

0.0182

0.0314

N

10

10

10

10

10

10

Ovaries

 

Mean

0.1487

0.0745

0.1010

0.0712

0.0701

0.0660

SD

0.2579

0.0188

0.0830

0.0193

0.0219

0.0148

N

10

10

10

10

10

10

Uterus

 

Mean

0.3818

0.4245

0.3543

0.4044

0.3376

0.3829

SD

0.1290

0.2232

0.1606

0.1534

0.1126

0.0608

N

0.3818

10

10

10

10

10

Brain

 

Mean

0.8971

0.8833

0.9251

0.9360

0.8322

0.8977*

SD

0.0856

0.0493

0.0636

0.1017

0.0714

0.0723

N

0.8971

10

10

10

10

10

*Significant at p ≤ 0.05 level with group 1&1R

 

   SUMMARY OF INCIDENCE OF MACROSCOPIC FINDING

MALE                                                                                                                     

GROUP

G1

G2

G3

G4

G1R

G4R

DOSE (mg/kg bw)

(0)

(100)

(300)

(750)

(0)

(750)

ANIMALS EXAMINED

10

10

10

10

10

10

ANIMALS WITHOUT ABNORMALITY

10

10

08

01

10

10

ANIMALS AFFECTED

00

00

02

09

00

00

STOMACH: Thickened, forestomach

00

00

02

09

00

00

STOMACH: Red foci, glandular stomach

00

00

00

06

00

00

 

FEMALE

GROUP

G1

G2

G3

G4

G1R

G4R

DOSE (mg/kg bw)

(0)

(100)

(300)

(750)

(0)

(750)

  ANIMALS EXAMINED

10

10

10

10

10

10

 ANIMALS WITHOUT ABNORMALITY

08

06

07

04

08

06

 ANIMALS AFFECTED

02

04

03

06

02

04

UTERUS: Distended with watery content

02

04

02

02

02

04

STOMACH: Thickened, forestomach

00

00

01

04

00

00

STOMACH: Red foci, glandular stomach

00

00

00

01

00

00

  

 

 

 

SUMMARY OF INCIDENCE OF MICROSCOPIC FINDINGS

SEX: MALE

GROUP

G1

G2

G3

G4

G1R

G4R

DOSE (mg/kg bw)

(0)

(100)

(300)

(750)

(0)

(750)

NUMBER OF ANIMALS

10

10

10

10

10

10

ADRENAL GLANDS

(10)

-

-

(10)

-

-

Dilatation, sinusoidal

01

 

 

00

 

 

Vacuolation

01

 

 

00

 

 

AORTA                     

0 (10)

-

-

0 (10)

-

-

AXILLARY LYMPH NODES

0 (10)

-

-

0 (10)

-

-

BONE MARROW (STERNUM)

0 (10)

-

-

0 (10)

-

-

BRAIN (3 LEVELS)

0 (10)

-

-

0 (10)

-

-

CECUM                                  

0 (10)

-

-

0 (10)

-

-

COLON                     

0 (10)

-

-

0 (10)

-

-

DUODENUM             

0 (10)

-

-

0 (10)

-

-

EPIDIDYMIDES

0 (10)

-

-

0 (10)

-

-

HEART                                   

 (10)

-

-

(10)

-

-

  Infiltrate, mononuclear cells

01

 

 

00

 

 

  Necrosis, myocardium

01

 

 

00

 

 

ILEUM WITH PEYER’S PATCHES

0 (10)

-

-

0 (10)

-

-

JEJUNUM                              

0 (10)

-

-

0 (10)

-

-

KIDNEYS

0 (10)

-

-

0 (10)

-

-

LIVER

0 (10)

-

-

0 (10)

-

-

LUNGS

(10)

-

-

(10)

-

-

Infiltrate, mononuclear cells

01

 

 

0

 

 

Histiocytosis

01

 

 

0

 

 

MESENTERIC LYMPH NODES

0 (10)

-

-

0 (10)

-

-

OESOPHAGUS                      

0 (10)

-

-

0 (10)

-

-

Key: Number in parenthesis indicates number of animals examined.


 

SEX: MALE

GROUP

G1

G2

G3

G4

G1R

G4R

DOSE (mg/kg bw)

(0)

(100)

(300)

(750)

(0)

(750)

NUMBER OF ANIMALS

10

10

10

10

10

10

PANCREAS

0 (10)

-

-

0 (10)

-

(1)

RECTUM

0 (10)

-

-

0 (10)

-

-

SKELETAL MUSCLE

0 (10)

-

-

0 (10)

-

-

SPINAL CORD

(CERVICAL, MID THORACIC, LUMBAR)

0 (10)

-

-

0 (10)

-

-

SPLEEN

0 (10)

-

-

0 (10)

-

-

STOMACH: FORESTOMACH

(10)

 (10)

(10)

(10)

(10)

(10)

  Hemorrhage, submucosal

00

00

02

02

00

00

  Infiltrate, polymorphonuclear and mononuclear cells

00

00

02

04

00

00

  Infiltrate, polymorphonuclear

00

00

00

04

00

00

  Necrosis, mucosal

00

00

02

04

00

00

  Hyperkeratosis

00

00

03

08

00

00

  Hyperplasia

00

00

03

09

00

00

STOMACH: GLANDULAR

(10)

(10)

(10)

(10)

(10)

(10)

  Hemorrhage, mucosal

00

00

02

07

00

00

TESTES

0 (10)

-

-

0 (10)

-

-

THYMUS

(10)

-

-

(10)

-

-

Apoptotic necrosis

04

-

-

05

-

-

Hemorrhage

01

-

-

01

-

-

THYROID WITH PARATHYROID

0 (10)

-

-

(10)

-

-

TRACHEA

0 (10)

-

-

0 (10)

-

-

URINARY BLADDER

0 (10)

-

-

0 (10)

-

-

Key: Number in parenthesis indicates number of animals examined.

 

 

SEX: FEMALE

GROUP

G1

G2

G3

G4

G1R

G4R

DOSE (mg/kg bw)

(0)

(100)

(300)

(750)

(0)

(750)

NUMBER OF ANIMALS

10

10

10

10

10

10

ADRENAL GLANDS

(10)

-

-

(10)

-

-

Dilatation

02

 

 

01

 

 

AORTA                     

0 (10)

-

-

0 (10)

-

-

AXILLARY LYMPH NODES

0 (10)

-

-

0 (10)

-

-

BONE MARROW (STERNUM)

0 (10)

-

-

0 (10)

-

-

BRAIN (3 LEVELS)

0 (10)

-

-

0 (10)

-

-

CECUM                                  

0 (10)

-

-

0 (10)

-

-

COLON                     

0 (10)

-

-

0 (10)

-

-

DUODENUM             

0 (10)

-

-

0 (10)

-

-

HEART                                   

0 (10)

-

-

0 (10)

-

-

ILEUM WITH PEYER’S PATCHES

0 (10)

-

-

0 (10)

-

-

JEJUNUM                              

0 (10)

-

-

0 (10)

-

-

KIDNEYS

(10)

-

-

(10)

-

-

Mineralization

01

 

 

00

 

 

LIVER

0 (10)

-

-

(10)

-

-

LUNGS

(10)

-

-

(10)

-

-

Infiltrate, mononuclear cells

01

 

 

01

 

 

Histiocytosis

01

 

 

00

 

 

Hemorrhage, alveolar

00

 

 

01

 

 

MESENTERIC LYMPH NODES

0 (10)

-

-

0 (10)

-

-

OESOPHAGUS                      

0 (10)

-

-

0 (10)

-

-

Key: Number in parenthesis indicates number of animals examined.

 

SEX: FEMALE

GROUP

G1

G2

G3

G4

G1R

G4R

DOSE (mg/kg bw)

(0)

(100)

(300)

(750)

(0)

(750)

NUMBER OF ANIMALS

10

10

10

10

10

10

OVARIES

0 (10)

-

-

0 (10)

-

-

PANCREAS

(10)

-

-

(10)

-

-

  Vacuolation

01

 

 

00

 

 

RECTUM

0 (10)

-

-

0 (10)

-

-

SKELETAL MUSCLE

0 (10)

-

-

0 (10)

-

-

SPINAL CORD

(CERVICAL, MID THORACIC, LUMBAR)

0 (10)

-

-

0 (10)

-

-

SPLEEN

0 (10)

-

-

0 (10)

-

-

STOMACH: FORESTOMACH

(10)

(10)

(10)

(10)

(10)

(10)

  Infiltrate, polymorphonuclear and  mononuclear cells

00

00

01

03

00

00

  Necrosis

00

00

01

01

00

00

  Hyperkeratosis

00

00

0

04

00

00

  Hyperplasia

00

00

0

06

00

00

  Congestion

00

00

0

01

00

00

  Hemorrhage

00

00

0

01

00

00

STOMACH: GLANDULAR

(10)

(10)

(10)

(10)

(10)

(10)

  Hemorrhage

00

00

00

02

00

00

THYMUS

(10)

-

-

(10)

-

-

  Apoptotic Necrosis

01

 

 

06

 

 

Hemorrhage

02

 

 

01

 

 

Hyperplasia, epithelial

00

 

 

02

 

 

THYROID

0 (10)

-

-

0 (10)

-

-

TRACHEA

0 (10)

-

-

0 (10)

-

-

URINARY BLADDER

0 (10)

-

-

0 (10)

-

-

UTERUS

(10)

(04)

(02)

(10)

(02)

(04)

Dilatation, luminal

02

04

02

02

02

04

Key: Number in parenthesis indicates number of animals examined.

Conclusions:
The administration of HOSTAPON TPHC to Wistar rats by oral gavage, at dose levels of 100, 300 and 750 mg/kg/day, resulted in no treatment-related changes in the body weights, feed consumption, haematology, clinical biochenistry and organ weights. Treatment related effects of clinical signs, macroscopic findings and microscopic effects observed in high dose group (750 mg/kg/day) were not considered adverse and were reversible as demonstrated in the recovery animals. Therefore, a ‘No Observed Adverse Effect Level` (NOAEL) is considered to be highest dose employed in the study i.e.,750 mg/kg/day.
Executive summary:

This study was conducted based on OECD Tèst Guideline 408 `Repeated Dose 90-Day Oral Toxicity Study in Rodents` and followed current principles of GLP. The purpose of this study was to assess the toxicity of HOSTAPON TPHC when administered once daily to Wistar rats by oral gavage for a period of 90 consecutive days. The reversibility of treatment-related changes was assessed after a treatment free 28 day recovery period. The doses selected for main study were 100, 300 and 750 mg/kg body weight. The test item was formulated in distilled water and administered once daily for 90 days to Wistar rats at dose levels of 100 (low dose, G2), 300 (intermediate dose, G3) and 750 (high dose, G4) mg/kg body weight/day. Vehicle control group 0 mg/kg (G1) was administered with distilled water for 90 days. Each group consisting of 10 males and 10 females were used for this study. In addition, 10 males and 10 females each were allocated to G1R (Control Recovery) and G4R (High Dose Recovery) respectively.

No mortalities were observed during the treatment period of 90 days and recovery period of 28 days. No clinical signs of toxicity were observed in control (G1) and low dose group (G2) animals during the study. Dullness was observed in few animals of intermediate group (G3). In addition to dullness, burrowing was observed in all animals of high dose group (G4) and high dose recovery group (G4R). All animals recovered from these signs after treatment withdrawal. This indicates that these clinical signs attribute to treatment. No adverse effects on food consumption were detected for treated animals when compared with vehicle control. No adverse effect on body weight and bodyweight gain (%) was detected for treated animals when compared with vehicle control. Opthalmological examiniation did not reveal any abnormalities in the vehicle control and high dose treated animals at the end of treatment period. No toxicologically releavant findings were noted in hematology, clinical biochemistry and urine alaysis parameters at the end of treatment and recovery period. No significant test item-related differences in absolute and relative organ weights were observed. Post-mortem examinations revealed thickened fore stomach and reddish foci in glandular stomach of animals of high dose (750 mg/kg) and intermediate dose (300 mg/kg) groups. Based upon the total sperm count, testes weights, gross and microscopic examination it was found that test item had no effect on the spermatogenic function of the testes under the present conditions of the study. Macroscopic examination revealed relevant findings to that of gross pathological changes as hemorrhage in forestomach and glandular stomach. Histopathologically, in forestomach polymorphonuclear and/or mononuclear cells infiltrate, necrosis, hyperkeratosis and hyperplasia of squamous epithelium were observed. These changes were reversible as demonstrated in the recovery animals.  The histopathological changes observed in the forestomach of the treated animals may be owed to the local irritant effect of the test item at mentioned doses and treatment period and considered as port of entry effects and are considered to be of minor or no relevance for human health hazard / risk assessment as elaborated hereafter in more detail.

Application of rodent forestomach effect data for predicting risk or even hazard for humans is in general not justified, given that a human counterpart for the rodent forestomach does not exist. In assessing the relevance of such findings in rodents aspects like the method of administration (e. g. gavage versus feeding) and the applicability of the forestomach to human organs (e. g. tissue concordance) have to be considered. Direct doses to the forestomach as it is the case via oral gavage, promotes the irritation of the epithelial lining of the forestomach due to a physical trauma by the gavage needle and/or chemical-induced irritation associated with the deposition of high doses of test material which then comes directly in contact with the forestomach epithelium for long periods of time. Beside the absence of a forestomach in humans, both aspects are neither representative of natural pathways of exposure nor relevant to human exposure conditions. Local irritation to the forestomach mucosa as such may cause local injury that stimulates compensatory cell proliferation. As an

example, forestomach tumors from repeated gavage dosing of ethyl acrylate were demonstrated to be an effect related specifically to the gavage administration rather than a substance specific effect (NTP, 2000).

Tissue concordance issues are also of importance when discussing specific toxicological responses of chemicals. In this context, dose-response profiles for a chemical are tissue specific (Klaassen, 1996). When assessing the applicability of rodent forestomach effects to humans, it is therefore important to consider that humans do not have a functional analogue of a rodent forestomach. Although the human esophagus and glandular stomach are histologically similar organs, there are fundamental functional and anatomical differences to the rodent forestomach. As the rodent forestomach has primarily storage and predigestion functions, tissue doses and tissue exposure times are not equivalent to a. m. organs in humans. Additionally, the forestomach mucosa in rodents has potent regenerative power, as it is to be expected for tissues which are exposed for long periods to irritant stimuli like acids and/or other aggressive food constituents. Abnormal continuous stimulation of this regenerative capacity by simple mechanical and chemical irritation can result in extensive hyperplasia of the rodent forestomach (Harrison, 1992). Structural differences like the lack of a protective lining in combination with higher pH levels after dosing may also contribute to a greater susceptibility and vulnerability of the rodent forestomach. In fact, several attempts to demonstrate similar reactivity for the esophageal and stomach mucosa in various species were unsuccessful, suggesting that these organs are less sensitive than the rodent forestomach (Wilbourn et al., 1986; Wester and Kroe, 1988). This view was also reached by the European Food Safety Authority (EFSA) during a re-evaluation of butylated hydroxyanisole (BHA), an antioxidant for which long-term gavage studies have demonstrated proliferative changes in the rodent forestomach. In its assessment, the EFSA expert panel concluded that forestomach hyperplasia in rodents may no longer be considered relevant for human risk assessment (EFSA, 2011). This view is also reflected by the endpoint specific guidance given by ECHA (Chapter R.7.2.1).

Based on the findings from this guideline conform 90-day subchronic oral toxicity study, the `no observed adverse effect level` (NOAEL) is considered to be 750 mg/kg body weight per day. This result is in good agreement with findings from a subacute 28 -day oral toxicity study in rats, where a NOAEL of 1000 mg/kg body weight per day was revealed.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
In the available GLP conform OECD TG 408 oral toxicity study, a NOAEL of 750 mg/kg body weight per day was derived for male and female rats. Forestomach lesions observed at this dose level are regarded to be local ("point-of-entry") effetcs due to irritation and not relevant for the assessment of systemic toxicity. No other specific target organ was identified. A comparable NOAEL of 1000 mg/kg body weight for the oral route was also derived in a supporting 28-day subacute oral toxicity study.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The available data allows for `route-to-route` extrapolation.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The available data allows for expert judgement concerning this endpoint.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The available data allows for route-to-route extrapolation and/or expert judgement.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The available data allows for expert judgement concenring this endpoint.

Additional information

Oral administration of the registered substance via gavage to Wistar rats at doses of 100, 300 and 750 mg/kg/day, for 90 days did not result in significant findings with regard to mortality, body weight gain, clinical symptoms, neurobehaviour, clinical biochemistry, urinalysis or gross pathology. An increase in white blood cell counts in some animals from the highest dose group is considered to be secondary in nature due to changes seen in the forestomach of high dose animals. The only test item-related findings were restricted to microscopic changes in the forestomach of animals at 750 and 300 mg/kg/day. These lesions consisted of acanthosis, parakeratosis, inflammation, ulceration and erosions and are considered to be local irritative effects. Comparable forestomach changes were not seen in the rats from the low-dose groups. Comparable forestomach findings were revealed also in a subacute 28 -day oral toxicity study in rats at a dose level of 1000 mg/kg body weight. Since the forestomach effects are considered to be `site of first contact` and thus local effects, the NOAEL of the registered substance with regard to systemic toxicity was established at 750 mg/kg body weight per day. This view is also reflected by the endpoint specific guidance given by ECHA (Chapter R.7.2.1). The forestomach changes in rats are considered to be of minor or no relevance for human health hazard / risk assessment as elaborated hereafter in more detail.

Application of rodent forestomach effect data for predicting risk or even hazard for humans is in general not justified, given that a human counterpart for the rodent forestomach does not exist. In assessing the relevance of such findings in rodents aspects like the method of administration (e.g. gavage versus feeding) and the applicability of the forestomach to human organs (e.g. tissue concordance) have to be considered.

Direct doses to the forestomach as it is the case via oral gavage, promotes the irritation of the epithelial lining of the forestomach due to a physical trauma by the gavage needle and/or chemical-induced irritation associated with the deposition of high doses of test material which then comes directly in contact with the forestomach epithelium for long periods of time. Beside the absence of a forestomach in humans, both aspects are neither representative of natural pathways of exposure nor relevant to human exposure conditions. Local irritation to the forestomach mucosa as such may cause local injury that stimulates compensatory cell proliferation. As an example, forestomach tumors from repeated gavage dosing of ethyl acrylate were demonstrated to be an effect related specifically to the gavage administration rather than a substance specific effect (NTP, 2000).

Tissue concordance issues are also of importance when discussing specific toxicological responses of chemicals. In this context, dose-response profiles for a chemical are tissue specific (Klaassen, 1996). When assessing the applicability of rodent forestomach effects to humans, it is therefore important to consider that humans do not have a functional analogue of a rodent forestomach. Although the human esophagus and glandular stomach are histologically similar organs, there are fundamental functional and anatomical differences to the rodent forestomach. As the rodent forestomach has primarily storage and predigestion functions, tissue doses and tissue exposure times are not equivalent to a.m. organs in humans. Additionally, the forestomach mucosa in rodents has potent regenerative power, as it is to be expected for tissues which are exposed for long periods to irritant stimuli like acids and/or other aggressive food constituents. Abnormal continuous stimulation of this regenerative capacity by simple mechanical and chemical irritation can result in extensive hyperplasia of the rodent forestomach (Harrison, 1992). Structural differences like the lack of a protective lining in combination with higher pH levels after dosing may also contribute to a greater susceptibility and vulnerability of the rodent forestomach. In fact, several attempts to demonstrate similar reactivity for the esophageal and stomach mucosa in various species were unsuccessful, suggesting that these organs are less sensitive than the rodent forestomach (Wilbourn et al., 1986; Wester and Kroe, 1988). This view was also reached by the European Food Safety Authority (EFSA) during a re-evaluation of butylated hydroxyanisole (BHA), an antioxidant for which long-term gavage studies have demonstrated proliferative changes in the rodent forestomach. In its assessment, the EFSA expert panel concluded that forestomach hyperplasia in rodents may no longer be considered relevant for human risk assessment (EFSA, 2011).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Guideline study according to GLP. No derivations and/or confounders. Klimisch rating 1 representing reliability without restrictions. Information is valid and meet data requirements.

Justification for classification or non-classification

Based on the results from the available repeated dose toxicity data and given that no human relevance is deducible from the effects on the forestomach seen in rats, it is concluded that the registered substance is not subject to classification and labelling according to the criteria of the EU Dangerous Substances Directive (67/548/EEC) (DSD) and of the EU Classification, Labelling and Packaging Regulation (1972/2008/EC) (CLP).