Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01/12/2000 - 19/09/2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
(1999)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF 59 NohSan No. 4200 (1985)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
Purity 98.9%
Batch No. 5400810

Test animals

Species:
rat
Strain:
other: Hanlbm: WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Source: RCC Ltd, Füllinsdorf, Switzerland
Age/weight at study initiation: 5-6 weeks old, P animals weighing 130-161 g for males and 93-126 g for females

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
See Table A6.8.2.2-1 for animal assignment to dosage groups.
Duration of exposure in general P, F1,F2 males, females: 10 weeks prior to mating through to weaning of the F1 offspring. Groups of 25 male and 25 female F1 generation offspring were then similarly treated
Details on mating procedure:
Duration of mating: up to 14 days
See Table 1.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item content was determined at the start of the pre-pairing periods and at the end of gestation/start of lactation periods for both P and F1 generations using HPLC.
Duration of treatment / exposure:
Duration of exposure before mating: 10 weeks
Frequency of treatment:
Daily
Details on study schedule:
Groups of 25 male and 25 female P generation Wistar rats were treated orally, by diet administration, with dinotefuran at concentrations of0, 300, 1000, 3000 or 10000 ppm for 10 weeks prior to mating through to weaning of the F1 offspring. Groups of 25 male and 25 female F1 generation offspring were then similarly treated. Overall achieved dose levels were within the ranges 16.5 - 47.9, 54.7 - 161.8, 162.7- 477.7 and 525.2 - 1653.9 mg/kg/day. In P and F1 parental animals, clinical signs were recorded daily, body weight and food consumption were recorded approximately weekly, oestrous cyclicity was monitored, the duration of gestation was recorded and the F1 animals were examined for sexual development landmarks. Pregnant females were allowed to litter normally and the litters were examined for live births, stillbirths and external abnormalities. The sexes and body weights of pups were recorded. Litters were not standardised by culling. Anogenital distance was measured in all F2 generation pups on day 1 of lactation. At 6 weeks of age, F1 animals selected for functional investigations were subjected to assessment of behavioural function, grip strength and locomotor activity.
Doses / concentrations
Remarks:
Doses / Concentrations:
P and F1 generation: 0, 300, 1000, 3000 or 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
25 males and 25 females per group (P generation)
Control animals:
yes, plain diet
Positive control:
No

Examinations

Parental animals: Observations and examinations:
Clinical signs:
Both generations were observed at least twice daily for clinical signs of a reaction to treatment. Dams were observed daily for survival and abnormalities in nesting or nursing behaviour.

Bodyweight:
Body weights were recorded weekly except during pairing. After mating, females were weighed on days 0, 7, 14 and 21 of gestation and days 1, 4, 7, 14 and 21 post partum.

Food/water consumption:
Food consumption was measured weekly throughout the study until day 14 post partum, except during mating.
Oestrous cyclicity (parental animals):
Oestrous cycles were monitored by vaginal smear for at least 3 weeks before mating and mean oestrous cycle duration calculated. The vaginal smears were prepared on the day of necropsy and determined the stage of estrous. Mating, fertility and conception indices were calculated. The duration of gestation was recorded. The age and body weight at which vaginal patency or preputial separation occurred was recorded for F1 generation parental animals.
Sperm parameters (parental animals):
Testis weight, epididymis weight, sperm motility, cauda epididymal sample examined for motility (all groups) and morphology (0 and 10000ppm), and one epididymis and one testis were retained for the determination of homogenisation-resistant spermatids and caudal epididymal sperm reserve (0 and 10000ppm). Additional testicular histopathology, qualitative sperm staging, was performed on PAS-stained sections.
Litter observations:
Clinical signs:
Both generations were observed at least twice daily for clinical signs of a reaction to treatment.

Pups were examined daily during the lactation period for clinical signs and mortality. Day 0 of lactation was the day of completion of parturition. Pregnant females were allowed to litter normally and the litters were examined for live births, stillbirths and external abnormalities. The sexes of pups were recorded on days 0, 4 and 21 of lactation. Litters were not standardised by culling. Pup weights were recorded on days 0/1, 4, 7, 14 and 21. Anogenital distance was measured in all F2 generation pups on day 1 of lactation.
Litters were examined as soon as possible after birth for litter size, live births, still births and gross abnormalities. The sex ratio was determined on days 0, 4 and 21 of lactation. Pups were weighed on days 0/1, 4, 7, 14 and 21 of lactation.
F1 animals for functional observation battery:
Commencing at 6 weeks of age, the F1 animals selected for functional investigations (20 animals/sex/group) were subjected to a modified Irwin screen test battery comprising qualitative and semi-quantitative assessment of appearance, motor activity, behaviour, respiration, reflexes and general autonomic functional observations. Observations were made “blind” in a standard arena. Grip strength and locomotor activity were measured quantitatively.
Postmortem examinations (parental animals):
Organ weights:
P generation: major organs, including testes/ovaries, uterus, prostate, epididymides and seminal vesicles, were weighed.

Histopathology:
Histopathological examination of reproductive organs, pituitary and adrenal glands was performed on all both generation parental animals treated at 0 or 10000ppm. Additional testicular histopathology, qualitative sperm staging, was performed on PAS-stained sections. Additional ovarian histopathology, comprising quantitative primordial follicle counts in 10 levels/ovary and a comparison with secondary/tertiary follicles, was performed on 10 females/group in F1 parental animals treated at 0 or 10000ppm. Histopathology was also performed on the reproductive organs of any animals treated at 300, 1000 or 3000ppm that failed to mate.
Postmortem examinations (offspring):
Organ weights:
F1 and F2 generation: the brain, spleen and thymus from one pup/sex/litter (randomly selected) from both F1 and F2 generation weanlings were weighed at necropsy.

Histopathology:
Histopathological examination of reproductive organs, pituitary and adrenal glands was performed on all both generation parental animals treated at 0 or 10000ppm. Additional testicular histopathology, qualitative sperm staging, was performed on PAS-stained sections. Additional ovarian histopathology, comprising quantitative primordial follicle counts in 10 levels/ovary and a comparison with secondary/tertiary follicles, was performed on 10 females/group in F1 parental animals treated at 0 or 10000ppm. Histopathology was also performed on the reproductive organs of any animals treated at 300, 1000 or 3000ppm that failed to mate.

Histopathology F1 not selected for mating, F2:
Dead pups, except where excessively cannibalised, were subjected to necropsy. Excess F1 pups not selected for further study on day 21 were also subjected to necropsy, post mortem examination and retention of the carcass in fixative. All F2 generation pups were killed shortly after weaning and subjected to necropsy and the carcasses retained in fixative.
Statistics:
Where appropriate, normally distributed variables were subjected to the Dunnett many-to-one t-test based on a pooled variance and the Steel many-to-one rank test was used for non-normally distributed variables. Fisher’s exact test was applied if the data could be dichotomised without loss of information.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
confined to soft feces during lactation in all P generation females at 10000ppm
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
of both sexes at 10000ppm was reduced
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
of both sexes at 10000ppm was reduced
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: Declines slightly during the course of the pre-pairing period.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

One P generation females at 10000 ppm died prematurely on day 21 post-partum (pp) after showing clinical signs during lactation of soft feces, ruffled fur and blood-stained urine. Macroscopic and histopathological examination showed renal changes that were considered causal to death. Although death may have been incidental to treatment with dinotefuran, a treatment-related etiology cannot be precluded because of its occurrence at the highest dose level at a time of greatly increased dosage. All other P and F1 generation parental animals survived the scheduled treatment period. Treatment-related clinical signs were confined to soft feces during lactation in all P generation females at 10000ppm and one F1 generation female treated at 10000ppm. The observation is considered to be treatment-related since its occurrence was confined to the lactation period at which time high dose levels were ingested. No treatment-related clinical signs occurred in males at 10000ppm or in either sex at lower dose levels in either parental generation.

Minor and transient effects occurred on food consumption during the pre-pairing period which were suggestive of reduced diet palatability. P and F1 generation parental animals of both sexes showed significantly reduced food consumption during weeks 1 and/or 2 of treatment (Table 3 and Table 4).

The group mean body weight gains during the pre-pairing period were reduced in P generation males and females at 10000ppm. Thus, group mean body weights at the start of mating were 5.8 and 4.0% lower than the controls, respectively (Table 5 and Table 6). The treatment-related reduction in pre-weaning body weight gains of F1 generation males and females at 10000ppm persisted during the pre-pairing treatment period. Thus, group mean body weights at the start of mating were 9.3 and 4.3% lower than the control values, respectively. The body weight gains of P and F1 generation animals of both sexes at 300 - 3000ppm were unaffected by treatment with dinotefuran.

The slightly lower body weights of P and F1 generation females at 10000ppm persisted during the gestation and lactation periods (Table 6). The group mean body weights at 21 days pp were 6.4 and 7.9% lower, respectively, than control values. The body weights of P and F1 generation females treated at 300 - 3000ppm were comparable to control values throughout gestation and lactation.

P generation males at 300 and 1000ppm also showed slightly lower food consumption than the controls during the first week of treatment only. Thereafter, there was no clear treatment-related effect on the food consumption of either sex at any dose level during the pre-pairing period. The food consumption during gestation of the P generation females was not affected by treatment at any dose level, but was significantly reduced by 11.3% during the first week of gestation in F1 generation females at 10000ppm. During lactation, the food consumption in P and F1 generation females treated at 3000 and 10000ppm was slightly reduced, but since the effect at 3000ppm was not accompanied by an effect on body weight gain, the effect is considered not to be adverse at 3000ppm.

There was no effect of treatment at any dose level in either generation on the duration of the estrous cycle. The mean duration of the estrous cycle in P generation females was 5.3, 5.6, 4.8, 5.1 and 4.9 days, in order of ascending dose level, and 4.9, 4.9, 4.9, 5.0 and 5.0 days, in order of ascending dose level in the F1 generation. There were no treatment-related effects at any dose level in either generation on fertility and mating performance, duration of gestation, number of implantations, post-implantation loss, litter size at birth, pup mortality, litter size at weaning and sex ratio (Table 7). With the exception of two non-dose-related occurrences of statistical significance (higher neonatal pup mortality at 300ppm and higher number of empty implantation sites at 3000ppm), all reproductive data in the treated groups were comparable to, and not significantly different from, the control group.

There were no treatment-related effects on sperm motility, morphology and counts in either P or F1 generation males at any dose level. The proportions of non-motile, stationary and progressively motile sperm in all treated P generation groups were similar to, and not significantly (p > 0.05) different from, the control group. In the F1 generation, statistically significant variation from the control values was observed for progressively motile and stationary sperm in the group treated at 10000ppm and for progressive sperm in the group treated at 1000ppm (Table 11). Males at 10000ppm showed 40% stationary and 50% progressively motile sperm compared with the control group that showed 32% stationary and 58% progressively motile sperm. The proportion of non-motile sperm in both groups was 10%. The group treated at 1000ppm also showed a significantly (p < 0.05) lower proportion of progressively motile sperm. Since the differences in the mean values were numerically small and showed no clear dose dependency, the small differences recorded are considered to be incidental to treatment. P generation males at 10000ppm showed a slightly, but significantly higher incidence of sperm abnormality type D (normal head but abnormally curved hook). However, the finding occurred in association with a high percentage incidence of normal sperm (95.0% versus 96.2% in the control) and the numerical difference from the control for this abnormality (type D) was small (2% affected versus 1.1% in the control). Furthermore, the F1 generation control incidence of type D abnormality was 1.7%. Therefore, the higher incidence in the P generation males at 10000ppm is considered to be incidental to treatment. In the F1 generation, there were minor differences from the control in the sperm morphology data, but the differences occurred in association with a high percentage incidence of normal sperm (95.4% versus 96.4% in the control) and the differences are considered to be incidental to treatment. There was no significant effect on epididymal sperm count for the P or F1 generation males, but the testicular sperm counts of the P generation males, showed a slight reduction at 10000ppm which was statistically significant (p < 0.01). However, as the magnitude of the difference was small, occurred in the absence of a significant effect on epididymal sperm count and was not repeated in the F1 generation males, the difference is considered incidental to treatment.

There were no treatment-related effects at any dose level in either generation on the nature and incidence of pup abnormalities during the pre-weaning period. The anogenital distance of F2 progeny of both sexes was unaffected by treatment at all dose levels (Table 7). The significantly greater anogenital distances of F2 pups at 1000ppm are considered incidental to treatment since a dose-relationship was not evident. Pre-weaning pup growth in both the F1 and F2 generations was retarded at 10000ppm. Group mean male and female pup weights were significantly (p < 0.01) reduced from day 14 pp, except for female F1 pups which were significantly (p < 0.05) reduced on day 21 pp only (Table 8). Thus at weaning, pup weights were 11.6 - 15.1% lower than control values. Pup weights were unaffected by treatment at lower dose levels.

Sexual maturation of F1 generation pups, based on preputial separation or vaginal patency, was unaffected by treatment at all dose levels. The group mean age at which these events occurred was 27.9, 28.0, 27.8, 28.2 and 28.2 days (males) and 34.3, 34.1, 34.5, 33.9 and 35.5 days (females), in order of ascending dose level. None of the values was significantly (p > 0.05) different from the control values. Quantitative locomotor activity of F1 progeny at 6 weeks of age was not affected by treatment at any dose level. There were no statistically significant (p > 0.05) differences in the low beam counts recorded for control and treated groups of either sex. There were no direct treatment-related effects at any dose level on motor capability as assessed by grip strength. Quantitative measurement of grip strength showed significantly (p < 0.05) lower absolute values at 10000ppm for male forelimb grip strength and female hindlimb grip strength (Table 9). However, grip strength to body weight ratios, were not significantly (p > 0.05) different from the controls, suggesting the differences were due to lower body weight/smaller size at 10000ppm, rather than a specific effect of dinotefuran on motor capability. Absolute grip strength at 3000ppm and below were not affected by treatment. None of the animals at any dose level showed any behavioural, postural, motor, respiratory or reflex anomalies in the modified Irwin screen, and all animals were of normal appearance.

The types and frequencies of gross lesions at necropsy in F1 and F2 pups shortly after weaning gave no indication of treatment-related effects. The most common finding in both generations was renal pelvic dilation, but the group incidences did not indicate an effect of treatment. The overall incidences of renal pelvic dilation were 11.0, 7.7, 11.5, 14.3 and 10.0% (F1 generation) and 8.0, 2.1, 17.0, 12.5 and 4.1% (F2 generation), in order of ascending dose level. All other gross lesions occurred at very low incidences and their distribution did not suggest an effect of treatment.

Direct treatment-related effects on organ weights were confined to the spleen in both the F1 and F2 generations. The mean absolute spleen weight (both sexes) and mean spleen weight relative to body weight (females only) were significantly (p < 0.05 or 0.01) reduced by up to 25.6% in F1 generation pups treated at 10000ppm (Table 10). The mean brain weight relative to body weight was also significantly (p < 0.01) elevated in these animals, but is considered to be due to the lower body weights of the group. There was no effect on thymus weights at any dose level. Absolute and relative spleen weights were significantly (p < 0.05 or 0.01) reduced to a similar extent in both sexes of the F2 generation at 10000ppm. Absolute brain and thymus weights were significantly (p < 0.05 or 0.01) reduced and relative brain weights were significantly increased in F2 animals exposed to 10000ppm. However, the pattern of response is indicative of a body weight effect rather than a specific effect of dinotefuran on these organs. There were no effects on any of the measured organ weights in F1 and F2 generation male and female pups at 300 - 3000ppm.

There were no treatment-related gross findings at necropsy in the male and female P and F1 generation parental animals at any dose level, but significantly (p < 0.05 or 0.01) reduced spleen weights (absolute and brain weight ratios, 10.0 - 16.0% reduced) occurred in both sexes of the P generation treated at 10000ppm. The effect was not evident at lower dose levels or in F1 generation parental animals at any dose level. Female F1 generation parental animals at 10000ppm showed significantly reduced thyroid weights (absolute, body weight and brain weight ratios, 20.0 - 25.0% reduced). Other minor, but statistically significant, differences in organ weights at 10000ppm are considered to be secondary to lower terminal body weights.

All histopathological findings recorded in the reproductive organs, pituitary and adrenal glands of P and F1 generation males and females were considered to be within the range of background lesions commonly observed in rats of this strain and age. The incidences of all individual findings at 10000ppm did not indicate an effect of treatment. There were no treatment-related findings during staging analysis of the testes. All cycles were complete and there were no indicators for maturation arrest. There were no treatment-related, biologically relevant effects on the quantitative evaluation of ovarian follicular stages. Although ovary staging revealed a significantly (p<0.025, 2-test) lower number of primordial follicles in 10000ppm animals (Table 12), the finding is deemed to have no biological significance because the numbers of antral follicles were markedly higher at 10000ppm and the numbers of corpora lutea were also slightly higher than the controls. The numbers of pre-antral follicles were comparable in the treated and control groups.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Direct treatment-related effects on organ weights were confined to lower spleen weight in both the F1 and F2 generations at 10000ppm. There was no effect on thymus weights at any dose level.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Pre-weaning pup growth in both the F1 and F2 generations was reduced at 10000ppm, resulting in lower pup weight at weaning. There were no treatment-related effects on the nature and incidence of pup abnormalities during the pre-weaning period and the anogenital distance of F2 progeny was unaffected by treatment at all dose levels. Sexual maturation of F1 generation pups was unaffected by treatment at all dose levels. There were no effects of treatment on behavioural function, locomotor activity and grip strength of 6 week old F1 progeny.

Pathological examination (F1/F2 pups):
There were no treatment-related gross lesions at necropsy in F1 and F2 pups. Direct treatment-related effects on organ weights were confined to lower spleen weight in both the F1 and F2 generations at 10000ppm. There was no effect on thymus weights at any dose level.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 2: Mean achieved dose levels

Sex / generation

Study period

300ppm

1000ppm

3000ppm

10000ppm

mg/kg bw/day

Male / P

Pre-mating

24.1

79.9

241.0

822.1

 

Post-mating

16.9

56.6

166.7

577.3

Female / P

Pre-mating

26.8

90.1

267.9

907.0

 

Gestation

21.9

75.1

226.1

767.5

 

Lactation (weeks 1 & 2)

47.8

161.8

466.1

1628.8

Male / F1

Pre-mating

27.2

90.5

269.0

934.7

 

Post-mating

16.5

54.7

162.7

575.3

Female / F1

Pre-mating

29.6

96.5

292.6

1004.8

 

Gestation

21.1

70.5

211.9

725.2

 

Lactation (weeks 1 & 2)

47.9

158.8

477.7

1653.9

 

Table 3: Summary of food consumption – P and F1 generation parental males

Generation

Study period

Group mean food consumption (g/day) at:

 

 

0ppm

300ppm

1000ppm

3000ppm

10000ppm

P

Week 1

22.9

21.9

21.0**

21.7*

20.6**

 

Week 2

23.3

22.9

22.4

22.9

22.5

 

Mean (weeks 1 - 10)

24.4

24.1

23.4

23.7

23.8

F1

Week 1

12.8

13.0

13.3

13.6

11.3

 

Week 2

17.4

17.8

18.0

17.2

14.6**

 

Mean (weeks 1 - 10)

20.3

21.3

21.4

21.2

19.4

* p < 0.05;

** p < 0.01

Table 4: Summary of food consumption – P and F1 generation parental females

Generation

Study period

Group mean food consumption (g/day) at:

 

 

0ppm

300ppm

1000ppm

3000ppm

10000ppm

P

Week 1

15.7

16.2

15.3

15.5

14.6**

 

Week 2

16.3

16.4

16.1

15.9

15.3*

 

Mean (weeks 1 - 10)

17.0

16.8

16.7

16.5

16.4

 

Mean during gestation

21.3

20.2

20.9

20.7

20.4

 

Mean during lactationa

44.7

43.3

44.2

41.7

41.2

F1

Week 1

12.2

11.7

12.2

12.2

11.2

 

Week 2

15.1

14.7

15.2

14.0*

13.3**

 

Mean (weeks 1 - 10)

14.8

15.5

15.5

15.4

15.0

 

Mean during gestation

19.6

18.5

18.9

18.6

18.5

 

Mean during lactationa

41.2

40.4

42.2

40.6

40.0

aweeks 1 and 2 only;

* p < 0.05;

** p < 0.01

Table 5: Summary of body weights – P and F1 generation parental males

Generation

Study period

Group mean body weight (g) at:

 

 

0ppm

300ppm

1000ppm

3000ppm

10000ppm

P

Week 1 (study start)

145

145

143

145

144

 

Week 10 (start of mating)

397

391

384

380

374*

 

Week 14 (post-mating)

450

443

436

431

424*

F1

Week 1 (study start)

61

62

64

65

52*

 

Week 10 (start of mating)

344

347

351

344

312**

 

Week 14 (post-mating)

421

423

427

422

387*

* p < 0.05;

** p < 0.01

Table 6: Summary of body weights – P and F1 generation parental females

Generation

Study period

Group mean body weight (g) at:

 

 

0ppm

300ppm

1000ppm

3000ppm

10000ppm

P

Week 1 (study start)

112

113

109

110

109

 

Week 10 (start of mating)

225

227

226

223

216

 

Day 0 (gestation)

224

225

225

222

217

 

Day 7 (gestation)

245

243

244

240

233*

 

Day 21 (gestation)

338

332

331

327

317**

 

Day 1 (lactation)

245

244

246

243

230*

 

Day 21 (lactation)

282

281

282

274

264**

F1

Week 1 (study start)

58

56

60

59

52*

 

Week 10 (start of mating)

207

207

211

209

198

 

Day 0 (gestation)

211

208

216

211

203

 

Day 7 (gestation)

229

227

234

230

220

 

Day 21 (gestation)

319

319

322

315

307

 

Day 1 (lactation)

233

230

235

232

225

 

Day 21 (lactation)

280

275

279

269

258**

* p < 0.05;

** p < 0.01

dp < 0.05 for total no. of empty implantation sites;

ep < 0.05 for pup mortality days 1 - 4pp;

* p < 0.05;

** p < 0.01


Applicant's summary and conclusion

Conclusions:
For the rat, the no adverse effect level for all effects in P and F1 generation adults was determined to be 3000 mg/kg, equivalent to dose levels of 241, 269, 267.9 and 292.6 mg/kg/d for P males, F1 males, P females and F1 females, respectively, based on the occuremce of reduced food consumption, body weight gain and spleen and thyroid weights at 10000 mg/kg.
The no adverse effect level for reproductive effects and effects on pup behaviour was established as 1000 mg/kg equivalent to dose levels of 822.1, 934.7, 907 and 1004.8 mg/kg/d for P males, F1 males, P females and F1 females, respectively, based on the absence of reproductive effects and effects on pup behaviour at 10000 mg/kg
The no adverse effect level for effects on pup development was established as 3000 mg/kg, equivalent to 241, 269, 267.9 and 292.6 mg/kg/d for P males, F1 males, P females and F1 females, respectivley, based on the occirence of reduced pup weight gain during lactation at 1000 mg/kg, equivalent to dose levels of 1628.8 and 1653.9 mg/kg/d for P females and F1 females, respectively.
Executive summary:

P/F1 parental animals:

There were no treatment-related deaths. Treatment-related clinical signs were confined to soft feces during lactation in all P generation females at 10000ppm and one F1 generation female treated at 10000ppm. Minor and transient effects occurred on food consumption during the pre-pairing period which were suggestive of reduced diet palatability. During lactation, the food consumption in P and F1 generation females treated at 10000ppm was slightly reduced, The body weight gain of P and F1 generation animals of both sexes at 10000ppm was reduced. There were no treatment-related effects at any dose level in either generation on fertility and mating performance, duration of gestation, number of implantations, post-implantation loss, litter size at birth, pup mortality, litter size at weaning and sex ratio.

F1/F2 offspring:

Pre-weaning pup growth in both the F1 and F2 generations was reduced at 10000ppm, resulting in lower pup weight at weaning. There were no treatment-related effects on the nature and incidence of pup abnormalities during the pre-weaning period and the anogenital distance of F2 progeny was unaffected by treatment at all dose levels. Sexual maturation of F1 generation pups was unaffected by treatment at all dose levels. There were no effects of treatment on behavioural function, locomotor activity and grip strength of 6 week old F1 progeny.

Pathological examination (parental animals):

There were no treatment-related gross findings at necropsy in the P and F1 generation parental animals at any dose level, but significantly reduced spleen weights occurred in both sexes of the P generation treated at 10000ppm. The effect was not evident at lower dose levels or in F1 generation parental animals at any dose level. Female F1 generation parental animals at 10000ppm showed significantly reduced thyroid weights. There were no treatment-related histopathological findings in the reproductive organs, pituitary and adrenal glands of P and F1 generation animals. There were no treatment-related findings during staging analysis of the testes. There were no treatment-related effects on sperm motility, morphology and counts in either P or F1 generation males at any dose level. There were no treatment-related, biologically relevant effects on the quantitative evaluation of ovarian follicular stages.

Pathological examination (F1/F2 pups):

There were no treatment-related gross lesions at necropsy in F1 and F2 pups. Direct treatment-related effects on organ weights were confined to lower spleen weight in both the F1 and F2 generations at 10000ppm. There was no effect on thymus weights at any dose level.