dinotefuran (ISO); 1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

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 occurrence 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, respectively, based on the occurrence 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.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

(1999)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

(1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 59 NohSan No. 4200 (1985)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Hanlbm: WIST (SPF)

Sex:

male/female

Details on test animals or test system 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

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.

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

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.

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

Dose descriptor:

NOEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

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

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.

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

Reproductive effects observed:

not specified

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 lactation^a	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 lactation^a	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

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

241 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effects on developmental toxicity

Description of key information

For the rabbit, the no adverse effect level of dinotefuran was determined to be 52 mg/kg/d for dams in terms of general toxicological effects and 300 mg/kg/d for embryonic development or teratogenicity.

For the rat, the no adverse effect level of dinotefuran was estimated to be 300 mg/kg/d in pregnant females in terms of general toxicological effects and 1000 mg/kg/d in foetuses in terms of embryonic development and teratogenicity.

The LOAEL for maternal toxic effects is 500 mg/kg bw/day, based on the occurrence of one death, tachypnea, reduced food consumption, body weight loss and increased incidence of premature delivery/abortion.

A no-observed-effect-level (NOEL) for pregnant rabbits was established as 175 mg/kg bw/day.

A no-observed-effect-level (NOEL) for embryonic development was established as 500mg/kg bw/day, based on no effects on embryonic development and no excess incidences of skeletal and visceral abnormalities and variants at this dose level.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15/10/1997 - 08/06/1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1984)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 59 NohSan no. 4200 (1985)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crj:CD(SD) IGS (SPF)

Details on test animals or test system and environmental conditions:

Source: Charles River Japan, Inc., Kawasaki, Japan
Age/weight at study initiation: 10-12 weeks old, weighing 212.22-269.37 g for females
Number of animals per group: 24 mated females per group. See Table 1
Mating period: 12 – 13 days

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

Total volume applied: 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical results for homogeneity showed concentration being 98.2 to 101%.
Analytical method not reported.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation:
- Proof of pregnancy: sperm in vaginal smear

Duration of treatment / exposure:

Duration of exposure: Rat, day 6-15, post-mating
Post-exposure period: 5 days

Frequency of treatment:

Daily

Duration of test:

Approximately 30 days

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

24 mated females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: to find appropriate dose levels for the present study, "a dose finding teratogenicity study of MTI-446 given orally to rats (report no. H-97162)" was performed (dose levels: 30, 100, 300 and 1000 mg/kg). Dams in the 1000 mg/kg group showed decreased bodyweight gains and food consumption in the early period of treatment. In the other groups, there were no treatment-related changes. Therefore, the 1000 mg/kg group was selected as the high dose. Other dose levels were 300 and 100 mg/kg with a common decremntal ratio of about 3.

Maternal examinations:

Body weight: Yes, on days 0 and 3 of gestation and then daily from day 5 of gestation until sacrifice.
Food consumption: Yes, on days 0 and 3 of gestation and then daily from day 6 of gestation until sacrifice.
Clinical signs: Yes, at least once daily on non-treatment days and at least twice daily during the treatment period.

Ovaries and uterine content:

Examination of uterine content: Yes, the uterine tract and ovaries were removed and pregnancy was confirmed. If implantations were not visible macroscopically, the uterus was immersed in ammonium sulphate to aid visualisation. Maternal organs of the cranial, thoracic and abdominal cavities, and ovaries (including corpora lutea count) and uteri (implantation site count) were examined macroscopically. The uterine contents were classified as live fetuses, embryo/fetal deaths, placental remnants, early or late resorptions, or macerated fetuses.

Fetal examinations:

General: Fetuses were sexed, examined for external malformations, and weighed.
Skelet: Yes, approximately half of the foetuses from each litter were subjected to skeletal evaluation using a dual staining technique for cartilage and bone and examined for skeletal malformations and variations including counting the number of ossification centers in vertebrae, metacarpals, metatarsals, proximal and medial phalanges.
Soft tissue: Yes, approximately half of the foetuses were examined for soft-tissue malformations and variations by fixation in Bouin’s solution and subsequent micro-dissection of the cranial and abdominal cavities by Wilson’s method and of the thoracic cavity by the method of Nishimura .

Statistics:

Where appropriate, data were analysed for homogeneity of variance using Bartlett’s test followed by one-way ANOVA for homogeneous data. If significant, Dunnett’s test was performed. Non-homogeneous data and percentage/dam data were analysed using the Kruskal-Wallis H-test followed by Dunnett’s test if significant.

Indices:

Not applicable

Historical control data:

Not applicable

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There were no deaths during the study. With the exception of a single animal treated at 1000mg/kg bw/day that showed transient hypoactivity on days 8 to 10 of gestation, there were no clinical signs of toxicity at any doe level. The body weight gain from day 6 to 11 of the group treated at 1000mg/kg bw/day was significantly reduced by 21% (Table 2). Thereafter, weight gain was not significantly different from the controls and on day 20 of gestation the group mean body weights of all treated groups were not significantly different from control values. The mean food consumption of the group treated at 1000mg/kg bw/day was significantly reduced by 10.5 to 13.0% on days 6, 7 and 9 of gestation. The mean water consumption of this group was significantly increased by 19.8 to 23.8% on days 10 to 12 of gestation. On other occasions during the treatment period the food and water consumption at 1000mg/kg bw/day were comparable to control values. There were no treatment-related effects on food and water consumption in the groups treated at 100 or 300mg/kg bw/day.

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment-related macroscopic findings in the maternal animals at any dose level. Litter parameters as assessed by pregnancy incidence, numbers of corpora lutea, implantations and live fetuses, post-implantation loss, external anomalies, fetal weights and sex ratio, were unaffected by treatment at all dose levels (Table 3). Although pre-implantation loss in the group treated at 1000mg/kg bw/day was high (24.0%) in relation to the control group (9.2%), it was not significantly different from the control value and is considered incidental to treatment with dinotefuran since implantation was complete at the initiation of treatment. The mean number of implantations in the 1000mg/kg bw/day group was slightly lower than, but not significantly different from, the control group as a consequence of higher pre-implantation loss.
There were no external fetal abnormalities in any of the treated or control groups. There were no treatment-related or statistically significant differences between treated and control groups on the incidence and nature of skeletal and visceral abnormalities and variations. No skeletal abnormalities occurred in any group and the incidences of visceral abnormalities, thymic remnant, microphthalmia, ectopic ovary, pyeloectasia, ureteroectasia and left umbilical artery, were similar in all groups (Table 4). Delayed ossification, as assessed by the number of vertebral and phalangeal ossification centers, was not apparent at any dose level.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 2: Group mean body weights and weight gains of pregnant animals

Treatment group	Mean body	Mean body weight gain (g) on days:			Mean body weight
(mg/kg bw/day)	weight on day 6 (g)	6 -11	6 -15	6 -20	on day 20 (g)
0	280	21.9	41.2	107	386
100	281	22.0	40.3	110	390
300	281	22.2	43.0	109	390
1000	276	17.3*	37.3	97	373

* p < 0.05

 

Table 3: Group mean caesarean data

Parameter	0 mg/kg	100mg/kg	300mg/kg	1000mg/kg
No. pregnant / no. mated	22 / 24	22 / 24	20 / 24	20 / 24
No. corpora lutea±SD (mean/dam)	15.6±2.0	16.2±1.6	15.3±2.7	15.7±2.1
No. implantations±SD (mean/dam)	14.2±3.0	14.8±1.6	14.1±3.5	12.2±5.3
Pre-implantation loss (%)	9.2	8.3	10.1	24.0
Total embryofetal loss (%)	5.1	5.1	3.4	3.6
- Implant remnant (%)	0.0	0.0	0.0	0.0
- Retained placenta (%)	4.3	3.7	3.4	2.7
- Early death (%)	0.0	1.1	0.0	0.9
- Late death (%)	0.8	0.0	0.0	0.0
- Macerated fetuses (%)	0.0	0.3^b	0.0	0.0
No. live fetuses±SD (mean/dam)	13.5±3.1	14.0±2.1	13.6±3.4	11.8±5.3
Sex ratio (% males)^a	53.0	55.7	50.4	42.8
Mean body weight±SD (g) - males	3.73±0.29	3.72±0.19	3.83±0.23	3.71±0.25
Mean body weight±SD (g) - females	3.55±0.23	3.51±0.22	3.65±0.23	3.47±0.35
Live fetuses with external abnormality (%)	0.0	0.0	0.0	0.0

^are-calculated by reviewer;

^bconjoined twin macerated fetuses

 

Table 4: Group mean skeletal and visceral examination data

Parameter	0 mg/kg	100mg/kg	300mg/kg	1000mg/kg
No. litters examined	22	22	19	20
No. fetuses examined (skeletal)	143	150	132	114
Total no. abnormal fetuses (skeletal):	0	0	0	0
Skeletal variations (mean %)±SD: Total variations - cervical rib - 14^thrib - shortened 13^thrib	18.4±17.5 1.2±4.0 13.6±16.6 3.6±11.2	7.6±12.4 0.0±0.0 7.6±12.4 0.0±0.0	10.5±14.9 0.7±2.9 9.1±14.6 0.8±3.3	12.1±23.3 0.6±2.5 11.5±23.5 0.0±0.0
Mean no. ossification centres±SD: - caudal centra - caudal arches - forelimb phalanges - hindlimb phalanges	2.7±1.0 0.8±0.5 2.7±1.0 2.5±1.2	2.6±0.7 0.8±0.4 2.8±0.5 2.4±1.1	2.8±0.8 0.9±0.4 2.8±0.6 2.5±1.2	2.7±0.8 0.8±0.3 2.5±1.1 1.9±1.4
No. fetuses examined (visceral)	155	159	140	122
No. abnormal fetuses (visceral): Total abnormal fetuses (mean %)±SD - thymic remnant - microphthalmia - ectopic ovary - pyeloectasia - ureteroectasia - left umbilical artery	5.0±9.0 3.1±7.3 0.6±2.7 0.8±3.6 0.0±0.0 0.0±0.0 1.3±4.3	3.5±11.1 3.5±11.1 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0	8.4±22.9 2.7±7.2 0.0±0.0 0.0±0.0 5.7±22.4 5.0±22.4 0.0±0.0	5.1±11.9 4.6±11.9 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.6±2.5

 

Conclusions:

The no adverse effect level of dinotefuran was estimated to be 300 mg/kg/d in pregnant females in terms of general toxicological effects and 1000 mg/kg/d in foetuses in terms of embryonic development and teratogenicity.

Executive summary:

There were no premature deaths during the study. With the exception of a single animal treated at 1000 mg/kg bw/day that showed transient hypoactivity on days 8 to 10 of gestation, there were no clinical signs of toxicity at any dose level.

The body weight gain from day 6 to 11 of the group treated at 1000mg/kg bw/day was significantly reduced by 21%. Thereafter, weight gain was not significantly different from the controls. The mean food consumption of the group treated at 1000 mg/kg bw/day was reduced up to day 9 of gestation. The mean water consumption of this group was increased on days 10 to 12 of gestation. There were no treatment-related effects on food and water consumption in the groups treated at 100 or 300mg/kg bw/day.

There were no treatment-related macroscopic findings in the maternal animals at any dose level. Litter parameters were unaffected by treatment at all dose levels. The mean number of implantations in the 1000mg/kg bw/day group was slightly reduced as a consequence of a non-treatment-related higher pre-implantation loss.

There were no external fetal abnormalities in any of the treated or control groups. There were no treatment-related or statistically significant differences between treated and control groups in the incidence and nature of skeletal and visceral abnormalities and variations. Delayed ossification, as assessed by the number of vertebral and phalangeal ossification centers, was not apparent at any dose level.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/01/1998 - 03/12/1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1984)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 59 NohSan No. 4200 (1985)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Source: Ichikawa Inc., Tokyo, Japan
Age/weight at study initiation: 5 – 6 months old, weighing 2.7-3.6 kg

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

Total volume applied: 10mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical results for homogeneity showed concetration being 95.6 to 100%.
Analytical method not reported.

Details on mating procedure:

Rabbits received already mated.

Duration of treatment / exposure:

Day 6-18, post mating
Post-exposure period: 10 days

Frequency of treatment:

Daily

Duration of test:

Approximately 22 days

Remarks:

Doses / Concentrations:
52 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
125 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

22 mated females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: To find appropriate dose levels for the present study "a dose finding teratogenicity study of MTI-446 given orally to rabbits (report no. H-97165)" was performed (dose levels: 30, 100, 300 and 1000 mg/kg). Abortion was observed in 4/6 of the 1000 mg/kg group. In the same group, panting, prone position and tremors were observed. In above the 300 mg/kg group, hypoactivity, sedation and flash of nose and auricle were observed, however the clinical signs were reduced or disappeared in the 5-6 days of administration. By necropsy, in above the 100 mg/kg group, gray white plaques in stomach, pale brown discoloration and enlargement of liver were observed. Therefore, the 300 mg/kg was selected as the high dose. Other dose levels were 125 and 52 mg/kg with a common decremental ratio of about 2.4.

Maternal examinations:

Body weight: Yes, recorded on day 0 (not reported), daily from day 6 to day 19, days 21, 23, 25 and 27 of gestation and on the day of necropsy.
Food consumption: Yes, food and water consumption were recorded daily from day 6 of gestation until necropsy.
Clinical signs: Yes, recorded at least once daily on non-treatment days and at least twice daily during the treatment period.

Ovaries and uterine content:

Yes, the uterine tract and ovaries were removed and pregnancy was confirmed. If implantations were not visible macroscopically, the uterus was immersed in 2% potassium hydroxide to aid visualisation. Maternal organs of the cranial, thoracic and abdominal cavities, and ovaries (including corpora lutea count) and uteri (implantation site count) were examined macroscopically. Gross lesions were preserved for subsequent histological examination. The uterine contents were classified as live or dead fetuses, placental remnants, early or late resorptions, or macerated fetuses.

Fetal examinations:

General: Live fetuses were weighed and examined for external and oral cavity abnormalities. The thoracic and abdominal organs were examined macroscopically and sexes recorded by examination of the internal reproductive organs.
Skelet: Yes, the carcasses of all fetuses were subjected to skeletal evaluation using a dual staining technique for cartilage and bone and examined for skeletal malformations and variations including enumeration of ossification centers in vertebrae, metacarpals, metatarsals, and right limb proximal and medial phalanges.
Soft tissue: Yes, The heads of approximately one half of the fetuses from each litter and the thoracic viscera from all fetuses were examined for soft-tissue malformations and variations by fixation in Bouin’s solution and subsequent micro-dissection of the cranial cavities by Wilson’s method and of the thoracic cavity by the method of Nishimura .

Statistics:

Where appropriate, data were analysed for homogeneity of variance using Bartlett’s test followed by one-way ANOVA or Dunnett’s test and the Kruskal-Wallace H-test. Percentages were analysed using the Kruskal-Wallace H-test and Dunnett’s test.

Indices:

Not applicable

Historical control data:

Not applicable

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There were no treatment-related deaths or abortions during the study, but a control animal died on day 7 of gestation due to mal-dosing. One animal to be treated at 300mg/kg bw/day was excluded from the study on day 6 due to body weight loss prior to the start of treatment. Clinical signs of toxicity were confined to the group treated at 300mg/kg bw/day. This group showed hypoactivity, prone position, panting, flushing of the nose and ears and tremors from the start of treatment until day 14 of gestation. The mean body weight gain of the group treated at 300mg/kg bw/day was significantly (p < 0.05 or p < 0.01) reduced by 50% during the treatment period (Table 2). Subsequently, the group gained weight at a greater rate than the controls and at termination body weights were similar to the control group. The mean body weight gain of the group treated at 125mg/kg bw/day was slightly but significantly (p < 0.05) reduced on day 8 of gestation only. The mean weight gain of the group treated at 52mg/kg bw/day was unaffected by treatment with dinotefuran. The mean food consumption during treatment of the group treated at 300mg/kg bw/day was reduced by 22.7% and water consumption was significantly (p < 0.05) reduced by up to 34.2% on days 14 to 16 of gestation. The food and water consumption of the groups treated at 125 or 52mg/kg bw/day were unaffected by treatment with dinotefuran.

Dose descriptor:

NOEL

Effect level:

52 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Treatment-related macroscopic findings in maternal animals occurred at 125 and 300mg/kg bw/day. Pale brown discoloration of the liver and gray/white plaque formation in the fundic region of the stomach occurred in most animals treated at 300mg/kg bw/day and in a smaller proportion of animals treated at 125mg/kg bw/day (Table 3). One animal at 300mg/kg b.w./day also showed liver enlargement. Histological examination of representative maternal liver and stomach lesions revealed no correlative histopathological alterations. No macroscopic changes occurred at 52mg/kg bw/day.
Litter parameters as assessed by pregnancy incidence, numbers of corpora lutea, implantations and live fetuses, post-implantation loss, external anomalies, fetal weights and sex ratio, were unaffected by treatment at all dose levels (Table 4).
There were no treatment-related effects on the incidence and nature of skeletal and visceral abnormalities and variants at any dose level (Table 6). The incidences of skeletal and visceral abnormalities and skeletal variants were not significantly different (p > 0.05) from the control group. Although one litter from a dam treated at 300mg/kg bw/day had 3 fetuses with hydrocephalus, this abnormality occurs spontaneously in rabbits of the strain and source employed. Delayed ossification, measured by the number of vertebral and phalangeal ossification centers, was not apparent at any dose level.

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 2: Group mean body weights and weight gains of pregnant rabbits

Treatment group	Mean body weight (kg) on day:				Mean weight gain (kg):
(mg/kg bw/day)	6	8	19	28	Day 6-18	Day 19-28
0	3.31	3.33	3.53	3.71	0.22	0.18
52	3.27	3.28	3.51	3.67	0.24	0.16
125	3.42	3.39	3.61	3.78	0.19	0.17
300	3.37	3.33	3.48	3.73	0.11	0.25
	Mean food consumption (g/animal/day) on day:
	6	8	18	28	Day 6-18	Day 19-28
0	155	154	149	114	154	124
52	154	161	159	115	158	123
125	159	138	137	123	147	123
300	152	118*	143	136	119	142

* p < 0.05

 

Table 3: Nature and incidence of treatment related macroscopic findings in maternal animals

Macroscopic finding	Incidence of macroscopic findings at necropsy at (mg/kg bw/day):
	0	52	125	300
Number examined*	22	22	22	22
Pale brown discoloration of liver	0	0	8	20
Liver enlargement	0	0	0	1
Gray-white discoloration of gastric mucosa	0	0	0	1
Gray-white plaque in fundus of stomach	0	0	15	20
Thickening of gastric mucosa	0	0	0	2

* including non-pregnant animals

Table 4: Group mean caesarean data

Parameter	Group value at (mg/kg bw/day):
	0	52	125	300
No. pregnant / no. mated	20 / 22	19 / 22	21 / 22	19 / 22
Mean no. corpora lutea±SD	8.6±1.9	8.5±2.1	9.0±2.3	8.8±1.7
Mean no. implantations±SD	7.9±2.0	8.3±2.3	8.7±2.7	8.4±1.6
Pre-implantation loss (%)	7.7	3.2	5.0	4.4
Total dead fetuses (%)	5.1	2.1	6.9	6.9
- Implant remnant (%)	0	0	0	0
- Retained placenta (%)	4.1	2.1	2.9	1.8
- Early death (%)	0	0	1.3	0.5
- Late death (%)	0.5	0	2.1	3.6
- Macerated fetuses (%)	0.5	0	0.6	1.0
Mean no. live fetuses±SD	7.6±2.2	8.1±2.2	8.1±2.8	7.7±1.6
Sex ratio (M / F x 100)	59.2	43.9	52.0	51.6
Mean body weight±SD (g) - males	42.7±5.9	40.6±6.2	39.7±7.1	40.4±5.3
Mean body weight±SD (g) - females	42.1±6.3	39.9±6.2	38.7±6.6	39.9±6.0
No. (%) fetuses with external abnormality	0 (0.0)	1 (0.9)	2 (2.1)	1 (0.7)
- omphalocele	0 (0.0)	1 (0.9)	0 (0.0)	0 (0.0)
- gastroschisis	0 (0.0)	0 (0.0)	1 (1.1)	0 (0.0)
- cleft palate + hydrocephalus	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.7)
- club foot + arthrogryposis (fore limbs)	0 (0.0)	0 (0.0)	1 (1.1)	0 (0.0)

 

Table 5: Group mean skeletal and visceral examination data

Parameter	Group value at (mg/kg bw/day):
	0	52	125	300
No. litters	20	19	21	19
No. fetuses examined (skeletal)	151	153	170	147
No. abnormal fetuses (%) mean±SD: - separation of sternebrae - flexion of caudal sternebrae	0.9±2.8 0.9±2.8 0.0	0.0 0.0 0.0	0.6±2.7 0.0 0.6±2.7	0.0 0.0 0.0
Skeletal variants (%) mean±SD: Total variants - lumbar rib - sacralisation - 8^thlumbar - asymmetrical sternebrae	58.9±24.8 49.4±25.5 0.0 42.5±25.3 0.0	70.2±30.5 55.7±33.2 2.3±6.2 66.3±28.1 0.0	60.7±27.0 60.7±27.0 0.6±2.7 57.3±33.4 0.0	67.6±21.0 55.4±23.1 1.9±6.1 58.3±25.3 0.6±2.5
Ossification centers (%) mean±SD: - caudal centra - caudal arches - forelimb phalanges - hindlimb phalanges	16.1±0.5 8.1±0.3 13.9±0.2 12.0±0.2	16.0±0.3 8.1±0.3 14.0±0.1 12.0±0.2	16.0±0.4 8.0±0.3 13.9±0.2 12.0±0.1	16.1±0.5 8.0±0.4 14.0±0.0 12.0±0.0
No. fetuses examined (visceral)	151	153	170	138
No. abnormal fetuses: Total (%) mean±SD hydrocephalus dilatation of lateral ventricle dilatation of 3^rdventricle supernumerary coronary ostium ventricular septal defect patent ductus arteriosus persistent truncus arteriosus hydronephrosis ureteroectasia	1.6±5.1 0.0±0.0 0.0±0.0 0.6±2.8 1.0±4.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0	0.7±2.9 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.7±2.9 0.0±0.0 0.7±2.9 0.0±0.0 0.0±0.0	2.9±8.2 0.0±0.0 0.7±2.9 0.0±0.0 0.5±2.3 0.0±0.0 0.0±0.0 0.0±0.0 1.8±7.6 1.8±7.6	3.0±9.5 2.2±9.1 0.0±0.0 0.0±0.0 0.8±3.5 0.0±0.0 0.8±3.5 0.0±0.0 0.0±0.0 0.0±0.0

 

Conclusions:

The no adverse effect level of dinotefuran was determined to be 52 mg/kg/d for dams in terms of general toxicolgical effects and 300 mg/kg/d for embryonic development or teratogenicity.

Executive summary:

There were no treatment-related deaths or abortions during the study. Clinical signs of toxicity were confined to the group treated at 300mg/kg bw/day that showed hypoactivity, prone position, panting, flushing of the nose and ears and tremors from the start of treatment until day 14 of gestation. The mean body weight gain of the group treated at 300mg/kg bw/day was markedly reduced during the treatment period. Subsequently, the group gained weight at a greater rate than the controls and at termination body weights were similar to the control group. The mean body weight gain of the group treated at 125mg/kg bw/day was slightly but significantly reduced on day 8 of gestation only. The mean food consumption during treatment of the group treated at 300mg/kg bw/day was reduced and water consumption was reduced on days 14 to 16 of gestation. The food and water consumption of the groups treated at 125 or 52mg/kg bw/day were unaffected by treatment with dinotefuran.

Treatment-related macroscopic findings in maternal animals occurred at 125 and 300 mg/kg bw/day. Pale brown discoloration of the liver and gray/white plaque formation in the fundic region of the stomach occurred in most animals treated at 300 mg/kg bw/day and in some animals at 125 mg/kg bw/day. One animal at 300 mg/kg b.w./day also showed liver enlargement. There were no correlative histopathological findings.

Litter parameters were unaffected by treatment at all dose levels. There were no treatment-related effects on the incidence and nature of skeletal and visceral abnormalities and variants at any dose level. Delayed ossification, measured by the number of vertebral and phalangeal ossification centers, was not apparent at any dose level.

Deficiencies: Yes, a dual staining technique for cartilage and bone was employed as specified in OECD revised draft guideline no. 414(1996). The deviation does not affect the validity or integrity of the study.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 20, 2012 - January 28, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Notification no. 12-Nousan-8147 of the APB, MAFF (November 2000) and its latest amendment no. 22-Shouan-10015 (April 2011).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

other: Kbl:JW

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kitayama Labes Co. Ltd.
- Age at study initiation: 20 weeks old
- Weight at study initiation: 3.27 – 4.25 kg

- Housing: Metal bracket cages (350 W×500 D×350 H, mm) with wire mesh floors. One male or one female per cage except during mating. Cage trays were changed at least once every two weeks.
- Diet: available ad libitum
- Water: available ad libitum
- Acclimation period: for 14 days before the start of mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20ºC ± 3ºC
- Humidity (%): 50% ± 20
- Air changes (per hr): 12 to 18 times/hour
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: From: July 23, 2012 To: October 17, 2012

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous carboxymethyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For each dose level, the test substance was accurately weighed out and homogeneously ground in a mortar, to which a small amount of the vehicle was gradually added and suspended using a mortar. The suspension was transferred to a graduated glass or plastic volumetric cylinder, and then the vehicle was added to obtain the prescribed concentration, rinsing the mortar with the vehicle. Test suspensions were homogeneously mixed using a magnetic stirrer. The 100 mg/mL test suspension was prepared using plastic containers.

The test suspensions were prepared at least every 4 days.

VEHICLE
- Concentration in vehicle: 100 mg/mL
- Total volume applied: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test suspensions of all concentrations, prepared at the first and final preparations and used for dosing, were analyzed for the concentration of the test substance. The concentrations in all analysed formulations were 100.3% to 109.0% relative to the prescribed concentrations, and the relative standard deviations were within the range 0.0% to 1.0%. The results confirmed adequate concentrations of the test substance.

Details on mating procedure:

After the quarantine and acclimatization period, animals showing no abnormalities in general appearance or body weights during the quarantine and acclimatization period, and in the results of habituation of males’ mating behavior and sperm test were selected and mated at 22−24 and 20−22 weeks of age in males and females, respectively.

Mating was performed as follows: Males and females were placed in cages for natural mating on a 1:1 basis and observed for mating. After visual confirmation of mating, vaginal smears were prepared by the stamp-smear method, and microscopically examined. Successful mating was confirmed by the presence of sperm in a vaginal smear, and the day was defined as gestation day 0.

On the day of confirmation of mating, females were weighed and assigned to 4 study groups: control, low dose, middle dose, and high dose groups, in a manner to equalize group means and standard deviations of body weights as far as possible. Females mated by the same male were distributed across the groups. Mating was performed over 11 days until 25 successfully mated females per group were obtained. Males and females were observed for general appearance once daily during the mating period. After the completion of group assignment, all males, and females that were not used in the study, were excluded from the study and treated according to the Standard Operating Procedure.

Duration of treatment / exposure:

Gestation days 6-27

Frequency of treatment:

Daily on gestation days 6-27

Duration of test:

22 days of exposure with daily treatment

Remarks:

Doses / Concentrations:
0 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
12 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
35 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
100 mg/L
Basis:
nominal conc.

No. of animals per sex per dose:

25 mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the dose range finding study of Dinotefuran at the dose levels of 0, 60, 200, 600 and 1000 mg/kg/day in mated rabbits (Safety Research Institute for Chemical Compounds, study no. SR12004)1), daily dosing of 600 and 1000 mg/kg produced overt acute clinical signs in pregnant females. In the 600 and 1000 mg/kg/day groups, premature delivery and/or abortion occurred in 1 and 2 pregnant females out of 8 females, respectively, and treatment-related decreases in body weight gain and food consumption were noted. Overall weight loss at 1000 mg/kg/day and markedly reduced weight gain at 600 mg/kg/day occurred during the dosing period. Autopsy revealed large-sized liver at 600 mg/kg/day or more and discoloration of the kidney medulla at 200 mg/kg/day or more. No adverse effects were detected at 60 mg/kg/day.

Based on the above results, 500, 175, and 60 mg/kg were selected as the high, middle, and low dose levels, respectively, in this study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: From gestation day 0 to the day of autopsy
- Cage side observations checked: All animals were observed for mortality, behavior, external appearance, excretion, and other changes by inspection and palpation at least twice a day before and after dosing (28 to 63 minutes after dosing) during the dosing period, and once a day during the other periods. Any abnormal findings were recorded with their duration. Females found dead during the course of the study were autopsied immediately after discovery and those showing signs of abortion or premature delivery were autopsied on the day when such evidence was observed, following euthanasia by exsanguination under anesthesia with thiamylal sodium. Delivered fetuses or uterine contents were macroscopically examined, and then fixed and preserved in 10% neutral buffered formalin. As to the animals found dead, the uterine of the animal No. 460 as being difficult to separate the uterine contents was preserved. In addition, the lung and trachea of animal No. 254 and the lung, trachea and heart of animal No. 470 were preserved in 10% neutral buffered formalin.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured on gestation days 0 (the day of successful mating) and 3, and daily from gestation days 6 to 28 (the day of autopsy). Females found dead or euthanatized during the course of the study were weighed on the day of discovery of the death or euthanasia.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- Organs examined: all major organs and tissues were subjected to macroscopic examination

Ovaries and uterine content:

The uterine tract and ovaries were removed and pregnancy was confirmed. If implantations were not visible macroscopically, the uterus was stained with 10% ammonium sulphide to aid visualization of very early resorptions. All major maternal organs and tissues were examined macroscopically, including ovaries (with corpora lutea count) and uteri (implantation site count). Gravid uterine weight was recorded. The uterine contents were removed and embryos, fetuses and placentae examined and classified as live or dead fetuses, placental remnants, early or late resorptions, or macerated fetuses. Fetuses were labeled to uniquely identify location in uterus.

Fetal examinations:

All live fetuses were weighed and examined for external and oral cavity abnormalities including all orifices. Placentae were weighed, and fixed in 10% neutral buffered formalin. The sexes were recorded by examination of the internal reproductive organs.

The carcasses of all fetuses were subjected to skeletal evaluation after staining with alizarin red S and examined for skeletal malformations and variations including enumeration of ossification centers in vertebrae, sternebrae, and all limb phalanges.

Approximately one half of the fetuses/litter had the head severed at the atlas and preserved for free-hand razor sectioning (Wilson’s technique1). In the other fetuses, an incision was made in the skull to observe the ventricles. All fetuses were necropsied and examined with tissues and organs in situ. The thoracic and abdominal organs were removed, preserved and then examined for soft-tissue abnormalities and variations. The heart was examined for abnormalities by Nishimura’s microdissection method2.

Statistics:

Where appropriate, data were analysed for homogeneity of variance using Bartlett’s test followed by one-way ANOVA, followed by Dunnett’s test if significant or the Kruskal-Wallis test when variances were not homogeneous, for differences among groups. Steel’s test was used to detect differences between treated and control groups.

Historical control data:

Referred to but not provided in the report.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There was one treatment-related death and an increased incidence of premature delivery or abortion (3/24 = 12.5%) at 500 mg/kg/day. Premature delivery or abortion was considered to be related to markedly reduced food consumption from the onset of treatment. Two dams at 175 mg/kg/day and one control dam also showed premature delivery or abortion during the study, but the incidence at 175 mg/kg/day (2/25 = 8.0%) remained within the historical control range of 0 – 8.3%. One dam at 60 mg/kg/day died on day 25 following premature delivery, but was considered not to be test substance related because at necropsy there was evidence of gavage dosing error.

All dams at 500 mg/kg/day showed tachypnea on days 6 and 7, and increased incidences of small amount or no feces and reduced urine output.

Body weight gain at 500 mg/kg/day was significantly reduced throughout the treatment period, resulting in slight overall group mean body weight loss from day 6 to day 28. (Table 1). Body weight gain was not affected at lower dose levels. Food consumption at 500 mg/kg/day was lower than control consumption throughout the dosing period, and was significantly lower on gestation days 6–24 (up to 47% lower on gestation days 12-15). Food consumption was not affected at lower dose levels (Table 1).

There were no maternal, treatment-related necropsy findings at any dose level. All necropsy findings in treated animals occurred in isolation or at comparable incidence to the controls.

There was no effect on pregnancy incidence, 22, 23, 23 and 20 pregnant animals survived to scheduled necropsy at 0, 60, 175 and 500 mg/kg/day, respectively. Live litters were obtained from all animals with the exception of one animal at 175 mg/kg/day with one resorbed implantation site and one at 500 mg/kg/day with two resorbed implantation sites.

The NOAEL for maternal effects was 175 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

175 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter parameters as assessed by gravid uterine weight, numbers of corpora lutea, implantations and live fetuses, post-implantation loss, external abnormalities, fetal and placental weights and sex ratio, were not significantly different from the controls and were unaffected by treatment at all dose levels (Table 3). Although post-implantation losses at 175 and 500 mg/kg/day (11.3 and 14.2%) were higher than the control incidence of 6.1%, the differences were due almost entirely to a single incidence of 100% post-implantation loss in each of the two groups. The mean number of live foetuses was unaffected by treatment at all dose levels.

There were no treatment-related effects on the incidence and nature of fetal external abnormalities, skeletal and visceral abnormalities and variants at any dose level (Table 3). The incidences of skeletal and visceral abnormalities and skeletal variants were not significantly different (p > 0.05) from the control group. Delayed ossification was not apparent at any dose level, as assessed by the number of vertebral, sternebral and phalangeal ossification centres Table 4).
The NOAEL for embryofetal effects was 500mg/kg bw/day, the highest dose level tested.

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Group mean body weights and weight gains of pregnant rabbits

Treatment group	Mean body weight (kg) on day:					Adjusted body weight (kg)
(mg/kg bw/day)	6	9	15	24	28
0	3.86	3.84	3.91	3.96	4.02	3.62
60	3.91	3.91	3.98	4.03	4.08	3.64
175	3.85	3.83	3.84	3.91	3.98	3.56
500	3.84	3.77	3.73*	3.69**	3.83	3.45
	Mean food consumption (g/animal/day) on days:
	6 – 9	9 – 12	12 – 15	15 – 18	18 – 21	24 – 27
0	156	142	136	136	136	105
60	165	152	131	143	143	101
175	155	136	108	127	127	96.2
500	120**	98.7**	63.9**	87.7**	87.7**	82.1

* p < 0.05; ** p < 0.01

Table 2: Summary of group caesarean data

Parameter	Group value at (mg/kg bw/day):
	0	60	175	500
No. pregnant / no. mated	22 / 25	23 / 25	23 / 25	20 / 25
Mean no. corpora lutea±SD	10.0±1.8	9.9±2.1	10.0±2.4	10.0±2.5
Mean no. implantations±SD	7.7±2.7	8.6±2.8	8.6±2.8	8.2±2.9
Pre-implantation loss index^c(%)±SD	22.0±24.8	14.5±21.5	14.9±19.1	17.9±20.6
Dead/resorbed embryos – means/litter
- early stage^a	0.3	0.5	0.7	0.5
- late stage^b	0.1	0.2	0.0	0.5
- total	0.4	0.7	0.7	0.9
- incidence (%)	6.1	8.9	11.3	14.2
Mean no. live fetuses±SD	7.4±2.8	7.9±2.8	7.9±2.7	7.3±2.9
Sex ratio (% M)	50.1	56.2	46.9	52.2
Mean body weight±SD (g) - males	38.9±6.1	39.8±6.4	38.3±3.8	36.8±6.6
Mean body weight±SD (g) - females	37.7±3.8	38.8±4.0	36.5±4.4	35.7±5.8
Mean placental weight (g)	5.46	5.79	5.10	5.30
No. litters examined	22	23	22	19
No. foetuses examined	162	181	182	145
No. foetuses with external abnormality	1	0	0	0
-         omphalocele	1	0	0	0

^aincludes implantation sites and placental remnants

^bincludes macerated and dead term foetuses

^c[(no. corpora lutea – no. implantations)/no. corpora lutea] x 100

Table 3: Group mean skeletal and visceral examination data

Parameter	Group value at (mg/kg bw/day):
	0	60	175	500
SKELETAL:
Heads: Litters examined Fetuses examined No. fetuses with skeletal abnormality	22 81 0	23 92 0	22 89 0	19 74 0
Bodies: Litters examined Fetuses examined No. (%) fetuses with skeletal abnormality: -         lumbar hemivertebra -         bifurcation of rib cartilage -         supernumerary sternebrae -         fusion of sternebrae	22 162 1 (0.51) 0 0 0 1 (0.51)	23 181 0 0 0 0 0	22 182 5 (2.65) 1 (0.41) 4 (2.24) 4 (2.24) 0	19 145 0 0 0 0 0
Heads: Litters examined Fetuses examined No. fetuses with skeletal variation	22 81 0	23 92 0	22 89 0	19 74 0
Bodies: Litters examined Fetuses examined No. (%) fetuses with skeletal variation: -         8 lumbar vertebrae -         Lumbarisation of sacral vertebra -         Cervical rib -         Lumbar rib -         13^thribs -         Shortening of 12^thrib -         Absence of 12^thrib -         Unossified sternebra(e) -         Incompletely ossified sternebra -         Bipartite ossification of sternebra -         Supernumerary ossification site in sternum	22 162 72 (45.74) 2 (0.91) 0 0 46 (28.68) 4 (3.64) 1 (1.14) 1 (1.14) 16 (9.35) 1 (1.14) 4 (2.47)   7 (3.35)	23 181 62 (33.98) 0 4 (1.59) 1 (0.48) 36 (21.30) 6 (3.42) 0 0 14 (6.11) 0 2 (2.66)   6 (3.43)	22 182 73 (38.61) 6 (2.79) 2 (0.92) 2 (1.02) 36 (18.89) 4 (2.35) 0 0 24 (12.72) 3 (1.53) 0   4 (1.84)	19 145 49 (34.21) 2 (1.32) 1 (0.66) 1 (0.53) 25 (16.36) 5 (3.50) 0 0 16 (12.50) 0 2 (1.32)   2 (1.58)
VISCERAL:
Heads fixed in Bouins: Litters examined Fetuses examined No. fetuses with visceral abnormality	22 81 0	22 89 0	22 93 0	19 71 0
Fresh heads: Litters examined Fetuses examined No. fetuses with visceral abnormality	22 81 0	23 92 0	22 89 0	19 74 0
Bodies: Litters examined Fetuses examined No. foetuses with visceral abnormality -         persistent ductus arteriosus -         narrowed aorta -         ventricular septal defect -         abnormal lung lobation -         abnormal liver lobation	22 162 0 0 0 0 0 0	23 181 2 0 1 1 1 1	22 182 1 1 0 1 0 0	19 145 0 0 0 0 0 0
No. foetuses with visceral variations: Litters examined Fetuses examined No. fetuses with visceral variation	22 162 0	23 181 0	22 182 0	19 145 0

Table 4: Group mean fetal skeletal development data

Parameter	Mean no. ossification centres^aat (mg/kg bw/day):
	0	60	175	500
Litters examinedFetuses examined	22 162	23 181	22 182	19 145
Sacrovertebral body	19.22	19.17	19.18	19.06
Sternebra	5.91	5.94	5.87	5.88
Forelimb phalanges – proximal (L / R)	4.97 / 4.97	5.00 / 5.00	5.00 / 4.99	5.00 / 5.00
Forelimb phalanges – medial (L / R)	3.94 / 3.91	3.99 / 3.98	3.95 / 3.95	3.96 / 3.95
Forelimb phalanges – distal (L / R)	5.00 / 5.00	5.00 / 5.00	5.00 / 5.00	5.00 / 5.00
Hindlimb phalanges – proximal (L / R)	4.00 / 4.00	4.00 / 4.00	4.00 / 4.00	4.00 / 4.00
Hindlimb phalanges – medial (L / R)	3.99 / 3.97	4.00 / 4.00	4.00 / 4.00	4.00 / 4.00
Hindlimb phalanges – distal (L / R)	4.00 / 4.00	4.00 / 4.00	4.00 / 4.00	4.00 / 4.00

^aThe litter is the unit evaluated

Conclusions:

The LOAEL for maternal toxic effects is 500 mg/kg bw/day, based on the occurrence of one death, tachypnea, reduced food consumption, body weight loss and increased incidence of premature delivery/abortion.

A no-observed-effect-level (NOEL) for pregnant rabbits was established as 175 mg/kg bw/day.

A no-observed-effect-level (NOEL) for embryonic development was established as 500mg/kg bw/day, based on no effects on embryonic development and no excess incidences of skeletal and visceral abnormalities and variants at this dose level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

52 mg/kg bw/day

Study duration:

chronic

Species:

rabbit