spirodiclofen (ISO);3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutyrate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A GLP- and guideline-compliant (OECD 416) reproductive toxicity study with spirodiclofen is identified as Key for this endpoint.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 1997 - 15 February 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: “Guidance on Toxicology Study Data for Application of Agricultural Chemical Registration”; Society of Agricultural Chemicals, Japan (MAFF Requirements)

Version / remarks:

1985

Deviations:

yes

Remarks:

additional investigations recommended in newly issued draft guidelines such as sperm tests, estrous cycle determination, registration of developmental milestones and measurement of organ weights in weanlings were performed.

GLP compliance:

yes (incl. QA statement)

Justification for study design:

The concentrations for this two-generation study were based on the results of a one-generation rangefinder study (Eiben, R., 1997) in which groups of 10 male and 10 female rats were exposed to spirodiclofen at concentrations of 0 (control), 250, 2500, or 10000 ppm in their diet.

Species:

rat

Strain:

other: Crl: WI (WU) BR

Details on species / strain selection:

As in the case of the previous one-generation study with spirodiclofen, this study was conducted with rats, a species recommended in guidelines for reproduction studies. In this study Wistar rats (strain Crl: Wl (WU) BR of the breeder Charles River, Germany/Sulzfeld) were used. The rats had been sorted by the breeder in such a manner that no siblings were present in the animals used. Wistar rats have been used for reprotoxicological studies at Bayer AG for a number of years. Historical data of Wistar rats on the test parameters are available. Historical data of reproductive parameters in Wistar rats are given in the Report Part 2.

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Housing: During the acclimatization period and study rats were housed singly under conventional conditions in Type Ha Makrolon® cages, which are larger Type II cages, (as described by SPIEGEL, A., and GONNERT, R., Z. Versuchstierkd. 1, 38 (1961) and MEISTER, G., Z. Versuchstierkd. 7, 144 (1965)). During the mating period females were kept (up to insemination) in Typ III cages and were co-housed overnight with their males.
As bedding material low-dust soft-wood shavings were used. When parturition was near cages of females were provided with a special nesting material such as coarse wood shavings. Both, bedding and nesting material were supplied by Ssniff GmbH, Soest, and tested for contaminants on a random basis (the results are held on file at BAYER AG). The cages containing the experimental animals were separated by groups and placed on shelves in ascending order of animal number.
- Diet (e.g. ad libitum): The diet consisted of a fixed-formula standard diet (Altromin®1321 meal, supplied by Altromin GmbH, Lage) and tap water during the acclimatization period and throughout the study. Food and water were available for the animals ad libitum.

Environmental conditions:
The animal room had a standardized climate:
-Room temperature: 23 ± 2°C
-Air humidity: 55 ± 5%
-Light/ dark cycle: 12 hour rhythm from 6 a.m. to 6 p.m. GET (artificial illumination: approx. 250 lux, for work in the room approx. 450 lux). From 6 p.m. to 6 a.m. GET
- orientation light, approx. 3-5 lux
-Air exchange: approx. 15-20 passages per hour

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on mating procedure:

During the following mating period the first male was co-housed with the first female F0 animal within the group and so on over night at a maximum of 12 times during the three-week mating period. Inseminated females were not further co-housed. Insemination was established by investigating vaginal smears prepared in the morningF0 and F1 females found sperm-positive after the first mating day but not pregnant were co-housed again over one week with the same male without checking insemination or measuring body weight and food intake during possible further pregnancy. Litters born were nursed up to day 28 p.p.. All data recorded from these litters were included to the litter data of their groups. F1 females that were never sperm-positive during the 3 weeks mating period were cohoused with another male (with the next minor animal No.) proven to be fertile. Litters born were nursed up to day 4 p.p.. Data recorded from these litters were not included to the litter data of their groups.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Pre-mating exposure was at least 12 weeks.

Frequency of treatment:

Feeding

Details on study schedule:

The F0 animals were pretreated with the compound for about 12 weeks up to the cohabitation period. Within the last three weeks of this premating period investigations on estrus cycle were performed. During the following mating period the first male was co-housed with the first female F0 animal within the group and so on over night at a maximum of 12 times during the three-week mating period. Inseminated females were not further co-housed. Insemination was established by investigating vaginal smears prepared in the morning. After a gestation period of about 22 days litters were born and the dams were allowed to rear them. If necessary, four days after birth the F1 litters were reduced (= culled) to eight pups according to random lists. If possible, four male and four female pups remained per litter. Pups found in a moribund state at day 4 were excluded from lactation immediately after their body weight had been established. This was done to investigate possible malformations and to prevent cannibalism during the further rearing period. The remaining F1 pups were raised to an age of four weeks and then necropsied. F0 females were killed and necropsied when 28 day old F1 animals had been weaned. F0 males were killed after the mating period partly in the course of spermatological investigations. Twenty-five male and 25 female F1 rats per group were selected for further treatment and to breed the F2 generation. This was done by randomly selecting one male and one female as far as possible from each litter. The weaned F1 offspring was treated further with the compound for at least 13 weeks (including a three-week period for estrus cycle determinations), and then co-housed for mating (the third male with the
first female within the group) as described in the case of the FO animals. Sibling matings among the F1 generation were thereby excluded as far as possible by the following procedure: At weaning the F1 male out of the first litter of a group was appointed as the first F1 male of the same group. The F1 female of the same litter was appointed as the second F1 female within its study group. During mating period the first F1 male was co-housed with the first F1 female of the same group as done with F0 rats. The procedures during the mating, pregnancy and lactation period of F1 rats were the same as described for F0 rats. The F1 parent animals were killed as scheduled after their F2 litters had been weaned at day 28 p.p. as described for F0 rats.

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

70 ppm

Dose / conc.:

350 ppm

Dose / conc.:

1 750 ppm

No. of animals per sex per dose:

25 males and 25 females.

Control animals:

yes, plain diet

Positive control:

Not required for this study type

Parental animals: Observations and examinations:

Clinical signs, body weights, food intake, mating performance, fertility, duration of pregnancy, estrus cycling and sperm parameters (sperm motility, morphology, counts) were examined in F0 and F1 rats. Selected clinicochemical parameters were examined in F1 parent animals.

Sperm parameters (parental animals):

Spermatological parameters (sperm/spermatid count, sperm motility, morphology) were determined in F0 and F1 males.

Litter observations:

Litter size, relation of males to females and pup weight at birth as well as viability, lactation and body weight gain were studied in F1 and F2 offspring. Developmental milestones were examined in F1 weanlings.

Postmortem examinations (parental animals):

Necropsies were done in all rats. Selected organs were weighed (F0 and F1 adults as well as F1, F2 weanlings) and histopathological evaluations were performed on some organs of F0 and F1 rats.

Postmortem examinations (offspring):

Necropsies were done in all rats. Selected organs were weighed (F0 and F1 adults as well as F1, F2 weanlings) and histopathological evaluations were performed on some organs of F0 and F1 rats.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was decreased at >350 ppm for parental F0 males between weeks 12 to 15, and at 1750 ppm for F0 females mainly during the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was not impaired in the F0 parental generation.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 70 ppm no treatment-related histopathological changes were detected. A slight increase in
the severity of the adrenal vacuolation was seen at >350 ppm in F0 females and at 1750 ppm in F0 males. In the F0 males a higher incidence of foamy vacuolation of the jejunal villi epithelium than in the controls occurred. In the F1 rats a slight increase in the severity of the adrenal vacuolation was seen at >350 ppm in F0 females and at 1750 ppm in F1 males. In 1750 ppm males a higher incidence of testicular and epididymidal atrophy than in other groups was found (0-1-1-4). This was associated with epididymal oligospermia (0-1-1-4) and partly atrophic prostate and seminal vesicles. Testicular and epididymidal findings in the other groups were regarded as incidental and within the normal historical range. A higher degree of vacuolation and/or degeneration of luteal cells within normal luteal regression in 1750 ppm females was regarded as incidental due to the high variability of this parameter and due to the only slight deviation from the controls.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In the 1750 ppm F0 and F1 males no changes of the determined parameters occurred so that an evaluation of the 350 and 70 ppm groups was not necessary.

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

70 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

350 ppm

System:

endocrine system

Organ:

adrenal glands

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

350 ppm

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the F1 generation body weight development was impaired in 1750 ppm males in week 1, and in F1 females mainly during the lactation period

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the F1 generation 1750 ppm females had a slightly increased food consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Up to 350 ppm no compound-related effects were detected. At 1750 ppm cholesterol and triglyceride levels were decreased, which in some animals were below historical control data. Also the UFA (non-esterified fatty acid) values were reduced in the 1750 ppm group. Deviations in other groups were not regarded as adverse since they occurred only in one sex and were within historical variation ranges.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the F1 rats a slight increase in the severity of the adrenal vacuolation was seen at >350
ppm in F females and at 1750 ppm in F1 males. In 1750 ppm males a higher incidence of testicular and epididymal atrophy than in other groups was found (0-1-1-4). This was associated with epididymal oligospermia (0-1-1-4) and partly atrophic prostate and seminal vesicles. Testicular and epididymal findings.
in the other groups were regarded as incidental and within the normal historical range. A higher degree of
vacuolation and/or degeneration of luteal cells within normal luteal regression in 1750 ppm females was
regarded as incidental due to the high variability of this parameter and due to the only slight deviation from the
controls.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

The motility and morphology of sperms were unaffected at 1750 ppm F1 males. Sperm counts per mg epididymis and testicular spermatid head counts revealed no signs of a treatment effect at 350 ppm. Four spermless 1750 ppm males resulted in clearly reduced group means of these
parameters. These four males showed atrophic testes and epididymides associated
with oligospermia. In two of them prostate gland and seminal vesicles were atrophically changed as well. Additionally, these four males exhibited very small testes, epididymides, seminal vesicles and prostate, which resulted in (partly significantly) reduced means for the absolute weights of these organs. All these effects are a result of the extremely low body weight of these four rats.

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Due to the four very small 1750 ppm F1 males lacking sperm production reproductive parameters (number of litters; total number of pups; number of viable pups) were affected at the high dose.

Reproductive parameters were adversely affected by treatment at 1750 ppm due to four males without sperm. These four males showed atrophic testes and epididymides associated
with oligospermia. In two of them prostate gland and seminal vesicles were atrophically changed as well. Additionally, these four males exhibited very small testes, epididymides, seminal vesicles and prostate, which resulted in (partly significantly) reduced means for the absolute weights of these organs. All these effects are a result of the extremely low body weight of these four rats.

Key result

Dose descriptor:

NOAEL

Effect level:

350 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 750 ppm

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

There was no influence on the live birth index, ratio of males: females, and the mean litter size in any dose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The pup weights of the 70 ppm group
were not affected. At 350 and 1750 ppm body weight development was slightly retarded beginning with day 4 in
male pups whereas in the females the pup weights were reduced already on day 0.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

The examined developmental milestones determined in F1 weanlings (mean age at
preputial separation, mean age at vaginal opening) were not affected although preputial separation in the 1750 males
occurred slightly later.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

70 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Clinical signs:

no effects observed

Description (incidence and severity):

No significant clinical findings were made in F2 pups during the four week lactation period at levels of up to 1750 ppm. Malformations were not observed.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability (day 4 p.p.) of treated F2 pups was comparable with that of controls. Up to the dose of 1750 ppm there was no dose-dependent reduction in the lactation indices.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean litter weights at birth were not affected at 70 ppm. At higher doses (partly) significantly and/or dose-dependently lower litter weights were noted at birth and weaning. Mean fetal weights at birth were not significantly affected at 70 ppm, but dose-dependently and significantly reduced at higher concentrations. During lactation no adverse effect could be detected in the groups 70 and 350 ppm. At 1750 ppm a significant depression of pup weights occurred.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No remarkable organ weight differences occurred between the control and 70 ppm group. At higher concentrations there were some reduced organ weights explainable by the pup body weight differences in these groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

In F2 pups necropsied during the lactation period no macroscopic alterations due to the treatment were observed. No skeletal deviations were seen in the F2 pups that had died before postpartum day four, were killed in the process of culling, or were necropsied unscheduled during lactation at levels of up to 1750 ppm. No treatment-related gross pathological findings were made in F2 weanlings at scheduled necropsy.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Pup weight gain was retarded at 1750 ppm. The birth weights were inconspicuous at 70 ppm and decreased mostly in female pups at higher concentrations. The mean litter weights of F2 pups at birth and weaning were not changed significantly at 70 ppm and slightly reduced at higher
concentrations. No test substance-related clinical or gross pathological findings were observed in F2 offspring up to 1750 ppm. The skeletal development of the pups or weanlings was unaffected at dose levels of up to 1750 ppm. Organ weight changes reflected lower mean body weights

Key result

Dose descriptor:

NOAEL

Generation:

F2a

Effect level:

70 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

350 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

2-generation study in rats: Clinical chemistry (females)

Dose (ppm)	0	70	350	1750	0	70	350	1750
	males				females
Cholesterol (mmol/L)	2.55	2.30	2.07**	1.58**	2.24	2.33	2.20	1.69**
Triglyceride (mmol/L)	2.62	1.84**	1.68**	0.96**	1.36	1.22	1.02	0.58**
UFAa (mmol/L)	0.39	0.38	0.32	0.24**	0.54	0.47	0.44**	0.32**

a  non-esterified fatty acid

Multi-generation study in rats: Organ weights

Dose (ppm):	0		70		350		1750
males	F0	F1	F0	F1	F0	F1	F0	F1
Testes (absol., mg)	3583	3587	3554	3394	3574	3458	3721	3161
Testes (rel., mg/100g)	720	751	739	700	753	755	809**	768
Adrenals (absol., mg)	56	52	50**	52	50**	53	59	55
Adrenals (rel.,mg/100g)	11	11	10	11	11	12	13**	14**
females
Adrenals (absol., mg)	68	63	69	66	66	69	72	75**
Adrenals (rel.,mg/100g)	26	25	26	25	26	27	31**	31**

p <= 5%, ** p <= 1%

Multi-generation study in rats: Live Birth Index

Dose (ppm)	0		70		350		1750
	F1	F2	F1	F2	F1	F2	F1	F2
Mean	99.6	99.6	100.0	99.1	100.0	99.5	97.2	98.0

 

Multi-generation study in rats: Male pup weights (g)

Dose (ppm):	0		70		350		1750
	F1	F2	F1	F2	F1	F2	F1	F2
Birth	5.73	6.39	6.08**	6.44	5.66	6.10**	5.62	5.99*
Day 4(postcull)	9.61	10.68	10.00	10.48	9.01**	10.11	8.73**	9.90**
Day 7	14.52	15.27	14.71	16.23*	13.88*	15.59	12.80**	14.75
Day 14	28.77	30.09	30.40**	30.14	28.12**	29.50	23.64**	25.73**
Day 21	44.98	46.00	46.94*	46.94*	43.28*	45.58	35.32**	38.31**
Day 28	75.34	74.17	77.45	77.45	71.27**	72.19	58.20**	62.28**

* p < 5%, ** p < 1%

 

Dose (ppm):	0		70		350		1750
	F1	F2	F1	F2	F1	F2	F1	F2
Birth	5.59	6.17	5.66	6.06	5.41**	5.72**	5.30**	5.37**
Day 4(postcull)	9.40	10.44	9.41	10.03	8.55**	9.87	8.40**	8.97**
Day 7	14.32	15.39	14.05	16.06	13.17**	15.34	12.42**	13.52**
Day 14	28.51	30.03	29.49*	29.19	27.00**	29.31	23.19**	24.22**
Day 21	44.26	45.51	45.25	44.44	41.89**	44.74	34.85**	35.92**
Day 28	71.09	70.31	71.40	68.72	67.13**	69.07	56.66**	57.57**

 

Conclusions:

The 2-generation study is acceptable as it was initiated before March 2015. The study was performed to the current (2001) version of the guideline at dietary concentrations of 0, 70, 350 and 1750 ppm. The NOAEL for reproductive toxicity was 350 ppm, based on testicular effects and decreased spermatogenesis.

Executive summary:

In this two-generation study (OECD 416), spirodiclofen was administered to groups of 25 male and female Wistar rats at concentrations of 0, 70, 350, and 1750 ppm in their diet. Parental F₀ animals were treated over a period of about 12 weeks and allowed to mate over a period of up to three weeks. F₁ offspring were nursed up to an age of four weeks.  Offspring were selected for further treatment and for breeding a F₂ generation. F₂ offspring were weaned at an age of four  weeks. Clinical signs, body weights, food intake, mating performance, fertility, duration of pregnancy, estrus cycling and sperm parameters (sperm motility, morphology, counts) were examined in F₀ and F₁ rats. Litter size, relation of males to females and pup weight at birth as well as viability, lactation and body weight gain were studied in F₁ and F₂ offspring. Developmental milestones were examined in F₁ weanlings. Spermatological parameters (sperm/spermatid count, sperm motility, morphology) were determined in F₀ and F₁ males. Selected clinicochemical parameters were examined in F₁ parent animals. Necropsies were done in all rats. Selected organs were weighed (F₀ and F₁ adults as well as F₁, F₂ weanlings) and histopathological evaluations were performed on selected organs of F₀ and F₁ rats.  The average daily doses were 0, 5.2, 26.2 and 134.8 mg/kg bw/d in F₀ males, 0, 5.5, 27.6 and 139.2 mg/kg bw/d in F₀ females; 0, 6.4, 30.2 and 177.6 mg/kg bw/d in F₁ males, and 0, 7.0, 34.4 and 192.7 mg/kg bw/d in F₁ females.  In parental F₀ and F₁ animals no clinical signs were observed. Body weight gain was decreased at >350 ppm for parental F₀ males between weeks 12 to 15, and at 1750 ppm for F₀ females mainly during the lactation period. In the F₁ generation body weight development was impaired in 1750 ppm males in week 1, and in F₁ females mainly during the lactation period. Food consumption was not impaired in the F₀ parental generation. In the F₁ generation 1750 ppm females had a slightly increased food consumption. A compound-related effect on the cycle length of the F₀ females was not detected. A compound-related effect on insemination length, mating index, fertility index, gestation index or length and on birth index in the F₀ generation was not detected. Up to 350 ppm no compound-related effects were detected. At 1750 ppm, cholesterol and triglyceride levels were decreased, which in some animals were below historical control data. Also the UFA (non-esterified fatty acid) values were reduced in the 1750 ppm group. Deviations in other groups were not regarded as adverse since they occurred only in one sex and were within historical variation ranges. In the 1750 ppm F₀ and F₁ males no changes in the determined sperm parameters occurred; evaluation of the 350 and 70 ppm groups was not necessary.  In the F₀ adults relative adrenal weights in both sexes and testes weights were increased at 1750 ppm. Other spurious organ weight effects were most likely related to the differences in the body weights. At 70 ppm no treatment-related histopathological changes were detected. A slight increase in the severity of the adrenal vacuolation was seen at > 350 ppm in F₀ females and at 1750 ppm in F₀ males. In the F₀ males a higher incidence of foamy vacuolation of the jejunal villi epithelium than in the controls occurred. In the F₁ rats a slight increase in the severity of the adrenal vacuolation was seen at >350 ppm in F₀ females and at 1750 ppm in F₁ males. In 1750 ppm males a higher incidence of testicular and epididymal atrophy was seen, and was associated with epididymal oligospermia, partly atrophic prostate and seminal vesicles. Testicular and epididymal findings in the other groups were regarded as incidental and within the normal historical range. A higher degree of vacuolation and/or degeneration of luteal cells within normal luteal regression in 1750 ppm females was regarded as incidental due to the high variability of this parameter and due to the only slight deviation from the controls. There was no influence on the live birth index, ratio of males: females, and the mean litter size in any dose group.  No clinical signs were obvious in the F₁ pups during the lactation period. The pup weights of the 70 ppm group were not affected. At 350 and 1750 ppm body weight development was slightly retarded beginning with Day 4 in male pups whereas in the females the pup weights were reduced already on Day 0. The 4-day viability and the lactation index of the F₁ pups was not impaired. No  treatment-related gross findings were made in F₁ weanlings at scheduled necropsy. No skeletal deviations were detected in the F₁ pups, and no effect on the organ weights occurred. The examined developmental milestones determined in F₁ weanlings (mean age at preputial separation, mean age at vaginal opening) were not affected although preputial separation in the 1750 ppm males occurred slightly later. The NOAEL for parental toxicity is 70 ppm (equivalent to 5.2 mg/kg bw/d in males and to 5.5 mg/kg bw/d in females) based on body weight effects and histopathological findings in the adrenals (vacuolization) at 350 ppm. The NOAEL for reproductive toxicity is 70 ppm based on effects on pup weight at 350 ppm.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

26.2 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A GLP- and guideline-compliant (OECD 416) reproductive toxicity study with spirodiclofen is identified as Key for this endpoint, and is supported by a range-finding one-generation study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

GLP- and guideline- (OECD 414) compliant studies of developmental toxicity in the rat and rabbit are available for spirodiclofen.  A Weight of Evidence approach is used for this endpoint.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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: Japanese MAFF Guidelines ‘Teratogenicity Study’

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 88/302/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

22 (23 in the 300 mg/kg bw/d group) female Himalayan rabbits.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous suspension

Details on exposure:

22 (23 in the 300 mg/kg bw/d group) female Himalayan rabbits each were treated daily, orally by gavage, with spirodiclofen formulated in a suspension of 0.5% carboxymethylcellulose in demineralized water, from gestation day 6 to 28 at doses of 0, 100, 300 or 1000 mg/kg bw/d.

Duration of treatment / exposure:

Treated daily with test material concentrations from day 6 to 28 post coitum.

Frequency of treatment:

Daily

Duration of test:

29-day

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 (23 in the 300 mg/kg group) female Himalayan rabbits.

Details on study design:

22 (23 in the 300 mg/kg group) female Himalayan rabbits each were treated daily, orally by gavage, with Spirodiclofen, formulated in a suspension of 0.5% carboxymethylcellulose in demineralized water, from day 6 to 28 post coitum in doses of 0, 100, 300 or 1000 mg/kg bw per day. On day 29 of gestation the fetuses were delivered by cesarean section.

The doses were based on the results of a pilot study in which doses of 0, 100, 300, and 1000 mg/kg bw Spirodiclofen were tested. In this study one of the two pregnant females of the 1000 mg/kg group revealed marked transient body weight loss after start of treatment (133 g from day 6 to 7 post coitum). Maternal toxicity could not be excluded at the 1000 mg/kg level while there was no indication of developmental toxicity at this dose level.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the females was determined on day 0 p.c. and daily from day 6 to day 29 p.c. Corrected body weight gain was determined by subtracting the uterus weight on day 29 p.c. from the body weight gain from day 0 to 29 p.c.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The feed intakes of the animals were determined from the difference in weight between the feed offered and the feed not consumed for the following days of gestation: days 0-6, 6-9, 9-12, 12-15, 15-18, 18-21,21 - 24, 24 - 27 and 27 - 29 p.c.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was assessed by visual estimation of the quantities left over.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

The fetuses were eviscerated according to the modified Staples technique, including a transverse section through the brain in about 50% of the fetuses. After evaluation of the fetuses the viscera were discarded.

- Occurrence of findings of the skeletal system including the cartilaginous part in the fetuses (processing and evaluation were performed after cesarean section) Staining of the cartilage of fetuses (including about 50 % of the skulls) was performed by using Alcian blue. Afterwards the fetuses were cleared with diluted potassium hydroxide solution and were stained with alizarin red S according to the modified Dawson technique.

- occurrence of visceral findings in the fetal skull: evaluation of about 50% of the fetal skulls according to the modified Wilson technique; processing and evaluation were performed after cesarean section)

Statistics:

Females with uterine anomaly or without implantation sites as well as females which aborted or died were not taken into account for calculation of mean values. Differences between the control and treated groups were considered significant when p<0.05. Statistical significance was tested using the following methods:
a. ANOVA and Dunnett's test for
- feed intakes
- body weights and body weight gains and corrected body weight gains
- uterus weights
- number of corpora lutea per female
- number of implantations per female
- number of live fetuses per female and as percentage of implantations per female
- placental weights
- fetal weights

b. CHI-squared test (with Yates' correction according to Yates) for:
- fertility rate
- gestation rate
- number of fetuses or litters with malformations or with external or visceral deviations

c. 2 by N CHI-squared test; in case of significant differences Fisher's exact test with Bonferroni correction for
- number of implantations per group
- number of preimplantation losses per group
- number of post-implantation losses, early resorptions, late resorptions or dead fetuses per group
- number of live fetuses per group in percent of implantations
- number of male or female fetuses or fetuses with indeterminable sex per group
- number of fetuses or litters with skeletal findings

d. Kruskall-Wallis test; in case of significant differences Dunn's test for
- number of pre-implantation losses per female
- number of post-implantation losses, early resorptions, late resorptions or dead fetuses per female
- number of male or female fetuses or fetuses with indeterminable sex per female

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

an increased incidence of alopecia occurred at the
1000 mg/kg level, for which a treatment related effect cannot be excluded.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was transiently decreased after start of treatment at dose levels of 300
mg/kg and above. Overall body weight gain during the treatment and gestation period as well as corrected body
weight gain were also decreased at the 1000 mg/kg bw level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The feed intakes and correspondingly the amounts of feces were transiently decreased at >300 mg/kg bw.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

The feed intakes and correspondingly the amounts of feces were transiently decreased at >300 mg/kg bw.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy of the females on day 29 post coitum did not
reveal treatment-related findings at levels up to and including 1000 mg/kg bw.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

One female of the 1000 mg/kg bw group aborted on day 20 post coitum after it had shown
distinct systemic effects before (cold ears, nearly no feed intake and severe body weight loss after start of treatment as well as distinct liver lobulation at necropsy).

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

One female of the 1000 mg/kg bw group aborted on day 20 post coitum after it had shown distinct systemic effects before (cold ears, nearly no feed intake and severe body weight loss after start of treatment as well as distinct liver lobulation at necropsy). Furthermore, an increased incidence of alopecia occurred at the 1000 mg/kg level, for which a treatment related effect cannot be excluded. None of the females died due to treatment. The feed intakes and correspondingly the amounts of feces were transiently decreased at >300 mg/kg bw.
Decreased water consumption with subsequently decreased, concentrated and discolored urination occurred at the 1000 mg/kg bw level. The body weight gain was transiently decreased after start of treatment at dose levels of 300 mg/kg and above. Overall body weight gain during the treatment and gestation period as well as corrected body weight gain were also decreased at the 1000 mg/kg bw level. Necropsy of the females on day 29 post coitum did not reveal treatment-related findings at levels up to and including 1000 mg/kg bw.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

number of abortions

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Regarding the intrauterine development, the gestation rate was marginally decreased by one abortion at 1000 mg/kg bw. It cannot be excluded that this was a consequence of maternal toxicity. The remaining parameters of intrauterine development (external appearance and weight of placentas, the postimplantation loss in the females with viable fetuses on day 29 post coitum and correspondingly the number of fetuses, fetal sex distribution and fetal weight, fetal morphology including stage of ossification) were not affected by treatment at levels up to and including 1000 mg/kg bw. An effect on the incidence of malformations was not evident.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Conclusions:

A maternal NOAEL of 100 mg/kg bw/d was determined for this study based on abortion and clinical signs (at 1000 mg/kg bw/d), reduced body weight gain and food consumption (at 300 mg/kg bw/d).  A developmental NOAEL of 1000 mg/kg bw/d can be determined in the absence of any effects of treatment

Executive summary:

In this pre-natal developmental toxicity study, groups of 22 or 23 mated female Himalayan rabbits were treated daily, orally by gavage, with spirodiclofen in 0.5% carboxymethylcellulose from gestation day 6 to 28 at dose levels of 0, 100, 300 or 1000 mg/kg bw/d.  On gestation day 29, the fetuses were delivered by caesarean section.  Investigations were performed on general tolerance by the females as well as on its effect on intrauterine development.  One female of the 1000 mg/kg bw/d aborted on gestation day 20 after it had shown distinct systemic effects before (cold ears, nearly no feed intakes and severe body weight loss after start of treatment as well as distinct liver lobulation at necropsy).  Furthermore, an increased incidence of alopecia occurred at the 1000 mg/kg bw/d for which a treatment related effect cannot be excluded.  None of the females died due to treatment.  The feed intakes and correspondingly the amounts of faeces were transiently decreased at dose levels of 300 and 1000 mg/kg bw/d. Decreased water consumption and decreased, concentrated and discolored urination occurred in rabbits at 1000 mg/kg bw/d.  Body weight gain was transiently decreased after start of treatment at levels of 300 and 1000 mg/kg bw/d. Overall body weight gain during the treatment and gestation period as well as corrected body weight gain were also decreased at 1000 mg/kg bw/d.  Necropsy of females on gestation day 29 did not reveal treatment related findings at dose levels up to and including 1000 mg/kg bw/d. With respect to intrauterine development the gestation rate was marginally decreased by one abortion at 1000 mg/kg bw/d for which a treatment-related effect as consequence of maternal toxicity cannot be excluded.  The remaining parameters of intrauterine development (external appearance and weight of placentas, the postimplantation loss in the females with viable fetuses on gestation day and correspondingly the number of fetuses, fetal sex distribution  and fetal weight, fetal morphology including stage of ossification) were not affected by treatment at levels up to and including 1000 mg/kg bw/d. A maternal NOAEL of 100 mg/kg bw/d was determined for this study based on abortion and clinical signs (at 1000 mg/kg bw/d), reduced body weight gain and food consumption (at 300 mg/kg bw/d).  A developmental NOAEL of 1000 mg/kg bw/d can be determined in the absence of any effects of treatment.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 December 1997 to 28 March 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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: 88/302/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF Guidelines ‘Teratogenicity Study’

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann, Germany
- Age at study initiation: 13-16 weeks
- Weight at study initiation: 185-241 g
- Fasting period before study: None
- Housing: individual
- Diet: ad libitum
- Water: ad libitum:
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): ~50%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: 13 January 1998 To: 17 November 1998

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% (aqueous)

Details on exposure:

Test material was suspended in 0.5% aqueous carboxymethylcellulose, from day 6 to day 19 post coitum at doses of 0, 100, 300 and 1000 mg/kg bw per day.
The doses were based on the results of a pilot developmental toxicity study in rats in which doses of 0, 100, 300 and 1000 mg/kg bw were tested.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Investigations on the stability and homogeneity in samples of 10 mg/mL and 200 mg/mL performed before the first substance administration and covering the concentrations used in this study revealed no significant deviations from the content determined on the day of preparation after 3-day storage (10 mg/mL) to 5-day storage (20 - 200 mg/mL). A content check of the formulations of all concentrations was carried out during the in-life period of the study in Weeks 3 and 5 after initiation of treatment of the first animals. The results revealed no significant deviation of the test compound content from the nominal value in the formulations of any of the treatment groups.

Duration of treatment / exposure:

20 days.

Frequency of treatment:

The rats were treated daily.

Duration of test:

Rats were dosed on GD6-19, and were terminated on GD20

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

28 inseminated female Wistar rats

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 28 inseminated female Wistar rats each were treated daily orally by gavage with Spirodiclofen, suspended in 0.5% aqueous carboxymethylcellulose, from day 6 today 19 post coitum at doses of 0, 100, 300 and 1000 mg/kg bw per day. The fetuses were delivered by cesarean section on day 20 of gestation

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the animals were determined on GD0 and daily from GD 6 to 20. Corrected body weight gain was calculated by substracting the weight of the uterus on GD 20 from the body weight gain over the period from GD0 to 20.

FOOD CONSUMPTION: Yes
The feed consumption of the animals on gestation days 0-6, 6-9, 9-12, 12-15, 15-18 and 18-20 was determined based on the differences in weight of feed provided and feed which remained unconsumed.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was determined by estimation of the remaining quantities in the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

Ovaries and uterine content:

The parameters of intrauterine development, i.e. gestation rate, appearance and weight of placentas and number of fetuses were examined

Fetal examinations:

Cesarean sections were performed on gestation day 20. The females were killed using cardiotomy under deep carbon dioxide anesthesia. The following parameters
were determined and assessed at cesarean section:
- Number of Corpora lutea
- Number of implantations (in females without visible implantation sites after
staining of the uterus with a solution of 10 % ammonium sulfide
- Uterus weight
- Individual weight and appearance of the placentas
- Number of early resorptions (only implantation site visible), late resorptions (fetal or placental remnant visible) and dead fetuses (fetuses without signs of life, but without maceration)
- Number of live fetuses
- Sex of live fetuses
- Individual weights of live fetuses
- External malformations and other findings deviating from normal
- Visceral malformations and other findings deviating from normal (evaluation of about half of the fetuses by razor blade sectioning according to the modified Wilson technique (processing and evaluation after cesarean section)
- Findings in abdominal, pelvic and thoracic organs as well as skeletal and cartilage findings by the modified Dawson technique with the addition of cartilage staining: evisceration, cartilage
staining with alcian blue GX, clearing of the fetuses with diluted potassium hydroxide solution, staining of the skeletal system with alizarin red S and evaluation of the skeletal system including cartilaginous findings (processing and evaluation after cesarean section).

Statistics:

Females; without implantation sites and one female with an anomaly of the uterus were excluded from any statistical evaluation. Females; with total resorption were not taken into account for calculation of group mean values of body weights, body weight gains, organ weights and feed
consumption. Differences between the control and treated groups were considered significant when p <0.05. Statistical significance was tested using the following methods:

a.) ANOVA and Dunnett's test as posthoc test for:
- feed consumption
- body weights, body weight gains and corrected body weight gains
- uterine weights
- number of Corpora lutea per female
- number of implantations per female
- number of live fetuses per female and as a percentage of implantations per female
- placental weights per female
- fetal weights per female

b. CHI-square test (with Yates' correction) for:
- fertility rate
- gestation rate
- number of fetuses or litters with malformations, visceral deviations and fetal cartilaginous tissue observations

c.) 2 by N CHI-squared test; for significant differences Fisher's exact test with Bonferroni correction for:
- number of implantations per group
- number of pre-implantation losses per group
- number of post-implantation losses, early resorptions, late resorptions or dead fetuses per group
- number of live fetuses per group in percent of implantations
- number of male or female fetuses or fetuses with undeterminable sex per group
- number of fetuses or litters with skeletal findings

d.) Kruskal-Wallis test and in case of significant differences Dunn's test for:
- number of pre-implantation losses per female
- number of post-implantation losses, early resorptions, late resorptions or dead fetuses per female
- number of male or female fetuses or fetuses with undeterminable sex per female

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Maternal toxicity: Appearance, behavior, mortality as well as feed consumption, water intake, excreta (urine, feces) and body weight gain of the females were not affected by treatment at doses up to and including 1000 mg/kg bw/day. No substance-related findings were observed during necropsy for doses up to and including 1000 mg/kg bw/day.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Developmental toxicity: The parameters of intrauterine development, i.e. gestation rate, appearance and weight of placentas, number of fetuses, post-implantation loss, fetal sex distribution, fetal weight and fetal morphology (including stage of ossification), were not affected by treatment with up to and including 1000 mg/kg bw/day. The incidence of malformations was not affected by the treatment

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

There were no effects of treatment at any dose level in this study.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Developmental toxicity study in rats: Intrauterine development

Dose (mg/kg bw/day)	0	100	300	1000
Number of females
With implantations (a)	25	25	25	26
With viable fetuses (b)	24	25	25	26
Means per female
Placental weight in g (b)	0.59	0.60	0.60	0.59
Number of live fetuses (b)	11.5	11.6	10.9	11.5
Post implantation loss (a)	0.9	0.9	0.6	0.7
Post implantation loss (b)	0.8	0.9	0.6	0.7
% Males (b)	50.0	49.9	48.7	50.2
Fetal weight in g (b)	3.60	3.78	3.72

 

Developmental toxicity study in rats: Incidence of malformations

Malformation	Dose Dose [mg/kg bw/day]
	0	100	300	1000
Number of fetuses (litters) affected:
Macroglossia		1
Microphthalmia				1
Hydrocephalus Internus	1
Dysplasia of fore limb bones	2(2)		1
Combined vertebral and rib malformation	1
Supernumerary lumbar vertebra, shift of iliac bone		1
Fetuses per group (n)	277	291	272	300
Malformed fetuses (n)	4	2	1	1
„ (%)	1.4	0.7	0.4	0.3
Litters per group (n)	24	25	25	26
Litters with malformations (n)	3	2	1	1
'' (%)	12.5	8.0	4.0	4.0

Conclusions:

A teratogenic potential was not evident at doses up to and including the highest dose of 1000 mg/kg bw/d.  Maternal and developmental NOAELs of 1000 mg/kg bw/d were determined for this study, in the absence of any effects of treatment at the highest dose level.

Executive summary:

In this pre-natal developmental toxicity study, groups of 28 inseminated female Wistar rats were treated daily orally (by gavage) with spirodiclofen (suspended in 0.5% aqueous carboxymethylcellose) from GD 6-19 at dose levels of 0 (vehicle controls), 100, 300 and 1000 mg/kg bw/d.  The fetuses were delivered by Caesarean section on GD20.  Investigations were performed on general tolerance by the females as well as its effect on intrauterine development.  Appearance, behavior, mortality as well as feed consumption, water intake, excreta (urine, faeces) and body weight gain of the females were not affected by treatment at doses up to and including 1000 mg/kg bw/d. No treatment-related findings were observed during necropsy at doses of up to and including 1000 mg/kg bw/d.  The parameters of intrauterine development (gestation rate, appearance and weight of placentas, number of fetuses, post-implantation loss, fetal sex distribution, fetal weight and fetal morphology (including stage of ossification)) were not affected by treatment with spirodiclofen up to and including 1000 mg/kg bw/d.  A teratogenic potential was not evident at doses up to and including the highest dose of 1000 mg/kg bw/d.  Maternal and developmental NOAELs of 1000 mg/kg bw/d were determined for this study, in the absence of any effects of treatment at the highest dose level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

GLP- and guideline- (OECD 414) compliant studies of developmental toxicity in the rat and rabbit are available for spirodiclofen.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Spirodiclofen has a harmonised classification for reproductive toxicity (fertility) in Category 2.

Effects on fertility

RAC concluded that although there is only one adverse effect observed (aspermia/oligospermia), in combination with the effects on reproductive organs (more pronounced in dogs) there was sufficient evidence for classification for reproductive toxicity in Cat 2 (H361f: Suspected of damaging fertility).

Prenatal developmental toxicity

RAC concluded that it was not clear whether the effect of liver lobulation in the rabbit study was treatment-related, and therefore should not be considered for classification.  In the rat developmental toxicity study, no relevant effects were observed.  RAC therefore agreed that no classification was required for developmental toxicity.

Effects on or via lactation

RAC concluded that no classification is required regarding effects on or via lactation.  There is lack of data on the concentration of spirodiclofen in milk and therefore no conclusion can be drawn whether the effects observed during lactation are due to the transfer of spirodiclofen to the offspring via milk.