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

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2004

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 3

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.

Test material

Test material information

Constituent 1

Reference substance name:

3-(3,5-Dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutanoate

Cas Number:

148477-71-8

Molecular formula:

C21H24Cl2O4

IUPAC Name:

3-(3,5-Dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutanoate

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

70 ppm

Doses / concentrations 3

Dose / conc.:

350 ppm

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Effect levels (P0)

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

Target system / organ toxicity (P0)

1

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

2

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Details on results (P1)

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.

Effect levels (P1)

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

Target system / organ toxicity (P1)

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

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

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

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

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

Effect levels (F2)

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

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

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

Any other information on results incl. tables

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**

 

Applicant's summary and conclusion

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.