cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov. 2001 - Aug. 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by Landesanstalt für Pflanzenbau und Pflanzenschutz, Rheinland-Pfalz

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

EC Number:

266-719-9

EC Name:

cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Cas Number:

67564-91-4

Molecular formula:

C20H33NO

IUPAC Name:

(2R,6S)-4-[3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Test animals

Species:

rat

Strain:

Wistar

Remarks:

CrlGlxBrlHan:WI

Details on species / strain selection:

This strain was selected since extensive historical control data was available on Wistar rats and the rat is the preferred animal species for reproduction studies according to the different test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 34 (± 1) d; (F1) 22 - 29 d
- Weight at study initiation: (P) Males: 97.0 - 130.4 g; Females: 87.9 - 110.2 g
- Housing: individually during the study period
- Diet (ad libitum): ground Kliba maintenance diet mouse/rat meal
- Water (ad libitum): drinking water
- Acclimation period: about 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: (P) 06.11.2001 To: (P) 4./05./08.-10.04.2002, (F1) 10./11./17.07./09./12./13.08.2002

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): premating period - weekly (m+f); mating period - once (m+f); gestation period - weekly (m), once (f); lactation period - weekly (m), once (f); post weaning period - weekly (m), once (f)
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse/rat meal
- Storage temperature of food: ambient temperature

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. - not specified
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. single housing

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and concentration control analyses were performed from all dose levels at the beginning and toward the end of the premating periods, as well as during the periods after weaning. At least one analysis of the test substance preparations for the female animals had been carried out during the gestation and lactation periods.
A HPLC with external calibration was used.
Based on the low standard deviation the homogeneity analysis showed that the test substance was distributed homogeneously in the food.
The mean values of test substance in food were found to be in the range of 91.1 - 108.3 % of the nominal concentration. These results demonstrate the correctness of the concentrations of the test substance in the food.
The stability analyses demonstrated the stability of the test substance in food over a period of 42 d under the conditions chosen (storage at ambient temperature).

Duration of treatment / exposure:

- PGeneration: premating period (about 74 d) + gestation period + lactation period until weaning of F1 pups; 21 weeks in total
- F1-Generation: after weaning + premating period (about 74 d) + gestation period + lactation period until weaning of F2 pups; 21 weeks in total

Frequency of treatment:

continuously

Details on study schedule:

- Selection of parents from F1 generation after weaning of the pups
- Age at mating of the mated animals in the study: approx. 15-16 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

16 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

8 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

4 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: not specified
- Fasting period before blood sampling for clinical biochemistry: yes (for about 16 h)

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A check for mortality was made twice daily on working days or once daily (Saturdays, Sundays or on public holidays). All parental animals were checked daily for clinically evident signs of toxicity. The nesting, littering and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. The littering behavior of the dams was also inspected on weekdays (except public holidays) in the afternoons in addition to the evaluations in the mornings.
- Cage side observations: mortality, clinical observations, nesting, littering and lactation behavior of the dams

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the male and female parental animals was determined on the first day of test substance administration and then once a week at the same time of the day (in the morning) with the following exceptions for female animals:
During each mating period the P and the F1 generation parental females were
weighed on the day of positive evidence of sperm (day O p.c.) and on days 7, 14 and 20 post coitum.
Females showing no positive evidence of sperm in vaginal smears were not
weighed during the mating interval.
Females with litter were weighed on the day after parturition (day 1 p.p.) and on
days 4, 7, 14 and 21 post partum.
Females without litter were not weighed during the lactation phase.
After weaning of the last F1 or F2 pups the female P or F1 generation parental
animals were weighed again once weekly (parallel to the male P or F1 generation parental animals) until sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for examinations: In general, food consumption was determined once a week (each time for a period of 6 days). After the 10th test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for days 0-7, 7-14, 14-20 p.c. and during lactation period (animals with litter) weekly for days 1-4, 4-7, 7-14 p.p. Food consumption was not determined in females between days 14 and 21 after parturition and during the
mating periods, in the females without positive evidence of sperm during the
programmed gestation phase, or in the females without litters during the lactation phase.

WATER CONSUMPTION: No

OTHER: CLINICAL PATHOLOGY
- Blood was taken from all P and F1 parental animals shortly before sacrifice from the retroorbital venous plexus in the morning from fasted animals without anesthesia.
- The following parameters were determined:
• alanine aminotransferase
• aspartate aminotransferase
• alkaline phosphatase
• serum-y-glutamyltransferase
• glutamate dehydrogenase
• serum cholinesterase
• triglycerides

Oestrous cyclicity (parental animals):

Estrous cycle length and normality were evaluated daily for all P and F1 female parental rats for a minimum of 3 weeks prior to mating and were continued throughout the mating period until the female exhibited evidence of mating. At necropsy a vaginal smear was examined to determine the stage of the estrous cycle for each P and F1 female with scheduled sacrifice.

Sperm parameters (parental animals):

Parameters examined in P and F1 male parental generations:
testis weight, epididymis weight, sperm head count in testes, sperm head count in epididymides, sperm motility, sperm morphology

Sperm morphology and sperm head count (epididymis and testis) were evaluated
for the control and highest dose group, only.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Standardization of litters was not performed in litters with <=8 pups.

PARAMETERS EXAMINED
- The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain
- All female pups selected to become the F1 parental generation females (25/group) were evaluated daily for vaginal opening with examinations initiating on day 27 p.p. At the day of vaginal opening the body weights of the respective animals were additionally determined.
- All male pups selected to become the F1 parental generation males (25/group) were evaluated daily for preputial separation with examinations initiating on day 40 p.p. At the day of preputial separation the body weights of the respective animals were additionally determined.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals; after weaning of the pups
- Maternal animals: All surviving animals; after weaning of the pups

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

ORGAN WEIGHTS
- The following weight parameters of all P and F1 parental animals sacrificed at
scheduled dates were determined:
1. anesthetized parental animals
2. adrenal glands
3. brain
4. epididymides (cauda)
5. epididymides (total)
6. kidneys
7. liver
8. ovaries
9. pituitary gland
10. prostate gland
11. seminal vesicles with coagulating glands (and their fluids)
12. spleen
13. testes
14. thyroid glands (with parathyroid glands)
15. uterus (with oviducts and cervix uteri)

HISTOPATHOLOGY
- The following organs or tissues of all P and F1 parental animals sacrificed at scheduled dates were fixed in 4% formaldehyde solution (Fo) or in BOUIN's solution (B), respectively:
1. adrenal glands (Fo)
2. all gross lesions (Fo)
3. brain (Fo)
4. cervix uteri (Fo)
5. coagulating glands (Fo)
6. kidneys (Fo)
7. left epididymis (B)
8. left testis (B)
9. liver (Fo)
10. ovaries (B)
11. oviducts (Fo)
12. pituitary gland (Fo)
13. prostate gland (Fo)
14. seminal vesicles (Fo)
15. spleen (Fo)
16. thyroid glands, with parathyroid glands (Fo)
17. uterus (Fo)
18. vagina (Fo)
- microscopical examination was performed according to table 2 in "Any other information on materials and methods incl. tables"

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age. The F2 offspring were sacrificed at 4 (standardization) or 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

ORGAN WEIGTHS
- brain, spleen and thymus of 1 pup/sex and litter

SKELETAL AND HEAD EXAMINATION
- in pups with notable findings or abnormalities in the daily clinical observation, if it was deemed necessary

Statistics:

see table 1 in "Any other information on materials and methods incl. tables"

Reproductive indices:

For the males, mating and fertility indices were calculated for F1 and F2 litters according to the following formulas:

- Male mating index (%) = number of males with confirmed mating* / number of males placed with females x 100
*defined by a female with vaginal sperm or that gave birth to a litter or with pups/implantations in utero

- Male fertility index (%) = number of males proving their fertility* / number of males placed with females x 100
*defined by a female giving birth to a litter or with pups/implantations in utero

For the females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:

- Female mating index (%) = number of females mated* / number of females placed with males x 100
*defined as the number of females with vaginal sperm or that gave birth to a litter or with pups/implantations in utero

- Female fertility index (%) = number of females pregnant* / number of females mated** x 100
*defined as the number of females that gave birth to a litter or with pups/implantations in utero, **defined as the number of females with vaginal sperm or that gave birth to a litter or with pups/implantations in utero

- Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant* x 100
*defined as the number of females that gave birth to a litter or with pups/implantations in utero

- Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100

- Postimplantation loss (%) = (number of implantations - number of pups delivered) / number of implantations x 100

Offspring viability indices:

- Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth x 100
*before standardization of litters (i.e. before culling)

- Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth x 100
*after standardization of litters (i.e. after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

16 mg/kg bw/d:
- hematuria (one male)
- ataxia, a reduced nutritional state, piloerection and discolored feces, hypothermia and signs of anemia (one female)

8 mg/kg bw/d:
- malocclusion (one male)
-piloerection and a palpable mass in the region of the throat (one male); the histopathological examination of the palpable mass revealed an adenocarcinoma

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In premating week 9, one high dose female had to be sacrificed in a moribund state. This female showed ataxia, reduced nutritional state, piloerection and discolored feces from premating week 8 onwards and in addition hypothermia and signs of anemia shortly before it was sacrificed. Pathological examinations revealed a severe chronic progressive glomerulonephropathy (CPN) as the major finding being
responsible for its poor clinical condition. Although this animal belonged to the high dose group, its intercurrent death was not regarded to be treatment-related but as of spontaneous origin.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

16 mg/kg bw/d:
- reduced female body weights during premating (7% below controls in study week 10), gestation (6% below controls on gestation day 20) and lactation (8% below controls on lactation day 21), attaining statistical significance occasionally
- impaired female body weight gains during premating (12% below controls if calculated for study weeks 0 - 10), gestation (4 % below controls if calculated for gestation days 0 -20) and lactation (about 3% below controls if calculated for lactation days 1 - 21 ), attaining statistical significance occasionally

8 mg/kg bw/d:
- reduced female body weights during premating (6% below controls in study week 10), gestation (5% below controls on gestation day 20) and lactation (7% below controls on lactation day 21 ), attaining statistical significance occasionally
- impaired female body weight gains during premating (11 % below controls if calculated for study weeks 0 - 10 attaining statistical significance) and lactation (18% below controls if calculated for lactation days 1 - 21)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

16 mg/kg bw/d:
- slightly to moderately decreased food consumption in the females during premating (up to 9% below controls), gestation (up to 12% below controls) and lactation (up to 13% below controls), attaining statistical significance occasionally

8 mg/kg bw/d:
- slightly, but statistically significantly decreased food consumption in the females during gestation days 0 - 7 (7% below controls) and lactation days 1 - 4 (about 11 %
below controls if calculated for lactation days 1 - 14)

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):

16 mg/kg bw/d:
- statistically significantly decreased serum cholinesterase activities in both sexes (22% / 53% below controls in males / females) and glutamate dehydrogenase activity in both sexes (23% / 52% below controls in males / females)

8 mg/kg bw/d:
-statistically significantly decreased serum cholinesterase (23% / 47% below controls in males / females)

4 mg/kg bw/d:
- statistically significantly decreased serum cholinesterase activities in both sexes (19% / 44% below controls in males/ females)

2 mg/kg bw/d:
- statistically significantly decreased serum cholinesterase activities in both sexes (14% / 35% below controls in the males / females)

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The recorded gross lesions and microscopic findings were either single observations, or they occurred at a very low incidence, sometimes in control animals only, or they were recorded at comparable incidence and graded severity in control and high dose males and/or females, giving no indication of a relationship to treatment.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- After mating, 2 sperm positive females of control group, 2 of test group 01 (2 mg/kg body weight/day), 1 of test group 02 (4 mg/kg body weight/day), 2 of test group 03 (8 mg/kg body weight/day) and 1 of test group 04 (16 mg/kg body weight/day) did not deliver any F1 pups. Consequently, the fertility index varied between 92% (control and test groups 01 and 03) and 96% (test groups 02 and 04). All respective values were within the range of the historical control data. Moreover, there were no corroborative histopathological findings in the sexual organs of
those females which were not pregnant.
- 2 control males, 2 2 mg/kg group males, 1 4 mg/kg group male, 2 8 mg/kg group males and 1 16 mg/kg group male did not generate F1 pups. Thus, the male fertility index ranged between 92% and 96% without showing any relation to dosing. All respective values were within the range of the historical control data. For none of these male rats conclusive histopathological findings occurred which could account for the observed impaired fertility.
- The observable statistical significance concerning the duration of gestation in test group 03 (8 mg/kg body weight/day)[= 21.7 days instead of 22.0 days in the control] was considered as a by chance finding.
- The statistically significantly increased value in total postimplantation loss in test group 01 is considered to be accidental in nature and without any toxicological relevance.

Effect levels (P0)

1

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Remarks:

Fertility and reproductive performance

Effect level:

16 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to and including the highest tested dose

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

8 mg/kg bw/d:
- kinked tail (one male)
- reduced nutritional state, vaginal hemorrhage, red crust formation around its nose and piloerection (one female)

4 mg/kg bw/d:
- blood crusts around anogenital region, poor general state, piloerection and
diarrhea (one male)
- exophthalmos of right eye (one male)

2 mg/kg bw/d:
- lesion of right forelimb (one female)
- piloerection (one female)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

8 mg/kg bw/d:
- One female was sacrificed in a moribund state on gestation day 15, because it showed a reduced nutritional state, vaginal hemorrhage, red crust formation around its nose and piloerection. Severe chronic progressive glomerulonephropathy (CPN), (multi)focal calcification with foreign body reaction in the mucosa of the glandular stomach, detritus in the lumen of the uterus (as correlate to the grossly recorded dead fetuses in the left uterus horn) and marked amounts of pus in the lumen of the vagina (correlating to the grossly described bloody contents) were observed during the gross and/or histopathological examination.

4 mg/kg bw/d:
- One male had to be sacrificed moribund during premating week 0, because it was in a poor general state, had blood crusts around its anogenital region and showed furthermore piloerection and diarrhea. The gross and/or microscopic examination revealed a moderate dilation of cecum and jejunum, but no further findings, which could account for the moribund condition of this rat.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

16 mg/kg bw/d:
- reduced mean body weights in the males, attaining statistical significance from study week 9 until terminal sacrifice (7% below controls in week 16) and impaired body weight gains during the treatment period (8% below controls if calculated for weeks 0 - 16)
- marginally reduced female body weights during gestation and lactation (up to 7% below controls) and impaired body weight gains during gestation (38% below controls on gestation day 0 - 7, but only 2% below controls if calculated for gestation days 0 - 20)

8 mg/kg bw/d:
- marginally reduced mean body weights gains in the females during premating (6% below controls in study week 10), gestation (5% below controls on gestation day 20) and lactation (7% below controls on lactation day 21) with or without attaining statistical significance
- impaired female body weight gains during premating (9% below controls if calculated for weeks 0 - 10) and during gestation (30% below controls on days 0 - 7 p.c., but only 3% below controls if calculated for gestation days 0 - 20)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

16 mg/kg bw/d:
- statistically significantly decreased food consumption (up to 8% below controls) in the males during premating weeks 8 - 11 and in the females occasionally during some intervals of premating, gestation and lactation (up to 14% below controls)

8 mg/kg bw/d:
- occasionally statistically significantly decreased food consumption in the females
during parts of premating and gestation (up to 13% below controls)

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):

16 mg/kg bw/d:
- statistically significantly decreased serum cholinesterase activities in both sexes (23% / 59% below controls in males/ females) and glutamate dehydrogenase activity in the males (24% below controls)

8 mg/kg bw/d:
-statistically significantly decreased serum cholinesterase activities in both sexes (28% / 59% below controls in males / females)

4 mg/kg bw/d:
- statistically significantly decreased serum cholinesterase activities in both sexes (26% / 45% below controls in males/females)

2 mg/kg bw/d:
- statistically significantly decreased serum cholinesterase activities in both sexes ( 16% / 33% below controls in the males / females)

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- 1 high dose dam (16 mg/kg body weight/day) did not nurse their pups properly from lactation days 2-8. As a consequence, nearly all of her pups died and/or were cannibalized until day 9 p.p. (with the exception of 3 pups, which were sacrificed as scheduled).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

16 mg/kg bw/d:
- statistically significantly increased absolute and relative liver weights in both sexes (up to 18% above controls)

8 mg/kg bw/d:
- statistically significantly, up to 14% increased absolute (males) and up to 17%
increased relative liver weights (both sexes)

4 mg/kg bw/d:
- statistically significantly increased absolute and relative liver weights (up to 16%
above controls) in both sexes

2 mg/kg bw/d:
- statistically significantly increased absolute (males) and relative liver
weights in both sexes (up to 11 % above controls)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross lesions were recorded from adrenal cortex (enlarged), bone (fracture or kinky tail), cecum (dilation), epididymides (enlarged or organ size reduced), eyes (exophthalmia), glandular stomach (erosion/ulcer, hyperemia or thickening of wall, respectively), jejunum (dilation), kidneys (enlarged, pelvic dilation or retraction, respectively), liver (focal constriction), oviducts (cyst), renal lymph nodes (enlarged), seminal vesicles (organ size reduced), skin (lesion or sparse hair), testes (organ size reduced), uterus (dead fetus) and vagina (effusion).
With one exception (cyst in the oviducts of two females of the low and two other females of the high mid dose group), all these gross lesions occurred only once per group, with no obvious indication of a relationship to treatment.
Regardless of whether or not they had a microscopic correlate, all the gross
lesions - and their microscopic correlates - were considered to have developed
spontaneously and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The recorded gross lesions and microscopic findings were either single observations, or they occurred at a very low incidence, sometimes in control animals only, or they were recorded at comparable incidence and graded severity in control and high dose males and/or females, giving no indication of a relationship to treatment.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm motility of test group 12 (4 mg/kg body weight/day) and test group 14 (16 mg/kg body weight/day) were statistically significantly decreased in comparison to the concurrent control group. The marginal decreases were not considered as substance-induced effect, but as spurious findings based on the following facts:
- A clear relation to dosing was missing.
- The decreased values were predominantly induced by just one low mid dose and one high dose male each.
- All calculated mean values for sperm motility (92% / 90% / 82% ** / 89% and 85% * at 0, 2, 4, 8 and 16 mg/kg body weight/day) were fully within or even exceed the historical control range (81 - 89%).
- Histopathology of testes and epididymides of the F1 parental males showed only findings, which were regarded to have developed spontaneously and unrelated to treatment.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- 1 sperm positive female of test group 11 (2 mg/kg body weight/day) and 1 rat of test group 12 (4 mg/kg body weight/day) did not deliver any F2 pups. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values were within the range of the historical control data and do not show any relation to dosing. Gross and histopathological examinations of these females, which did not become pregnant failed to show a morphological correlate for the observed infertility.
- The following F1 male rats did not prove fertility: 1 control, 1 low dose male (2 mg/kg body weight/day) and 1 low mid dose male rat (4 mg/kg body weight/day). This reflects the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the
historical control data. Gross and histopathological examinations of these males, however, failed to show a relevant morphological correlate for the observed impaired fertility.
- In test groups 11 (2 mg/kg bw/d), 13 (8 mg/kg bw/d) and 14 (16 mg/kg bw/d), the mean duration of gestation was statistically significantly shorter than in the concurrent control group, but this was considered as a by chance finding without any toxicological relevance.

Effect levels (P1)

1

Dose descriptor:

LOAEL

Remarks:

General toxicity

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Remarks:

Fertility and reproductive performance

Effect level:

16 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to and including the highest tested dose

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- Some more pups were cannibalized in test group 02 (4 mg/kg body weight/day) in
comparison to the concurrent control group, but this was considered to be spontaneous in nature because of its scattered occurrence.
- The viability index reached 98% (control and test group 02 (4 mg/kg bw/d)), 99% (test groups 01 (2 mg/kg bw/d) and 04 (16 mg/kg bw/d) and 100% (test group 03 (8 mg/kg bw/d)), and thus showed no compound related changes.
- The lactation index was not affected by test substance administration in any of the groups. It was 99% / 100% / 98% / 99% / 99% in test groups 00-04 (0, 2, 4, 8, and 16 mg/kg body weight/day).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

16 mg/kg bw/d:
- statistically significantly lower mean pup body weights on day 7 p.p. (about 4% below [male+ female] controls on day 21 p.p.) and impaired body weight gains in these pups on days 4 - 7 p.p. (about 4% less weight gain than the controls, both sexes combined if calculated for days 4- 21 p.p.)

8 mg/kg bw/d:
-statistically significantly lower mean pup body weights on day 7 p.p. (about 7% below [male+ female] controls on day 21 p.p.) and impaired body weight gains in these pups on days 4 - 7 p.p. (about 7% less weight gain than the controls, both sexes combined if calculated for days 4 - 21 p.p.)

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:

no effects observed

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):

- The statistically significantly increased relative brain weights of the high mid dose male and female F1 pups (8 mg/kg body weight/day) might be a consequence of the slightly lower pup body weights/ body weight gains in this group. However, this weight increase did not occur in a dose dependent fashion. Furthermore, an increase in relative brain weight was not considered as an adverse effect. Finally, the statistically significantly increased relative brain weights of the high mid dose male and female F1 pups) were assessed as an incidental finding. This assumption is supported by the fact, that the mean relative brain weight of test group 02 (4 mg/kg body weight/day) was statistically significantly decreased. Moreover, the respective values are fully within historical control ranges.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- In the low (2 mg/kg body weight/day), the low mid (4 mg/kg body weight/day) and the high mid (8 mg/kg body weight/day) dose groups dilated renal pelvis occurred at statistically significantly rates. If expressed on an affected pup per litter basis the mean percentages amounted to 0.0 / 2.4* / 2.2* / 1.5* and 1.1 % at the 0, 2, 4, 8 and 16 mg/kg body weight/day dose levels. Because a dose-response relationship was lacking and the respective incidences were similar to the historical control range (0 - 2.3 %), this was considered as a by chance finding and not to be of substance-induced origin.
- A few of the examined F1 pups showed some further spontaneous findings at necropsy (e.g. post mortem autolysis, hemorrhagic thymus, diaphragmatic hernia and small testis), which were scattered throughout the test groups. These findings occurred without a clear relation to dosing and/or most of it can be found in the historical control data at comparable or even higher incidences.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

4 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

1 control F2 pup showed hydrocephaly and bilateral anophthalmia and 1 low dose pup had anophthalmia on its right eye. In another low dose F2 pup and in most of the low mid dose F2 pups from one dam a kinked tail was observed during their clinical examinations, but it consisted only in some pups of this dam until necropsy.
The scattered occurrence of these clinical findings in single pups of different test groups including the controls without a consistent pattern does not suggest any substance related origin.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- The viability index varied between 96% (control), 97% (16 mg/kg bw/d) and 98% (2 - 8 mg/kg bw/d), thus showing no compound related changes, but the usual biological variation inherent in the strain of rats used for this study.
- The lactation index was not affected by test substance administration in any of the test groups. It reached 99% / 100% / 98% / 99% and 98% at 0, 2, 4, 8 and 16 mg/kg body weight/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

16 mg/kg bw/d:
- lower mean body weights from day 4 p.p. up to weaning (about 7% below [male+
female] controls on day 21 p.p.) and impaired body weight gains in these pups until
weaning (about 6% less weight gain than the controls, both sexes combined, if
calculated for days 4 - 21 p.p.)

8 mg/kg bw/d:
- slightly lower mean body weights up to weaning (about 6% below [male+ female]
controls on day 21 p.p.) and impaired body weight gains in these pups until weaning
(about 6% less weight gain than the controls, both sexes combined, if calculated for
days 4 - 21 p.p.)

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):

The statistically significantly decreased absolute and/or increased relative brain weights of the high mid and/or high dose male and female F2 pups (8 or 16 mg/kg body weight/day) may be a consequence of the slightly lower pup body weights / body weight gains in these groups. However, as the respective values were fully within historical control ranges, these marginal weight changes were not considered by itself as adverse effects.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Findings, which occurred were e.g. post mortem autolysis, incisors sloped, anophthalmia, hydrocephaly, hemorrhagic or small thymus, empty stomach, dilated renal pelvis, hydronephrosis, small or malpositioned testis and kinked tail. These findings occurred without a clear relation to dosing and/or most of it can be found in the historical control data at comparable or even higher incidences.
- In the additionally performed head examinations according to WILSON's method
a low dose F2 pup showed hydrocephaly and anophthalmia on its right side.
- In the additional skeletal examinations according to a modified method of KIMMEL and TRAMMELL the following findings were confirmed: 1 low dose F2 pup and 3 low mid dose pups showed a kinked tail (indicated by misshapen or bipartite caudal vertebrae and caudal hemivertebrae).

Histopathological findings:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

.

Effect levels (F2)

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F2

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

8 mg/kg bw/day (nominal)

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