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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two-Generation Toxicity Study (OECD 416), rats:

NOAEL general toxicity (P1, females) = 20 ppm (equivalent to 2.2 mg/kg bw/day in P1 females)

NOAEL general toxicity (P0, males and females; P1 males) = 300 ppm (equivalent to 21.4 and 28.1 mg/kg bw/day in P0 males and females, respectively and 25.0 mg/kg bw/day in P1 males)

 

NOAEL reproductive toxicity (P0 and P1) >= 1800 ppm (highest dose tested) (equivalent to 138.9 and 204 mg/kg bw/day in P0 males and females, respectively and to 202 and 260 mg/kg bw/day in P1 males and females, respectively)

 

NOAEL offspring/developmental toxicity (F1 and F2) = 300 ppm (equivalent to 21.4 and 28.1 mg/kg bw/day in P0 males and females, respectively and 25.0 mg/kg bw/day in P1 males; correlated to maternal toxicity)

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Jan - 26 Nov, 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted 26 May 1983
Deviations:
yes
Remarks:
sperm parameters not evaluated
Qualifier:
according to guideline
Guideline:
other: U.S. EPA (Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Humans and Domestic Animals, Series 83-4: "Reproductive and Fertility Effects"
Version / remarks:
November 1984
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
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
ICO:WU (IOPS Cpb)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wistar rats ICO:WU (IOPS Cpb), IFFA, Credo/France, Belgium
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5 - 6 weeks; (F1) 4 weeks
- Weight at study initiation: (P) Males: 169 - 217 g; Females: 112 - 158 g
- Housing: individually in Type IIa Makrolon® cages during acclimatization period, females were housed individually in Typ III cages during the mating period; males were placed in the cages of the females overnight, Bedding: low-dust soft-wood shavings (Ssniff GmbH, Soest)
- Diet: Altromin® 1321 meal (Altromin GmbH Lage), ad libitum
- Water: tap water, ad libitum, analysis was performed
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was blended (using a mixing granulator, Loedige, Paderborn) with Altromin® 1321 feed containing 1% peanut oil (DAB 10) to minimize dust formation (including feed of the control group). The amount of test substance was calculated on the basis of an assumed 100% content of the test substance.

DIET PREPARATION
- Rate of preparation of diet: once weekly
- Mixing appropriate amounts with: Altromin® 1321 containing 1% peanut oil
- Storage temperature of food: at room temperature

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 3 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes (F0 and F1 females found sperm-positive on the first day of pairing but get not pregnant were co-housed over one week with the same male. Females not found sperm positive during the regular mating period were co-housed three times with the same male.)
- After successful mating each pregnant female was caged: Inseminated females were not further co-housed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical investigations on homogeneity and stability of the test compound in diet preparations were done prior to commencement of the study using samples from test mixtures. The test substance concentration within the feed was checked at regular intervals throughout the study (start of study, randomly each 3 month period, end of study). This was done by analyzing samples of the food mixes used. Per dose one sample of the food mixes was taken on the day the food was prepared, and another was taken after being kept under animal room conditions for the feeding period. All samples taken directly after diet preparation or at the end of the feeding period(s) were kept deep frozen (at temperatures of approximately -20°C) until examination. Reserve samples from each mixture were stored at least for 8 weeks at about -20°C. The analytical investigations proved that the test substance concentrations were in the specified concentrations ranges.
Duration of treatment / exposure:
P0 males and females: 12 weeks before mating
P0 males and females: up to 21 days during mating period
P0 females: about 22 days during pregnancy
P0 females: 28 days during lactation of F1 male and female litters, until weaning of F1 litters

P1 males and females: 10 weeks before mating (treatment up to an age of at least 14 weeks)
P1 males and females: up to 21 days during mating period
P1 females: about 22 days during pregnancy
P1 females: 28 days during lactation of F2 male and female litters up to an age of 4 weeks
Frequency of treatment:
once weekly up to necropsy (in the diet, ad libitum)
Details on study schedule:
- P1 parental animals not mated until an age of 14 weeks
- Age at mating of the mated animals in the study: at least 14 weeks
Dose / conc.:
20 ppm
Remarks:
corresponding to a mean test substance intake of (P0):
1.4 mg/kg bw/day (males)
1.8 mg/kg bw/day (females)

corresponding to a mean test substance intake of (P1):
1.6 mg/kg bw/day (males)
2.2 mg/kg bw/day (females)
Dose / conc.:
300 ppm
Remarks:
corresponding to a mean test substance intake of (P0):
21.4 mg/kg bw/day (males)
28.1 mg/kg bw/day (females)

corresponding to a mean test substance intake of (P1):
25.0 mg/kg bw/day (males)
32.1 mg/kg bw/day (females)
Dose / conc.:
1 800 ppm
Remarks:
corresponding to a mean test substance intake of (P0):
138.9 mg/kg bw/day (males)
204 mg/kg bw/day (females)

corresponding to a mean test substance intake of (P1):
201.8 mg/kg bw/day (males)
259.2 mg/kg bw/day (females)
No. of animals per sex per dose:
30 P0 males and females
30 P1 males and females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dosages were selected on the basis of the results of a one-generation study (T1060122) in Wistar rats (strain ICO:WU (IOPS Cpb) IFFA Credo /France, Belgium) in which the test substance was administered to groups of 10 males and 10 females at concentrations of 0, 50, 400, and 3200 ppm in the diet using a four week premating period (Bayer (b), 1997). In this study dietary concentrations of up to 400 ppm of the test substance were tolerated without adverse effects. The body weights and mortality of the parent animals were not affected up to 3200 ppm. In the group of 3200 ppm F0 rats consumed more food than controls and some lactating dams were found to be aggressive when placed into a new cage. Additionally, enhanced liver weights and a toxicologically relevant inhibition of the cholinesterase in the brain (F0 females) were seen in the 3200 ppm group. No adverse effects on pups were seen up to 400 ppm. At 3200 ppm body weights and survival of pups were reduced.

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and public holidays). In P0 and P1 parental animals a detailed evaluation of the general condition was made and recorded at the weekly during the premating periods, during gestation periods females were examined on day 0, 7, 14, 20, and during lactation on day 0, 4, 7, 11, 14, 21, and 28.
- Cage side observations checked in table 2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (once daily on weekends and public holidays). In P0 and P1 parental animals a detailed evaluation of the general condition was made and recorded weekly during the premating periods. During gestation periods females were examined on day 0, 7, 14, 20, and during lactation on day 0, 4, 7, 11, 14, 21, and 28. Laboratory tests on blood samples were performed in week 9 (P0 and P1) on 10 randomly selected animals per sex and group. Cholinesterase in plasma and erythrocytes were taken in the morning from the retro-orbital venous plexus of non-fasted animals anaesthetized with ether. During terminal necropsies brain samples (left half) were taken and deep frozen to be used for determination of cholinesterase activity. Since collecting of brains must be done over several weeks individual values were entered online under the fictitious week 20 (P0) or week 21 and 23 (P1).

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study, the male animals were weighed at weekly intervals up to week 21, and the female animals until week 11 (= end of premating period). After insemination had been established, the female animals were weighed on postcoital days 0, 7,14 and 20; and on days 0, 4, 7, 11, 14, 21 and 28 after birth of their pups. P0 and P1 animals were weighed on the date of necropsy to permit calculation of the relative organ weights.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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

WATER CONSUMPTION AND COMPOUND INTAKE: No

Oestrous cyclicity (parental animals):
Vaginal smears were taken daily during the two last weeks of premating period. Fifteen randomly selected P0 and P1 females per dose level were used. The vaginal smears were examined microscopically whether large serrated cells indicating estrus had occurred. Vaginal smears were evaluated to characterize the estrus cycle length and to determine if females were cycling properly.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- yes, maximum of 8 pups/litter (4 sex/litter as nearly as possible); excess pups were raised to an age of four weeks and then necropsied

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: number and sex of pups (including those of dead pups), individual body weights, clinical signs, malformations

GROSS EXAMINATION OF DEAD PUPS: yes, unless autolysis or cannibalism rendered examination impossible, pups that were found dead at birth, that died during the course of lactation as well as those killed in moribund condition were macroscopically inspected after opening the body cavities, with particular attention being paid to the organs of reproduction. A lung flotation in tap water was performed during the necropsy of pups found dead on the day of the first litter inspection. This was done to determine whether pups had breathed at birth or not.


Postmortem examinations (parental animals):
SACRIFICE
- Male animals: P0 males were killed after mating period.
- Maternal animals: All surviving animals, after weaning (28 days) of litters, animals that died or were killed in moribund condition during the study were necropsied and macroscopically examined.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The following organs of of the P0/P1 generation were fixed for analysis: adrenals (only P1), liver, pituitary gland, urinary bladder (only P1), vagina, uterus, ovaries, oviducts, mammary gland with skin, coagulation glands, seminal vesicles, prostate, tattooed ears, testes, epididymides and all organs/organ specimen exhibiting macroscopic changes.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues and organs were weighed: brain, liver, testes and ovaries were weighed in P0 animals.
The following organs of the control and high dose groups of both, the P0- and P1- generation were examined microscopically: coagulation glands, epididymides, mammary gland area, ovaries, oviducts, pituitary gland, prostate gland, seminal vesicles, testes, uterus including cervix uteri and vagina. Ovaries, oviducts, uteri and vagina were investigated additionally in low- and mid-dose P0 females. The adrenal glands and urinary bladder were examined in all groups of the P1 generation. The liver and gross lesions were examined from all parent animals of all groups. Organ weight determinations of the brain, liver, testes and ovaries were done during the scheduled necropsy of P0 and P1 rats.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 days of age, pups selected for litter reduction as well as pups delivered after the additional mating procedures were killed on postpartum day 4.
- Unscheduled necopsies: unless autolysis or cannibalism rendered examination impossible, pups that were found dead at birth, that died during the course of lactation as well as those killed (with carbon dioxide) in moribund condition were macroscopically inspected after opening the body cavities, with particular attention being paid to the organs of reproduction.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic and abdominal viscera and organs with macroscipically visible changes.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following tissues of the first F1 and F2 males and females per litter (day 28 p.p.) were fixed for analyses: liver and organs/organ specimen of all pups with macroscipically changes. The body weights and the weights of brain, liver, spleen and thymus were determined at scheduled necropsy.
The following organs of the control and high dose groups of both, the F0- and F1- generation were examined microscopically: coagulation glands, epididymides, mammary gland area, ovaries, oviducts, pituitary gland, prostate gland, seminal vesicles, testes, uterus including cervix uteri and vagina. Ovaries, oviducts, uteri and vagina were investigated additionally in low- and mid-dose F0 females. The adrenal glands and urinary bladder were examined in all groups of the F1 generation.
Statistics:
a. The Dunnett-Test in connection with a variance analysis was used for body weights of parent animals, organ weights of parent animals
b. A Kruskal-Wallis-Test with a Steel-Test was done on food consumption data
c. The U-Test was used for the evaluation of the pup weights, litter sizes, litter weights. The mean pup weight of each individual litter was used as a basis for calculation of the pup weight means of the dose groups. The litter size calculation was based on the number of female animals with live pups.
d. Fisher's exact probability test (two-tailed) at significance levels of a = 5 % and a 1 % was used for the evaluation of the insemination index (1), fertility index (1), gestation index (1), live birth index (1), viability index (1), lactation index (1)
e. The adjusted Welsh-Test was used for CHE-Data evaluation

(1) done if dose-related changes had occurred
Reproductive indices:
Insemination index (%) = No. of sperm positive females*/No. of females co-housed with a male* x 100
Fertility index (%) = No. of pregnant females/No. of sperm positive females** x 100
Gestation index (%) = No. of females with live pups/No. of pregnant females x 100

* excluding additional matings (those cases where mating was repeated with
another male)
** including pregnant females that were not sperm positive
*** moribund pups died during the course of culling were not included
# calculations were done by using number of pups per group
Offspring viability indices:
Live birth index (%)# = No. of live pups at birth/total No. of pups born x 100
Viability index (%)# = No. of live pups on day 4 pre-culling/No. of live pups born x 100
Lactation index (%)# = No. of live pups after three/four weeks/No. of live pups after four days (after culling)*** x100

# calculations were done by using number of pups per group
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No effects were observed on appearance, general conditions and behaviour of test animals up to the highest dose applied.
yes control and 300 ppm: loss of hair in one female (non adverse)
yes control: diarrhea in one female (non adverse)
Please refer to table 2 under "Any other information on results incl. tables".
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
yes
control: 1/30 female died on day 24 due to delivery problems (dam could not deliver their pups) (non-adverse)
20 ppm: 1/30 pregnant female had been found dead on day 11 of pregnancy with unknown cause of death (non-adverse)
300 ppm: 1/30 female died on day 28 of pregnancy due to delivery problems (dam could not deliver their pups) (non-adverse)
1800 ppm: no mortality occured
Please refer to table 2 under "Any other information on results incl. tables".
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects observed. The body weights of the male and female F0 animals receiving up to 1800 ppm did not differ significantly from those of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
yes 300 and 1800 ppm: females of the 300 and 1800 ppm groups consumed 12.1 to 32.7% more food per animal during the premating period than controls, respectively. Weekly group means were sgnificantly higher at several (300 ppm) or most (1800 ppm) time points during the premating period, as well as sporadically at 1800 ppm during pregnancy and lactation. The food intake (per animal and per body weight) of all treated males and of females receiving 20 ppm test substance was comparable with control data. Deviations to control data occurring in these groups are below 10%.
yes 1800 ppm: due to the increase in food consumption at 1800 ppm in females these rats had a somewhat higher test substance intake than that expected from the dosing factor.
The test substance intake in treated male and female groups up to 300 ppm roughly corresponded to the theoretical dose intervals. Please refer to table 2 under "Any other information on results incl. tables".
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):
yes 1800 ppm: toxicologically relevant cholinesterase inhibition was found in erythrocytes (30.9% in males and 40.3% in females) and the brain (39.1% in females only). Up to the dietary concentration of 300 ppm no biologically significant inhibition of cholinesterases was detected (adverse). Please refer to table 2 under "Any other information on results incl. tables".
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):
yes 1800 ppm: increase in the number of rats exhibiting cytoplasmatic changes in hepatocytes. Additionally, centrilobular hypertrophy was found in 17/30 females. This effect is interpreted as a rather adaptive response due to substance intake and metabolism than as adverse effect (non-adverse). There were no treatment-related alterations of the liver morphology at a dose of 300 ppm. All other morphological findings are considered to be spontaneous in nature and within the normal range of background pathology commonly seen in rats of this strain and age. Please refer to table 2 under "Any other information on results incl. tables".
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean of estrus cycle length and number of estri observed was comparable among the groups. Irregular estrus cycle and prolonged estrus was obersevd in individual females of all treatment groups without dose dependency and within the expected range of untreated animals. Thus this effect is considered as incidental and not treatment-related. Please refer to table 2 under "Any other information on results incl. tables".
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The insemination, fertility, gestation and mean duration of pregnancy did not differ to a toxicologically relevant extent from the pertinent control data at levels of up to 1800 ppm (non adverse). Please refer to table 2 under "Any other information on results incl. tables".
Reproductive performance additional matings:
There were some F0 females (3-2-2-4 in ascending concentration) which got not pregnant with their first male. To investigate the reason of the failed pregnancy additional matings were performed with males already shown to be fertile (with another female). None of the three control and both 20 ppm females co-housed with a fertile male got pregnant. 1/2 300 ppm females delivered pups. In the 1800 ppm group 3/4 remated females dropped a litter and the remaining 1800 ppm female exhibited implantation sites in the uterus. In summary, results of regular and additional matings do not indicate any adverse effect on male or female fertility. The mating performance was not affected by the treatment at levels of up to 1800 ppm.
Dose descriptor:
NOEL
Remarks:
general toxicity
Effect level:
20 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed at this dose level
Remarks on result:
other:
Remarks:
20 ppm corresponding to 1.8 mg/kg bw/day in females and 1.4 mg/kg bw/day in males
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose level
Remarks on result:
other:
Remarks:
300 ppm corresponding to 28.1 mg/kg bw/day in females and 21.4 mg/kg bw/day in males
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
1 800 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: 1800 ppm corresponding to 138.9 mg/kg bw/day in males and 204 mg/kg bw/day in females
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
1 800 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Remarks on result:
other: 1800 ppm corresponding to 138.9 mg/kg bw/day in males and 204 mg/kg bw/day in females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 800 ppm
System:
nervous system
Organ:
other: cholinesterase activity inhibited in erythrocytes and brain (Statistically significant inhibition by 20% or more in erythrocytes or brain is considered a clear adverse toxicological effect)
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No effects were observed on appearance and behaviour.
yes control, 20 and 300 ppm: loss of hair was observed in males and females of the control group, in one male of the 20 ppm group and in males and females of the 300 ppm group (non-adverse)
yes 20 ppm: one male with a poor general condition was observed (non-adverse)
yes 20 and 1800 ppm: at each dose level one animal showed agression (non-adverse)
For details, please refer to table 2 under "Any other information on results incl. tables".
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
no data
Mortality:
mortality observed, non-treatment-related
Description (incidence):
None of the F1 males died unscheduled. In the 20 ppm (pregnant rat showed poor general condition) and 1800 ppm groups (rat showed aggressive behavior) each one female were killed prescheduled. No evidence of a treatment-related increase in mortality was observed (non-adverse). Please refer to table 2 under "Any other information on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
yes 1800 ppm: statistically significant body weight depression in both male and female weanlings with a maximum (26% and 15%) in week two. Additionally, pregnant and lactating females of this group exhibited reduced body weights. Please refer to table 2 under "Any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
yes 1800 ppm: 12.0% (males) or 35.4% (females) higher food intake per rat was observed. Calculated per kg bw a 43.9% and 51.9% higher consumption was determined in males and females, respectively. Due to the increase in food consumption and lower body weights at 1800 ppm, males and females of this group had a somewhat higher test substance intake than that expected from the dosing factor. Please refer to table 2 under "Any other information on results incl. tables".
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):
yes 1800 ppm: significant cholinesterase inhibition was found in erythrocytes (males 48.3% and females: 72.8%) and the brain (41.7% females) in P1 rats (adverse). Please refer to table 2 under "Any other information on results incl. tables".
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
Description (incidence and severity):
yes 1800 ppm: absolute (12.7% in females) and relative (males: 6.6 and females 13.3%) liver weights were higher than in controls. This effect is interpreted as a rather adaptive response due to substance intake and metabolism than as adverse effect (non-adverse). Relative testes weight was also increased by about 12% in this group, since histopathological investigations revealed no morphological correlates and insemination index and fertility indices were not altered this finding is considered to have no toxicological relevance (non-adverse). All doses: ovary weights were sightly reduced in treated females without dose-response, reaching statistical significance at 20 and 300 ppm (non-adverse due to missing dose-response). Statistically significant lower absolute brain weight was observed. Statistically significant lower absolute brain weights were probably due to differences in body weight, which can differ more or less at the necropsy date due to weaning (non-treatment-related, non-adverse). Please refer to table 2 under "Any other information on results incl. tables".
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant gross pathological findings were observed at necropsy of male or female F1 animals at levels of up to 1800 ppm. Please refer to table 2 under "Any other information on results incl. tables".
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
yes 300 and 1800 ppm: cytoplasmatic change (both sexes) and hypertrophy (females only) in the centrilobular hepatocytes were seen more often than at 0 or 20 ppm This effect is interpreted as a rather adaptive response due to substance intake and metabolism than as adverse effect (non-adverse). Additionally, a fine and even cytoplasmatic vacuolation of cortical cells in the zona fasciculata and glomerulosa, however, without signs of degenerative processes was found in the adrenal glands with increasing incidence in females receiving 300 or 1800 ppm (adverse).
yes 1800 ppm: in males the incidence of a simple urothelial hyperplasia in the urinary bladder was higher than in the other groups (adverse). All other findings recorded in the remaining organs are considered to be spontaneous in nature and within the normal range of the background pathology commonly seen in rats of this strain and age. Please refer to table 2 under "Any other information on results incl. tables".
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrus cycle staging in F1 females showed no abnormalities up to 1800 ppm. Please refer to table 2 under "Any other information on results incl. tables".
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The reproduction parameters such as mating performance, insemination, fertility, gestation as well as the mean duration of pregnancy showed no treatment-related effect at levels of up to 1800 ppm. There were two F1 females (1-1-0-0 with ascending dose) which had been found to be sperm-positive after the day of co-housing but failed pregnancy. According to experience this could have happened if a male co-housed with a female for the first time had inseminated the female outside the estrus. This assumption was supported by the fact that all these females had pups when remated with the same male for one week that followed the three week co-housing period. Please refer to table 2 under "Any other information on results incl. tables".
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
20 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed at this dose level
Remarks on result:
other: 20 ppm corresponding to 2.2 mg/kg bw/day in females and 1.6 mg/kg bw/day in males
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed at this dose level
Remarks on result:
other: 300 ppm corresponding to 32.1 mg/kg bw/day in females and 25 mg/kg bw/day in males
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: 300 ppm corresponding to 32.1 mg/kg bw/day in females and 25 mg/kg bw/day in males
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
1 800 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: 1800 ppm corresponding to 259.2 mg/kg bw/day in females and 201.8 mg/kg bw/day in males
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
1 800 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on reproductive function/performance observed up to this dose level
Remarks on result:
other: 1800 ppm corresponding to 259.2 mg/kg bw/day in females and 201.8 mg/kg bw/day in males
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 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.:
1 800 ppm
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 800 ppm
System:
nervous system
Organ:
other: cholinesterase activity inhibited in erythrocytes and brain (Statistically significant inhibition by 20% or in erythrocytes or brain more is considered a clear adverse toxicological effect)
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No significant clinical findings were made in F1 pups during the four-week lactation period at levels of up to 1800 ppm. Please refer to table 2 under "Any other information on results incl. tables".
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
no data
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
PND 0: yes 1800 ppm: slightly more dead pups were found at birth resulting in a slightly reduced live birth index.
PND 4: mean viability indices were comparable among the groups up to and including 300 ppm. In the high-dose group, slightly fewer pups survived up to PND 4 (increased loss of pups as a result of cannibalism) resulting in a significantly reduced viability index of 82.1%. Please refer to table 2 under "Any other information on results incl. tables".
The total number of pups, litter size, and sex ratio did not differ to a toxicologically relevant extent from the pertinent control data at levels of up to 1800 ppm. Please refer to table 2 under "Any other information on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Birth weights were comparable among the test groups up to 1800 ppm; mean litter weight was significantly reduced on PND28 at 1800 ppm.
PND4 - PND28 : 1800 ppm: reduced pup weight in males (significant)
PND21 - PND28: 1800 ppm reduced pup weight in females (significant)
At the lower doses, sporadically, non-dose dependent altered pup weights were determined in males and females. Please refer to table 2 under "Any other information on results incl. tables".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Please refer to P1 (second 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
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
yes 1800 ppm: liver weights were inconspicuous up to 300 ppm, but in relation to body weights they were increased (16-17%) at 1800 ppm in both sexes. This effect is interpreted as a rather adaptive response due to substance intake and metabolism than as adverse effect (non- adverse). Brain, spleen, and thymus weights of treated pups were not changed in a toxicologically relevant manner. Deviations occurring in some cases are minor and are considered to be a result of differences in body weights. Please refer to table 2 under "Any other information on results incl. tables".
Gross pathological findings:
no effects observed
Description (incidence and severity):
In F1 pups necropsied during lactation period no macroscopical alterations due to the treatment were observed. No malformations were determined in the F1 pups, which died before PND 4, were killed in the process of litter reduction on PND 4, or were necropsied unscheduled during lactation at levels of up to 1800 ppm. No gross pathological findings were made in F1 weanlings at scheduled necropsy.
Histopathological findings:
not examined
Description (incidence and severity):
Please refer to P1 (second parental generation)
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no advers effects observed at this dose level
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
1 800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
Remarks on result:
other: correlated to maternal toxicity determined via cholinesterase inhibition
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 1800 ppm. Please refer to table 2 under "Any other information on results incl. tables".
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
yes 300 and 1800 ppm: the lactation indices were significantly reduced. The reduction observed at 300 ppm is not considered as treatment-related, since the indices were within the range of the control groups. The significantly reduced values at 1800 ppm are considered as treatment-related (adverse). Please refer to table 2 under "Any other information on results incl. tables".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
yes 1800 ppm: reduced litter weights were visible at birth and weaning; individual birth weights were not affected up to 1800 ppm; mean body weights of male and female pups was statistically significantly depressed starting on PND 7; 300 ppm: increased mean body weight starting on PND 14 (adverse). Please refer to table 2 under "Any other information on results incl. tables".
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
yes 1800 ppm: 12% elevated liver weight means (relative) were noted in female pups. This effect is interpreted as a rather adaptive response due to substance intake and metabolism than as adverse effect (non- adverse). Reduced spleen weights (absolute about 18% and relative about 12%) were calculated in pups of both sexes. These effects are interpreted as related to reduced body weight gain and are therefore not considered as adverse. However, regarding the mean weights of the brain and thymus no conspicuous differences could be detected between the controls and treatment groups. Please refer to table 2 under "Any other information on results incl. tables".
Gross pathological findings:
no effects observed
Description (incidence and severity):
In F2 pups necropsied during the lactation period no macroscopical alterations due to the treatment were observed. No malformations were determined in the F2 pups that had died before PND 4, were killed in the process of culling, or were necropsied unscheduled during lactation at levels of up to 1800 ppm. No gross pathological findings were made in F2 weanlings at scheduled necropsy.
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Litter observations at birth:
Live birth indices and viability indices are comparable among the groups. No significant alteration in sex distribution was determined. At 1800 ppm, fewer pups were born resulting in a significantly reduced mean litter size.
Behaviour (functional findings):
not examined
Description (incidence and severity):
no data
Developmental immunotoxicity:
not examined
Description (incidence and severity):
no data
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to this dose level
Key result
Dose descriptor:
LOAEL
Generation:
F2
Effect level:
1 800 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: reduced mean litter size; reduced lactation index
Remarks on result:
other: correlated to maternal toxicity determined via cholinesterase inhibition
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 800 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

   Table 1: Table for animal assignment for mating

 

Number of animals

Controls

20 ppm

300 ppm

1800 ppm

P0

1.4 mg/kg bw/day (males)

1.8 mg/kg bw/day (females)

 

21.4 mg/kg bw/day (males)

28.1 mg/kg bw/day (females)

 

138.9 mg/kg bw/day (males)

204.0 mg/kg bw/day (females)

 

P1

1.6 mg/kg bw/day (males)

2.2 mg/kg bw/day (females)

 

25 mg/kg bw/day (males)

32.1 mg/kg bw/day (females)

 

201.8 mg/kg bw/day (males)

259.2 mg/kg bw/day (females)

 

P0

m

30

30

30

30

f

30

30

30

30

P1

m

30

30

30

30

f

30

30

30

30

Table 2: Table for reproductive toxicity study

Parameter

Genera­tion

Controls

20 ppm

300 ppm

1800 ppm

 

♂

♀

♂

♀

♂

♀

♂

♀

 

Clinical Observations1)Incidence

 

appearance

P0

0/30

0/30

0/30

0/30

0/30

0/30

0/30

0/30

 

P1

0/30

0/30

0/30

0/30

0/30

0/30

0/30

0/30

 

behaviour

P0

0/30

0/30

0/30

0/30

0/30

0/30

0/30

0/30

 

P1

0/30

0/30

0/30

0/30

0/30

0/30

0/30

0/30

 

mortality

P0

0/30

1/30

0/30

1/30

0/30

1/30

0/30

0/30

 

P1

0/30

0/30

0/30

1/30

0/30

0/30

0/30

1/30

 

loss of hair

P0

/

1/30

/

0/30

/

1/30

/

0/30

 

P1

2/30

1/30

0/30

1/30

2/30

5/30

0/30

0/30

 

diarrhoea

P0

/

1/30

/

0/30

/

0/30

/

1/30

 

P1

0/30

0/30

0/30

0/30

0/30

0/30

0/30

1/30

 

poor general condition

P0

/

0/30

/

1/30

/

1/30

/

0/30

 

P1

0/30

0/30

0/30

1/30

0/30

0/30

0/30

0/30

 

aggression

P0

/

0/30

/

0/30

/

0/30

/

0/30

 

P1

0/30

0/30

0/30

1/30

0/30

0/30

0/30

1/30

 

Mean daily food intake g/kg bw/day

(premating period)

P0

70.6

82.9

71.9

89.4

71.3

93.8

77.2

113.3

 

P1

77.9

94.8

81.9

112.5

83.3

107.0

112.1

144.0

 

Mean daily test substance intake mg/kg bw/day

(premating period)

P0

0

0

1.4

1.8

21.4

28.1

138.9

204.0

 

P1

0

0

1.6

2.2

25.0

32.1

201.8

259.2

 

Inhibition of Cholinesterases %

plasma (week 9)

P0

/

/

/

/

4.9

/

9.8

/

 

P1

/

/

/

/

/

/

/

/

 

Inhibition of Cholinesterases %

erythrocytes (week 9)

P0

/

/

/

/

/

/

30.9

40.3

 

P1

/

/

/

/

2.3

16.3

48.3

72.8

 

Inhibition of Cholinesterases %

brain (terminal necropsy)

P0

/

/

/

/

/

7.2

7.4

39.1

 

P1

/

/

1.6

1.5

/

8.7

10.4

41.7

 

Relative Organ weights

[mg/100 g bw]

brain

P0

425

707

435

707

430

701

434

681

 

P1

399

703

404

669

404

675

418

667

 

liver

P0

3558

3639

3471

3643

3469

3813

3804**

4425**

 

P1

3430

4143

3409

4164

3403

4394

3658**

4696**

 

testes/ovaries

P0

709

64

721

64

741

63

765*

62

 

P1

717

62

735

54*

744

52**

806**

58

 

Absolut Organ weights

[mg]

brain

P0

2050

1795

2058

1769

2023

1786

2018

1765

 

P1

2008

1845

1986

1811

2023

1825

1916**

1727**

 

liver

P0

17234

9256

16482

9146

16405

 

9727

17773

11512**

 

P1

17378

10926

16862

11353

17140

11999

16893

12319*

 

testes/ovaries

P0

3418

164

3401

160

3496

160

3557

160

 

P1

3609

164

3626

145

3732

140*

3707

150

 

body weight [g]

F0

484

255

474

251

473

255

467

260

 

F1

506

264

495

272

503

273

462**

261

 

Histopathology Incidence

 

Centrilobular Hepatocytes:

cytoplasmatic change

 

P0

0/30

0/30

0/30

1/30

0/30

0/30

15/30

21/30

 

P1

0/30

0/30

0/30

0/30

8/30

4/30

21/30

23/30

 

Centrilobular Hepatocytes:

hypertrophy

 

P0

0/30

1/30

0/30

0/30

0/30

0/30

0/30

17/30

 

P1

0/30

0/30

0/30

0/30

0/30

2/30

0/30

13/30

 

Adrenal glands (cortex):

cytoplasmatic vacuolation

P0

0/30

0/30

0/30

0/30

0/30

0/30

0/30

0/30

 

P1

0/30

0/30

0/30

1/30

0/30

5/30

0/30

28/30

 

Urinary bladder: urothelial hyperplasia

P0

0/30

0/30

0/30

0/30

0/30

0/30

0/30

0/30

 

P1

1/30

0/30

0/30

0/30

0/30

0/30

3/30

1/30

 

Reproductive Performance

 

 

 

Generation

Controls

20 ppm

300 ppm

1800 ppm

Insemination index

%

P0

100

100

96.7

96.7

P1

100

100

100

100

Fertility index

%

P0

90

93.3

96.6

89.7

P1

93.3

93.3

96.7

93.1

Gestation index

%

P0

96.7

96.4

96.4

100

P1

100

100

93.1

100

females with irregular cycle/prolonged estrus

 

number

P0

0

3

1

3

 

P1

0

1

1

0

Number of pups

dead

 

F1

0

3

0

10

 

F2

2

2

3

2

Live birth index

%

F1

100

98.9

100

96.3

F2

99.1

99.3

99.0

99.2

Sex ratio

% Males

F1

50

50

46

52

F2

45

46

53

49

Viability index

%

F1

97.3

96.5

94.3

82.1**

F2

96.8

96.5

98.6

97.7

Lactation index Day 28

%

F1

77.9

80.3

77.7

78.1

F2

95.0

90.6

88.2*

73.6**

Litter size

(viable pups only)

Mean

P0

10.03

9.59

10.33

10.11

P1

11.28

10.48

10.51

9.51**

Litter weight

Day 28

Mean [g]

F1

485.50

434.98

460.59

381.80*

F2

567.47

508.17

538.72

383.89**

Pub weight at birth

 

sex

 

♂

♀

♂

♀

♂

♀

♂

♀

day 0

Mean

F1

6.4

5.95

6.26

5.94

6.30

6.19*

6.34

5.98

day 4

Mean

F1

10.04

8.90

9.70

9.46

9.35

9.60*

9.07**

8.75

day 7

Mean

F1

15.18

13.64

13.50**

13.91

13.75*

14.51

12.96**

12.95

day 14

Mean

F1

30.65

28.39

29.06

29.58

29.90

31.65**

27.38**

27.64

day 21

Mean

F1

49.55

46.18

47.51

46.87

48.02

49.51**

41.71**

41.59**

day 28

Mean

F1

80.75

72.66

74.75**

71.32

77.70

75.26

67.82**

65.45**

day 0

Mean

F2

6.35

6.01

6.25

6.06

6.34

5.95

6.44

6.26**

day 4

Mean

F2

10.39

9.98

10.33

10.05

10.59

10.24

10.26

9.83

day 7

Mean

F2

16.31

15.75

15.44

15.03

16.80

15.74

14.24**

14.17**

day 14

Mean

F2

31.50

31.13

31.34

31.04

33.87**

32.86*

29.52**

29.61**

day 21

Mean

F2

48.19

47.61

50.11*

48.75

51.80**

49.96**

43.51**

43.40**

day 28

Mean

F2

75.23

72.06

77.30

72.89

80.92**

75.15**

68.43**

66.55**

F1 weanlings

Relative Organ weights

mg/100 g bw:

brain

 

F1

1826

1893

1897

1979

1812

1778

1992

2075

liver

 

F1

4112

3997

4008

3970

4208

4042

4813

4638

spleen

 

F1

328

344

331

316

344

324

323

319

thymus

 

F1

397

474

405

479

415

432

411

457

body weight

[g]

F1

83

75

75

72

81

80

71

64

F2 weanlings

Relative Organ weights

mg/100 g bw:

brain

 

F2

1891

 

1911

1811

 

1888

1664

 

1813

1938

1899

liver

 

F2

4461

 

4183

 

4048

 

4011

 

4263

 

4273

 

4639

4695

spleen

 

F2

315

 

322

318

 

303

320

 

309

277

280

thymus

 

F2

433

 

459

423

 

444

385

 

430

413

429

body weight

[g]

F2

81

78

82

76

91

81

75

74

1)only effects occurring in more than one animal of the F0 or F1 generation are reported. Effects observed only in a single animal were not considered relevant.

Conclusions:
CLP: not classified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Additional information:

Toxicity to reproduction of the test substance was tested in a GLP-compliant Two-Generation Reproductive Toxicity study according to OECD 416 (1997o). 30 Male and 30 female Wistar rats (ICO:WU) per generation were fed with a diet containing 20, 300 and 1800 ppm test substance (corresponding to 1.4/1.8, 21.4/28.1 and 138.9/204 mg/kg bw/day in P0 males and females, respectively and 1.6/2.2, 25.0/32.1 and 201.8/259.2 mg/kg bw/day in P1 males and females, respectively. Parental animals (P0) were pretreated over a period of 12 weeks followed by a mating period of up to 3 weeks. F1 offspring was nursed up to an age of 4 weeks. Additional animals were selected for further treatment and to breed a F2 generation. F2 offspring were weaned at an age of 4 weeks. Control animals received standard diet (Altromin®1321) containing 1% peanut oil.

Examinations in parental P0 and P1/F1 animals included clinical signs, body weights, food intake, mating performance, fertility, duration of pregnancy, and estrus cycling. In the F1 and F2 offspring litter size, sex ratio and pup weight at birth as well as viability, lactation, and body weight gain were evaluated. All rats were necropsied and selected organs of P0, P1/ F1 and F2 rats were weighed and selected organs of P0 and P1/F1 rats were histopathological examined.

 

No treatment-related clinical effects, effects on behavior, mortality and body weights were observed in the first parental generation (P0) up to the highest dose tested. Females (P0) of the 300 and 1800 ppm group ingested more food and hence, test substance uptake was higher than planned. Analysis of cholinesterase activity in the brain, plasma and erythrocytes revealed significant cholinesterase inhibition in erythrocytes (30.9% in males and 40.3% in females) and the brain (39.1% in females) in P0 animals at 1800 ppm thereby representing a clear toxicological effect (ECHA, 2016). Thus, the tremendous effect on cholinesterase inhibition above 20% in the brain and erythrocytes indicates an adverse effect on the nervous system and subsequently on physiological homeostasis, which clearly indicates maternal toxicity. Further, changes in organ weights of P0 animals were observed at 1800 ppm and included an increase in relative liver weights in both sexes and a slight elevation of testes weights. Since testes weights were only slightly increased, histopathological investigations revealed no correlating morphological finding and the fertility of P0 males was not affected, the increased testes weight was considered to have no toxicological relevance. At1800 ppm histopathology revealed a remarkable increase in the number of rats exhibiting cytoplasmatic changes in hepatocytes. Additionally, in several females a centrilobular hypertrophy was observed. These findings reflect a changed liver function as a consequence of an adaptive response and are thus not considered as adverse. All other morphological findings are considered to be spontaneous in nature and within the normal range of background pathology commonly seen in rats of this strain and age. The estrus cycle was not affected in P0 rats up 1800 ppm. Reproduction parameters in P0 animals, including mating performance, insemination, fertility, gestation and mean duration of pregnancy were not affected up 1800 ppm. Further, total number of pups, litter size, and sex ratio were comparable among the groups. At 1800 ppm slightly more dead pups were found at birth resulting in a slightly reduced live birth index.

 

In the second parental generation (P1) no treatment-related clinical findings, effects on behavior and mortality were observed in animals exposed to up to 1800 ppm. An effect on body weight and food/compound intake was observed in P1 rats at a dose of 1800 ppm. Males of the highest dose group had significantly reduced body weights although food consumption and compound intake was enhanced. Also in the second parental generation reduced cholinesterase activity in erythrocytes (48.3 % males, 72.8% females) and the brain (41.7% females) was observed at 1800 ppm. Thus, the effects on body weight and cholinesterase inhibition clearly indicate maternal toxicity in the second parental generation (P1). In line with changes in organ weights in the first parental generation also in the second parental generation a significant increase in liver weights was observed in males and females. Further, testes weights were elevated at 1800 ppm. Since testes weights were only slightly increased, histopathological investigations revealed no morphological finding and fertility was not impaired, the observed elevation in testes weight was considered to have no toxicological relevance. Ovary weights were slightly reduced in treated females, reaching statistical significance at 20 and 300 ppm: Due to missing dose-response, these effects are not considered adverse. Statistically significant lower absolute brain weight was observed at 1800 ppm. Lower absolute brain weights were probably due to differences in body weight, which can differ more or less at the necropsy date due to weaning.

Similarly to the P0 generation, adverse effects including cytoplasmatic changes of the liver and hypertrophy were observed in both sexes at 300 and 1800 ppm. As these effects reflect a changed liver function as a consequence of an adaptive response, they are not considered as adverse. In addition, starting at 300 or 1800 ppm there was an increase of cytoplasmatic vacuolation detectable in adrenals of females. Further, a slightly increased incidence of urinary bladder urothelial hyperplasia was noted in the high dose males. No effect was observed on the estrus cycle in the second parental generation. The following reproduction parameters were not affected in the second parental generation up to 1800 ppm: mating performance, insemination, fertility, gestation and mean duration of pregnancy.

In F1 weanlings no significant clinical effects were observed up to 1800 ppm. However, slightly fewer pups survived up to PND4 resulting in a reduced viability index which might not be treatment-related since no similar effect was observed in the F2 generation. Furthermore, an increased loss of pups as a result of cannibalism was noted at 1800 ppm. At the same dose litter weights were reduced and lower pup weights were visible in males from PND4 and in females from PND 21 onwards. As the above mentioned effects observed in the offspring occurred at the dose level which induced maternal toxicity, the effects on pup viability, survival and body weight are considered to be caused by maternal toxicity and not as specific developmental effect. In F1 weanlings relative liver weights were significantly increased in males and females of the highest dose group. No gross pathological findings and no malformations were observed in F1 pups, which died before PND4, were killed in the process of litter reduction on PND4, or were necropsied unscheduled during lactation at levels of up to 1800 ppm.

 

In the F2 offspring, live birth indices, number of dead pups and sex ratio were comparable. In the 1800 ppm dose group fewer pups were born and the mean litter size was significantly reduced at the 1800 ppm dose level. In F2 weanlings no significant clinical effects were observed up to 1800 ppm. Litter weights at weaning and litter size was reduced in the high-dose group. The lactation indices were reduced in the 300 and 1800 ppm dose group. However, since the lactation index at 300 ppm was within the historical control range, only the effects at 1800 ppm were considered as treatment-related and of biological significance. However, as discussed before, the effects occur at the dose level which clearly induced maternal toxicity and hence, are rather considered to be caused by maternal toxicity and not as indicative for developmental toxicity. In F2 weanlings relative liver weights were significantly increased in females, which are interpreted as adaptive and not adverse. Reduced spleen weights were observed in both sexes of the highest dose group, which are considered as a result of the reduced body weight gain rather than an indication of adverse spleen toxicity. No gross pathological findings and no malformations were observed in F2 pups, which died before PND4, were killed in the process of litter reduction on PND4, were necropsied unscheduled during lactation or at scheduled necropsy at dose levels of up to 1800 ppm.

 

Based on the above mentioned results, a NOAEL of 1800 ppm is defined for reproductive toxicity for parental animals of the first and second generation (P0 and P1). Due to the inhibition of cholinesterase activity in the brain and erythrocytes, a NOAEL for general toxicity of 300 ppm is derived for P0 parental animals and P1 males. Based on the effects observed on the adrenals, a NOAEL of 20 ppm and a LOAEL of 300 ppm is set for general toxicity in the P1 parental females. Regarding the effects observed on pup and mean litter weight, viability and live-birth index, a NOAEL of 300 ppm and a LOAEL of 1800 pm is set for developmental toxicity in F1 offspring. Similar effect levels are defined for the F2 generation based on the effects observed on body weight, mean litter size and lactation index.

 

Overall, in F1 and F2 offspring, effects were observed at a dose level which in parallel induced a tremendous inhibition of cholinesterase activity in parental animals by more than 20% in erythrocytes and the brain, which represents a clear toxicological effect (ECHA, 2016) pointing to maternal toxicity. Thus, the observations found in F1 and F2 offspring are considered to be caused by maternal toxicity rather than being an indication of specific developmental toxicity.

 

Further, supporting data from a GLP-compliant one-generation study for testing of toxicity to reproduction in rats are available (1997p). This study was performed as pilot study for the above discussed two-generation study (1997o). The study was performed similar to OECD 416 and OECD 421.

10 male and 10 female Wistar rats (ICO:WU) were fed with a diet containing 50, 400 and 3200 ppm test substance (corresponding to 3.2/3.9, 25.4/31.0 and 251.8/357.4 mg/kg bw/day in males and females, respectively). Animals were exposed to the test substances starting 28 days prior to mating, during mating (up to 21 days), pregnancy (22 days) and lactation (28 days). As no vehicle was used control animals received standard diet (Altromin®1321) containing 1% peanut oil.

Examinations in parental P0 animals included clinical signs, body weights, food and compound intake, cholinesterase activity measurements in the brain, duration of pregnancy, insemination, fertility, gestation indices as well as gross pathological analysis. Litter examinations (F1) included live-birth indices, litter size, sex ratio, clinical findings, body weights, viability and lactation indices, malformations and macroscopical alterations. Histopathological examinations were not performed.In the parental generation no clinical findings, except aggression in 3 animals and diarrhea in individual animals were observed. None of the test animals died or were killed in moribund conditions. Body weights of P0 animals were not affected in any dose group, but at 3200 ppm males and females ingested more feed and therefore also had a higher test substance intake. Analysis of cholinesterase activity revealed a significant inhibition in the brain at 3200 ppm in P0 animals reaching statistical significance in females, which might be the cause of aggressive behaviour observed in 3/10 dams. Taking into consideration that cholinesterase activity is inhibited by 20%, a clear toxicological effect in maternal animals has to be taken into account (ECHA, 2016) indicating maternal toxicity. At the same dose liver weights of males and females were significantly increased. However, gross pathology did not reveal any alterations and hence, the effects are considered as rather adaptive than adverse. Reproductive performance parameters, including mating performance, duration of pregnancy, insemination, fertility and gestation were not affected up to the highest dose tested. Further, live-birth indices, number of pups, litter size and sex ratio were comparable among the groups.

In F1 pups no significant clinical findings were observed up to a dose level of 3200 ppm. The viability and lactation indices were reduced at 3200 ppm. In addition, depressed body weight evolution was observed starting on PND 4 in males and females reaching statistically significance on PND 4 for males and females and on PND 7 for males at 3200 ppm, a dose which caused maternal toxicity. Further, reduced body weight was noted in males at PND 21 and PND 28 at 400 ppm.

Since dams of the 50 and 400 ppm groups had had to nurse more pups per litter up to day 4 p.p. than those of group 0 ppm, these observations were not considered as an adverse effect. No test substance-related gross pathological findings were observed up to 3200 ppm. Skeletal development in the pups up to postpartum week 4 was unaffected.

In conclusion, based on the above mentioned results, a NOAEL of 3200 ppm is defined for reproductive toxicity. Due to the inhibition of cholinesterase activity in the brain, a NOAEL for general toxicity of 400 ppm is derived for the P0 generation. Regarding the reduced viability and lactation index and the depressed body weight development in the F1 generation, a NOAEL of 400 ppm is set for the F1 generation.

 

Overall, similar to the results obtained in the Two-Generation study, reproductive performance was not altered due to test substance intake. Effects observed in the offspring are considered to be caused by maternal toxicity indicated by cholinesterase inhibition in maternal animals.

 

In conclusion, no adverse effects on fertility haven been observed in the available studies on reproduction. Briefly, no effects on estrous cycle and reproductive performance including mating performance, insemination, fertility, gestation and duration of pregnancy have been observed in the One- and Two-Generation studies. In the offspring, effects indicative for developmental toxicity were observed, including reduced live birth and reduced viability index and/or reduced lactation index and reduced mean litter size leading to an overall NOAEL of 300 ppm for developmental toxicity. Moreover, depressed body weight and weight gain of the offspring was observed during the lactation periods. In the parental generations, an inhibition of cholinesterase activity and adaptive liver alterations were observed leading to a NOAEL of 300 ppm in P0 parental animals and P1 males (due to histopathological alterations in the adrenals, a NOAEL of 20 ppm was defined for P1 females).

Overall, effects indicating developmental toxicity were observed at dose levels which in parallel induced a tremendous inhibition of cholinesterase activity in parental animals, which represents a clear toxicological effect (ECHA, 2016) thereby indicating the presence of maternal toxicity. Thus, the effects observed in the offspring in the One- and Two-Generation studies are caused by maternal toxicity and are therefore not considered as indicative for specific developmental toxicity.

 

In conclusion, the test substance does not meet the classification criteria for reproductive toxicity according to Regulation EC No 1272/2008.

 

Additional reference:

ECHA, 2016: Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.7a: Endpoint specific guidance. Draft Version 6.0. May 2016. European Chemicals Agency, Helsinki, Finland

 

Effects on developmental toxicity

Description of key information

Developmental toxicity study (OECD 414), rats:

NOAEL maternal animals: >= 1000 mg/kg bw/day (highest dose tested)

NOAEL fetuses: >= 1000 mg/kg bw/day (highest dose tested

Developmental toxicity study (OECD 414), rabbits:

NOAEL maternal animals: 2.5 mg/kg bw/day

NOAEL fetuses: 640 mg/kg bw/day (highest dose tested)

Two-Generation Toxicity Study (OECD 416), rats (please refer to "Effects on fertility"):

NOAEL offspring/developmental toxicity (F1 and F2) = 300 ppm (equivalent to 21.4 and 28.1 mg/kg bw/day in P0 males and females, respectively and 25 mg/kg bw/day in P1 males; correlated to maternal toxicity)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March - 08 June 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 12 May 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese MAFF guidelines (Guidance on Toxicology Study Data for Application of Agricultural Chemical Registration, Requirements for Safety Evaluation of Agricultural Chemicals, Teratogenicity Study" )
Version / remarks:
January 28, 1985
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA guidelines (Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals", Series 83-3, "Teratogenicity Study"
Version / remarks:
November 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EEC guidelines (Comission Directive 88/302/EEC, Official Journal of the European Communities L 133)
Version / remarks:
May 30, 1988
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wistar rats Hsd/Cpb:WU, Harlan-Winkelmann GmbH, Borchen
- Weight at study initiation: > 300 g (males), 187 - 240 g (females)
- Housing: females were kept in groups in Type III Makrolon® cages during adaptation period and individually in Type II Makrolon® cages from gestation day 0 Makrolon®, low-dust wood shavings, males were kept individually in Type III Makrolon® cages
- Diet: Altromin® 1324 8 (Altromin Company, Lage, Germany), ad libitum
- Water: tap water (drinking water quality), ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 25
- Humidity (%): 35 - 60
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 March 1995 To: 08 June 1995
Route of administration:
oral: gavage
Vehicle:
other: 0.5% tylose suspension
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Administration formulations were prepared using a 0.5% tylose suspension in demineralized water, the administration formulations were stored for the duration of their use at room temperature

VEHICLE
- Justification for use and choice of vehicle: tylose suspension in demineralized water, the vehicle has no effect on the parameters investigated
- Concentration in vehicle: 0.5%

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Investigations on the stability of the active ingredient in samples of 1 mg/mL and 200 mg/mL (dose volume 10 mL/kg bw) covering the range of concentrations used in this study revealed no significant deviations after 8-day storage from the content determined on the day of preparation. The homogeneity of the administration formulations at the 1 mg/mL and the 200 mg/mL concentration also complied. A content check of the formulations of all concentrations was carried out during the inlife period of the study in week 1 and week 3 (April 4 and April 20, 1995 respectively) after initiation of treatment of the first animals. The results revealed no significant deviation (+ 20%) of the active ingredient content from the nominal value in the formulations in any of the three treatment groups.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 - 15 post mating
Frequency of treatment:
daily
Duration of test:
20 Days
Dose / conc.:
100 mg/kg bw/day
Remarks:
concentration: 10 mg/mL
volume: 10 mL/kg bw
Dose / conc.:
300 mg/kg bw/day
Remarks:
concentration: 30 mg/mL
volume: 10 mL/kg bw
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
concentration: 100 mg/mL
volume: 10 mL/kg bw
No. of animals per sex per dose:
28
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels used were selected according to a preceding developmental toxicity screening study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily from day 0 - 20 post mating
- The following cage side observations were checked: appearance and behavior, feed and water consumption, the appearance of their excretory products, the body weight development and mortality of the animals, as well as on the basis of pathological findings

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, daily from Day 6 - 15 and on Day 20 post mating, corrected body weight gain was calculated by subtracting the weight of the uterus on day 20 p.c. from the body weight gain over the period from day 0 to day 20 p.c.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, feed consumption of the animals on gestation days 0-6, 6-11, 11-16 and 16-20 was determined based on the differences in weight of feed provided and feed which remained unconsumed.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, 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 ovaries and uterine content was examined after termination: Yes
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
- Other: number of live fetuses, sex of live fetuses, individual weights of fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: [all per litter / half per litter / #? per litter ]
Statistics:
Differences between the control and test substance-treated groups were considered significant when p<0.05

Analysis of Variance (ANOVA) and in case of significant results Dunnett's t-Test as posthoc test for:
- feed intakes
- body weights and body weight gains
- uterus weights
- corrected body weight gains
- number of corpora lutea per dam
- number of implantations per dam
- number of live fetuses per dam and as percentage of implantantions per dam
- placental weights
- fetal weights

CH|2 test (correction according to Yates) for:
- fertility rate
- gestation rate
- number of fetuses or litters with malformations

2 by N CH|2 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 postimplantation 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

Kruskall-Wallis test and in case of significant differences Dunn's test for:
- number of preimplantation losses per dam
- number of postimplantation losses, early resorptions, late resorptions or dead fetuses per dam
- number of male or female fetuses or fetuses with undeterminable sex per dam


Historical control data:
Please refer to attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section and "Any other information on materials and methods incl. tables" section.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: red stains around the nose were observed in two animals during the treatment period (days 10 and 11) as this observations was occasionally observed in control animals in previous studies, the effect is considered as non-adverse.
yes 1000 mg/kg bw/day: 7 dams showed light discoloration of the feces which is attributed to the elimination of test material which is a white powder (non-adverse). Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data please refer to attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
no data
Mortality:
no mortality observed
Description (incidence):
no moratility observed. Please refer to table 1 in the "any other information on results incl. tables " section.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and weight gain were comparable among the groups. Please refer to table 1 in the "any other information on results incl. tables " section.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected. Please refer to table 1 in the "any other information on results incl. tables " section.
Food efficiency:
no effects observed
Description (incidence and severity):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was not affected to a toxicologically significant extent by the treatment at doses of up to and including 1000 mg/kg.
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:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: pre-implantation loss in the high dose group was slightly elevated when compared to controls, but well within the normal range of historical control values (non adverse)
Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1 and 2 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: in 2 females total resorption of the conceptus was observed, which is known to occur spontaneously in the rat strain used as demonstrated by historical data and a preceding developmental toxicity screening study (Supporting study, M-011047-01-1, Nihon Bayer Agrochem K.K., 1994). Thus, the effect is interpreted as incidental and non-adverse. Otherwise, the number of fetuses and the incidence of late resoprtions is comparable among the groups.
Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1 and 2 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1 and 2 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of fetuses per female was not affected by the treatment in any dose group. Please refer to table 1 in the "any other information on results incl. tables " section.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of pregnant females was not affected by treatment. Please refer to table 1 in the "any other information on results incl. tables " section.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: placental weights were slightly but statistically significantly increased when data were calculated on an individual basis but not when evaluated on a litter basis. Since this increase in mean placenta! weight was also within the range of historical control values it is not considered to be related to treatment. Necrotic placental borders were observed in all dose groups including the control animals without apparent dose-dependency. Since this very common finding which was also observed at only marginally lower levels in untreated animals of former studies, it is not considered to be related to the treatment with the test compound. A slightly increased number of engorged placentas was observed in the 1000 mg/kg dose group. Since there was no clear dose-response for this finding when data were evaluated on a per litter basis and considering that this finding was also observed in control groups of previous studies at similar incidence rates it is not assumed to be related to treatment with the test compound. (non adverse). Please refer to table 1 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1, 2 and 3 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Details on maternal toxic effects:
Total litter losses by resorption:
Total resorption of the conceptus was observed in two females at the 1000 mg/kg dose level. Historical data collected during 1991 to 1994 in this laboratory, indicate that single total resorptions occur by chance, apparently independent of treatment. Furthermore a preceding developmental toxicity screening study did not reveal any total resorptions in 20 treated animals even at dose levels of 2000 mg/kg bw/day (M-011047-01-1, Report No. RS94072,1994). It is therefore concluded that the two incidences of complete postimplantation loss observed in the high dose group of this study were incidental and that the gestation rate was unaffected by the treatment at doses of up to and including 1000 mg/kg bw/day.

Other effects:
The weight of the placentas was slightly but statistically significantly increased at 1000 mg/kg bw/day when data are calculated on an individual basis but not when evaluated on a litter basis. Since this increase in mean placental weight was also within the range of historical control values it is not considered to be related to treatment. Necrotic placental borders were observed in all dose groups including the control animals without apparent dose-dependency. Since this very common finding which was also observed at only marginally lower levels in untreated animals of former studies, it is not considered to be related to the treatment with the test compound. A slightly increased number of engorged placentas were observed in the 1000 mg/kg dose group. Since there was no clear dose-response for this finding when data were evaluated on a per litter basis and considering that this finding was also observed in control groups of previous studies at similar incidence rates it is not assumed to be related to treatment with the test compound.

General reproductive data:
Fertility rates (percentage of inseminated animals with implantations) in the dose groups did not differ to a statistically significant extent from the control dams and were also within the range of historical control data. Further, the number of corpora lutea and implantations was comparable among the groups.
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed at this dose
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
yes 1000 mg/kg bw/day: slightly statistically significantly elevated fetal weight, when calculated on an individual basis, but not when the data are calculated on a per litter basis and are within the range of historical controls (non adverse).
Please refer to table 2 in the "any other information on results incl. tables " section. For historical control data, please refer to table 1 and 2 in the "any other information on materials and methods incl. tables" section and the attached PDF (M-011586-02-1_Developmental Toxicity study in rats historical control data) in the "attached background material" section.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of fetuses per dam was not affected by treatment. Please refer to table 2 in the "any other information on results incl. tables " section.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentages of male and female fetuses did not differ from those in the control groups to any marked extent at levels up to and including 1000 mg/kg. Please refer to table 1 in the "any other information on results incl. tables " section.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size and weights were not affected by treatment.
Please refer to table 2 in the "any other information on results incl. tables " section.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
yes 300 mg/kg bw/day: slightly elevated malformation rate (external, skeletal and visceral) (non-adverse) Otherwise, the type and distribution of anomalies and deviations regarding external findings in the fetuses did not reveal compound-related effects in any of the dose groups. Please refer to table 2 in the "any other information on results incl. tables " section. For historical control data, please refer to table 4 in the "any other information on materials and methods incl. tables" section.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
yes 300 mg/kg bw/day: slightly elevated malformation rate (external, skeletal and visceral). Because dose-dependency was missing and similar malformation rates and types have been reported in control groups of this and previously conducted studies with the same rat strain, the elevated malformation rate was considered incidental (non-adverse). Please refer to table 2 in the "any other information on results incl. tables " section and to attached PDF (M-011586-02-1_Developmental Toxicity study in rats selected tables) in the "attached background material" section. For historical control data, please refer to table 4 in the "any other information on materials and methods incl. tables" section.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
yes 300 mg/kg bw/day: slightly elevated malformation rate (external, skeletal and visceral), which is considered incidental because of lack of dose-dependency. Further, similar malformation rates and types of malformations have also been reported in control groups of this and previously conducted studies with the same rat strain in this laboratory (non-adverse) Please refer to table 2 in the "any other information on results incl. tables " section and to attached PDF (M-011586-02-1_Developmental Toxicity study in rats selected tables) in the "attached background material" section. For historical control data, please refer to table 4 in the "any other information on materials and methods incl. tables" section.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal System Deviations (Variations, Retardations):
yes 1000 mg/kg bw/day: when elevated on a fetal basis, there was a dose-dependent increase in the incidence of dumbbell shaped 12th thoracic vertebral body, advanced ossification of the 4th sacral and 2nd caudal vertebral arches as well as the 6th caudal vertebral body, unossified hyoid body and enlarged fontanelle. None of those findings was statistically significantly different from control values when the data were calculated on a per litter basis except for-advanced ossification of the 4th sacral vertebral arch. All of the above mentioned findings have either also been reported at similar incidence levels in control fetuses of previous studies (dumbbell shaped 12th thoracic vertebral body, advanced ossification of the 4th sacral and 2nd caudal vertebral arches as well as the 6th caudal vertebral body and enlarged fontanelle; or do not display a clear dose-response (ossification of hyoid body) and are for those reasons not considered to be related to treatment.(non adverse) Please refer to attached PDF (M-011586-02-1_Developmental Toxicity study in rats selected tables) in the "attached background material" section.
Details on embryotoxic / teratogenic effects:
Other effects:
Skeletal System Deviations (Variations, Retardations):
There was a dose-dependent increase in the incidence of dumbbell shaped 12th thoracic vertebral body, advanced ossification of the 4th sacral and 2nd caudal vertebral arches as well as the 6th caudal vertebral body, unossified hyoid body and enlarged fontanelle- which were significantly different at the 1000 mg/kg dose level when evaluated on a fetal basis. None of those findings was statistically significantly different from control values when the data were calculated on a per litter basis except for-advanced ossification of the 4th sacral vertebral arches. All of the above mentioned findings have either also been reported at similar incidence levels in control fetuses of previous studies (dumbbell shaped 12th thoracic vertebral body, advanced ossification of the 4th sacral and 2nd caudal vertebral arches as well as the 6th caudal vertebral body and enlarged fontanelle) or do not display a clear dose-response (ossification of hyoid body) and are for those reasons not considered to be related to treatment. It is also highly unlikely that the same compound would elicit advanced and/or delayed ossification, respectively in different skeletal localizations at the same time and that only a few single localizations would be affected. It was therefore concluded that a substance-related effect on ossification (variations and retardations) can be excluded.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed on intrauterine development at this dose
Key result
Developmental effects observed:
no

Table 1: Maternal effects and caesarean section data  

Parameter

Controldata

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Number of dams examined

28

28

28

28

Clinical findings

 

 

 

 

Red strains around nose [no. animals]

0

0

0

2

Light discoloration feces [no. animals]

0

1

0

7

Mortality of dams [%]

0

0

0

0

Body weight gain [g]

Day 6-15 p.c.

32.0

34.8

32.1

30.5

Day 0-20 p.c.

105.5

104.6

101.8

101.9

Day 0-20 p.ccorrected

38.9

43.0

40.0

37.1

Food consumption, range [g/animal/day]

Day 0-6 p.c.

17.7

18.0

17.8

17.7

Day 6-11 p.c.

17.7

18.3

18.3

18.2

Day 11-16 p.c.

19.4

19.9

19.7

19.4

Day 16-20 p.c.

20.9

21.2

20.9

21.5

Inseminated animals

28

28

28

28

Animals with implantations (a)

Total

24

25

26

26

In [%] of those inseminated

85.7

89.3

92.9

92.9

Animals with viable fetuses (b)

Total

24

25

26

24

in [%] of animals with implantations

100

100

100

92.3

Mean values per dam with implantations (a) / viable fetuses (b)

Corpora Lutea (b)

14.3

14.0

13.7

14.6

Preimplantation loss (b)

1.7

2.4

2.2

2.9

Implantations (b)

12.6

11.5

11.5

11.7

Placental weight in [g] (b)

0.6

0.63

0.62

0.64

Number of fetuses (b)

11.7

10.9

10.7

11.0

Late resorptions (a)

0.9

0.6

0.8

1.1

Late resorptions (b)

0.9

0.6

0.8

0.7

Early resorptions (a)

0.0

0.0

0.0

0.1

Early resorptions (b)

0.0

0.0

0.0

0.0

Sex ratio [%] males

46.9

45.2

47.6

46.3

Fetal weight in [g] (b)

3.74

3.70

3.79

3.84

Postimplantation loss (a)

0.9

0.6

0.8

1.2

Postimplantation loss (b)

0.9

0.6

0.8

0.7

Dead fetuses (a)

0.0

0.0

0.0

0.0

Dead fetuses (b)

0.0

0.0

0.0

0.0

 Table 2: Examination of fetuses

Parameter

Controldata

100 mg/kg

300 mg/kg

1000 mg/kg

Number of fetuses per group

281

272

277

264

Total number of fetuses with malformations

3

3

11

4

[%] of fetuses with malformations

1.1

1.1

4.0

1.5

Litters per group

24

25

26

24

Litters with malformations

3

3

8

3

Litters with malformations [%]

12.5

12.0

30.8

12.5

Weight of live fetuses (litter basis) total mean [g]

3.74

3.70

3.79

3.84

Weight of live fetuses (individual basis) total mean [g]

3.73

3.67

3.76

3.84**

External, skeletal and visceral malformations - number of fetuses (litters) affected

Multiple Malformations

0

1

0

0

Microphthalmia/Anophthalmia

2(2)

1

3(2)

2(2)

Situs inversus totalis

0

1

0

0

Globular shaped heart; lung reduced

0

0

0

0

In size; slightly oedematous

0

0

1

0

Lobe of thyroid gland reduced/absent

1

0

2(2)

2(1)

Hernia, left body wall

0

0

1

0

Malformation of vertebra with or without

0

0

0

0

Supernumerary ribs

0

0

2(1)

0

Pelvis shift

0

0

1

0

Dysplasia of scapula

0

0

1

0

** p<0.01

 

Conclusions:
CLP: not classified
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 March - 20 June 1995 (M-011609-01-1), 4 Jan - 27 Feb 1996 (M-011343-01-1)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
M-011609-01-1
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 12 May 1981 (M-011609-01-1)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 15 June 2018 (M-011343-01-1)
Deviations:
yes
Remarks:
animal number less than 16 (only 4 animals per dose group), supplemantary study with specifc endpoints for maternal toxicity
Principles of method if other than guideline:
Developmental toxicity was tested in rabbits according to OECD 414 (main study, M-011609-01-1). Further, a supplementary study was performed to achieve additional data on maternal toxicity (supplementary study, M-011343-01-1). The doses applied are based on the results of a dose range finding study (dose range finding study, M-012513-01-1).
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals or test system and environmental conditions:
TEST ANIMALS (M-011609-01-1, main study)
- Source: Himalayan rabbits, strain CHBB:HM (Dr. Karl Thomae GmbH, Biberach, Germany)
- Weight at study initiation: 2165 - 2943 g (females)
- Housing: individually in Macrolon® cages with perforated cage sheets
- Diet: Ssniff® rabbit diet K - Z (Ssniff Spezialdiaten GmbH, Soest), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Mar 1995 To: 20 Jun 1995 (M-011609-01-1)


TEST ANIMALS (M-011343-01-1, supplementary study)
- Source: Himalayan rabbits, strain CHBB:HM (Dr. Karl Thomae GmbH, Biberach, Germany)
- Weight at study initiation: 2178 - 3302 g (females)
- Housing: individually in Macrolon® cages with perforated cage sheets
- Diet: Ssniff® rabbit diet K - Z (Ssniff Spezialdiaten GmbH, Soest), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 20 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

From: 4 Jan 1996 To: 27 Feb 1996 (M-011343-01-1)
Route of administration:
oral: gavage
Vehicle:
other: 0.5% tylose in demineralized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: test substance was dissolved in 0.5% tylose in demineralized water (M-011609-01-1, M-011343-01-1)

VEHICLE
- Justification for use and choice of vehicle: tylose in demineralized water, this vehicle is known to have no effect on the investigated parameters
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
(M-011609-01-1) Homogeneity and stability after 8 days storage of the active ingredient in samples of low (1 mg/mL) and high concentrations (200 mg/mL) covering the concentrations used in this study had been performed before the start of study. Homogeneity and stability were confirmed in all investigations. A content check of the formulations for the 40, 160 and 640 mg/kg bw groups was carried out in the second and seventh week after start of treatment. A content check of the formulation for the 10 mg/kg bw group, which was added to the study after evaluation of the results from the other three groups, was performed in the second week of treatment of this group. The results revealed no significant deviation of the active ingredient content from the nominal value in the formulations of any of the four treatment groups.

(M-011343-01-1)
Based on the analytical verification performed in the main study (M-011609-01-1), homogeneity and stability after 8 days storage for the lowest dose of 2.5 mg/kg bw/day (0.5 mg/ml) were performed in the supplementary study before the treatment of the animals of this group started. Homogeneity and stability were confirmed in all investigations. A content check of the formulations of the concentrations for the 10 mg/kg to 640 mg/kg groups was carried out in the second week of treatment of these groups. A content check of the formulation of the concentration for the 2.5 mg/kg group was also performed in the second week of treatment for this group. The results of the analyses revealed no significant deviation of the active ingredient content from the nominal value in the formulations of any of the five treatment groups.
Details on mating procedure:
- Impregnation procedure: cohoused (M-011609-01-1, M-011343-01-1)
- M/F ratio per cage: 1/1
- Length of cohabitation: mating was performed between 7 and 10 o'clock a.m. One male rabbit was mated with one female rabbit under obser vation. About one hour after the first mating had occurred the same animals were mated again.
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: observation of copulation referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 to 19 post mating (M-011609-01-1, M-011343-01-1)
Frequency of treatment:
daily (M-011609-01-1, M-011343-01-1)
Duration of test:
29 Days (M-011609-01-1), 20 Days (M-011343-01-1)
Dose / conc.:
10 mg/kg bw/day
Remarks:
M-011609-01-1, M-011343-01-1:
concentration: 2 mg/mL
dose volume: 5 mL/kg bw/day

Dose / conc.:
40 mg/kg bw/day
Remarks:
M-011609-01-1, M-011343-01-1:
concentration: 8 mg/mL
dose volume: 5 mL/kg bw/day

Dose / conc.:
160 mg/kg bw/day
Remarks:
M-011609-01-1, M-011343-01-1:
concentration: 32 mg/mL
dose volume: 5 mL/kg bw/day

Dose / conc.:
640 mg/kg bw/day
Remarks:
M-011609-01-1, M-011343-01-1:
concentration: 128 mg/mL
dose volume: 5 mL/kg bw/day

Dose / conc.:
2.5 mg/kg bw/day
Remarks:
(M-011343-01-1):
concentration: 0.5 mg/mL
dose volume: 5 mL/kg bw/day
No. of animals per sex per dose:
16 (M-011609-01-1)
4 (M-011343-01-1)
Control animals:
yes, concurrent vehicle
Details on study design:
M-011609-01-1:
- Dose selection rationale: The dose levels were selected according to a range finding developmental toxicity study in rabbits (M-012513-01-1/T8058032). In this study 3 mated female Himalayan rabbits each were daily treated orally by gavage with the test substance in 0.5 % tylose in demineralized water from day 6 to 28 post coitum (p.c.) in doses of 100, 200, 400, 600 or 1000 mg/kg bw/day. A further group of 3 mated rabbits received 800 mg test substance/kg body weight daily from day 6 to 19 p. c. Cesarean section was performed on day 29 p.c. Investigations were performed on the general tolerance of the test compound by the females as well as on its effect on intrauterine development. One animal of the 800 mg/kg group aborted on day 24 p.c., the second animal of this group was killed on day 24 p.c. in moribund conditions after severe weight loss. The third female of this group had to be killed on day 22 p.c. due to motionless of both and insensitivity of one hindlimb probably due to an injury. Two animals of the 1000 mg/kg group were killed in moribund conditions on day 14 or 17 p.c.; the third animal of this group was also killed due to severe clinical findings in the other two dams. Feed consumption was transiently (day 6 to 10 p.c.) decreased in the 600 mg/kg group and was decreased during most of the time of the treatment period at levels of 800 mg/kg and above. Water consumption was decreased at levels of 800 mg/kg and above. The urine of the females was discolored (light yellow) and the feces was reduced, light or soft at levels of 800 mg/kg and above. Transient body weight loss from day 6 to 9 p.c. (- 174 g) was evident in the 600 mg/kg group. Body weight gain during the treatment period and during the whole pregnancy period as well as corrected body weight gain were unaffected by treatment at levels up to and including 600 mg/kg. The animals of the 800 mg/kg and of the 1000 mg/kg group distinctly lost weight during the treatment period. Necropsy revealed marbled livers in one animal each in the 800 mg/kg and in the 1000 mg/kg group. Due to the severe maternal toxicity in the 800 mg/kg and in the 1000 mg/kg group there were no fetal data available for these groups. The pregnancy rate, the resorption rate and correspondingly the number of fetuses were unaffected at levels up to and including 600 mg/kg. Fetal weight was decreased at the 600 mg/kg level. There were no external and no visceral findings in the fetuses that were attributable to treatment at levels up to and including 600 mg/kg body weight/day. The skeletal evaluation of the fetuses revealed an increased incidence of fetuses with incompletely ossified hyoid bones or with supernumerary ribs (4 fetuses in 1 litter) in the 600 mg/kg group. The low number of evaluated fetuses, however, did not allow a final assessment of this retardation or variation, respectively. There were no treatment related skeletal malformations at levels up to and including 600 mg/kg. Thus, doses up to and including 400 mg/kg bw/day did not reveal signs of maternal or developmental toxicity. The dose of 600 mg/kg bw/day was maternally toxic (transient body weight loss and transient decrease in feed consumption) and revealed unspecific developmental toxicity (decrease in fetal weight). Doses of 800 mg/kg bw/day and above induced severe maternal toxicity. Therefore, dose levels of 40, 160 or 640 mg/kg bw/day are proposed as dose levels for the main developmental toxicity study in rabbits.

- Rationale for animal assignment: main study (M-011609-01-1) random for 40, 160 and 640 mg/kg dose groups, as one abortion had occurred and two females had to be killed in the 640 mg/kg group during the first two months of the study two further females were added to the 640 mg/kg group without randomisation. During the course of the study abortions were evident at all dose levels a treatment related effect for which could not be completely excluded even at the low dose. Therefore, an additional low dose group (10 mg/kg) of 16 animals was added to the study without randomisation.

M-011343-01-1: The dose levels were selected based on the main study (M-011609-01-1). Since the animals of the low dose (10 mg/kg) already revealed an inhibition of acetylcholinesterase in the erythrocytes an additional group treated with a lower dose (2.5 mg/kg) was included in the supplementary study together with an additional control group.
Maternal examinations:
M-011609-01-1
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily from day 0 - 29 post mating and once at weekends and on bank holidays
- The following cage side observations were included: appearance and behavior, feed and water consumption, the appearance of their excretory products, the body weight development and mortality of the animals, as well as on the basis of pathological findings

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily from day 0 - 29 post mating and once at weekends and on bank holidays

BODY WEIGHT: Yes
- Time schedule for examinations: day 0 and daily from day 6 to 19 post mating and on day 29 post mating

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, for the following days of gestation: day 0 - 6 , day 6-10, day 10 - 14, day 14 - 20, day 20 - 24 and day 24 - 29 post mating.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, water consumption was determined by estimation of the remaining quantities in the water bottles.

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

M-011343-01-1
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily from day 0 to 20 post mating and once at weekends and on bank holidays
- The following cage side observations were included: appearance and behavior, excretory products, feed and water consumption, the appearance of their excretory products,

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily from day 0 - 20 post mating and once at weekends and on bank holidays

BODY WEIGHT: Yes
- Time schedule for examinations: day 0 and daily from day 6 to 20 post mating

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, for the following days of gestation: day 0 - 6 , day 6-11, day 11 - 16 and day 16 - 20 post mating.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, water consumption was determined by estimation of the remaining quantities in the water bottles.

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

OTHER:
Hematology and Hemostaseology: Blood was withdrawn from the veins of the extremities of the animals on day 6 ost coitum (p.c.) (before the first administration), on day 7 p.c. (24 h after the 1st administration), on day 13 p.c. (24 h after the 7th administration) and on day 20 p.c. (24 h after the 14th administration). Hematology and hemostaseology were not performed for the 2.5 mg/kg group and the respective control group (control group II), as a no effect level had been achieved in part I of the study with respect to hematology and hemostaseology. The following parameters were evaluated: differential blood count, erythrocytes, hemoglobin, hematocrit, Heinz bodies, leucocytes, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocytes, platelet count and thromboplastin time.

Clinical chemistry of blood plasma and erythrocytes: blood was withdrawn from the veins of the extremities of the animals on day 6 post coitum (p.c.) (before the first administration), on day 7 p.c. (24 h after the 1st administration), on day 13 p.c. (24 h after the 7th administration) and on day 20 p.c. (24 h after the 14th administration). Clinical chemistry in the blood of the animals of the 2.5 mg/kg group and the respective control group (control group II) was limited to determination of the acetylcholinesterase in plasma and erythrocytes as with respect to the other parameters a no effect level was already achieved in part I of the study. The following parameters were evaluated (the samples taken on day 6 p.c. 3 h after the 1st administration were only examined for the activity of the acetylcholinesterase): alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, acetylcholinesterase in plasma and erythrocytes, creatine kinase, glutamate dehydrogenase, gamma glutamyltransferase, lactate dehydrogenase, total bilirubin, cholesterol, creatinine, triglycerides, urea, albumin and total protein.

Clinical chemistry in the liver: on day 20 after sacrifice of the animals, determination of triglycerides in the liver

Clinical chemistry in the brain: on day 20 after sacrifice of the animals, determination of the activity of the acetylcholinesterase in the brain


Ovaries and uterine content:
M-011609-01-1
The ovaries and uterine content was examined after termination: Yes
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
- Individual weights and appearance of placentas
- Number of live and dead fetuses or embryos
- Individual weights of live fetuses

M-011343-01-1
- Number of implantations: Yes
- Number of live fetuses

Fetal examinations:
M-011609-01-1
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

M-011343-01-1
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
M-011609-01-1:
Differences between the control and test substance-treated groups were considered significant when p<0.05. Statistical significance was tested using the following methods:

Analysis of Variance (ANOVA); in case of significances Dunnett's test for: feed intakes, body weights, number of corpora lutea per dam, implantations per dam, live fetuses per dam and as percentage of implantantions per dam, placental weights, fetal weights

F-test and t-test or t-test according to Welch for: body weight gains, corrected body weight gains

CHI2 test (correction according to Yates) for: fertility rate, gestation rate, number of fetuses or litters with malformations

2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for number of: implantations per group, preimplantation losses per group, postimplantation losses, early resorptions, late resorptions or dead fetuses per group, live fetuses per group in percent of implantations, male or female fetuses or fetuses with undeterminable sex per group, fetuses or litters with skeletal findings

Kruskall-Wallis test; in case of significant differences Dunn's test for number of: preimplantation losses per dam, postimplantation losses, early resorptions, late resorptions or dead fetuses per dam, number of male or female fetuses or fetuses with undeterminable sex per dam

Animals with uterine anomaly or abortion were not taken into account for statistical evaluation. Animals with total resorption were not taken into account for calculation of group mean values of body weights, body weight gains and feed intakes. The mean values in the tables calculated by computer are the rounded results of the calculations with unrounded raw data values.

M-011343-01-1:
Animals with uterine anomaly or without implantation sites were not included into the calculation of mean values. Statistical evaluation was not performed due to the low number of 3 to 4 females with viable fetuses per group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1:
yes 160 and 640 mg/kg bw/day: an increased incidence of females with soft feces (treatment-related, adverse)
yes 640 mg/kg bw/day: light color of feces appeared in several animals of the 640 mg/kg dose group (treatment-related, adverse)
yes starting at 40 mg/kg bw/day: in females which aborted or were killed in moribund conditions, distinct systemic signs were observed for several days before abortion or sacrifice (severely decreased feed intakes, decreased amount of feces, soft feces including diarrhea, decreased water intakes, decreased urination, light-yellow discoloration of urine, body weight loss) (treatment-related, adverse)
Please refer to attached PDF (M-011609-01-1 Developmental Toxicity study in rabbits selected tables) in the "attached background material" section.

M-011343-01-1:
yes 160 mg/kg bw/day: in one female reduced amount of feces, reduced water intake and light-yellow discoloration of urine was observed. Since similar findings were observed in individual animals at levels of 40 mg/kg body weight/day and above in the previous main developmental toxicity study (M-011609-01-1) a treatment-related effect is assumed (treatment-related, adverse)
Please refer to attached PDF (M-011343-01-1 Supplementary study for maternal toxicity, selected tables) in the "attached background material" section.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
M-011609-01-1:
yes 640 mg/kg bw/day: two females were killed in moribund conditions on day 17 and 18 p.c. They had shown their head lying on their forelimbs three or one days before they were k
illed (adverse) Please refer to attached PDF (M-011609-01-1 Developmental Toxicity study in rabbits selected tables) in the "attached background material" section.

M-011343: No mortalities were observed (non-treatment-related, no effects observed).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1:
yes 640 mg/kg bw/day: body weight gains of the females which had viable fetuses at cesarean section were decreased. Females of this dose group lost weight during the first days of treatment (from day 6 to 9 p.c.) which resulted in a decreased weight gain during the treatment period. The two females of this group which were killed in moribund conditions had shown distinct body weight loss from day 6 p.c. until sacrifice. (adverse)
yes 40, 160 and 640 mg/kg bw/day: females which aborted also showed weight loss from day 6 p.c. until abortion which was slight at the 40 mg/kg level and distinct at the 160 mg/kg and 640 mg/kg levels. (adverse) Please refer to table 3 in the "any other information on results incl. tables " section.

M-011343:
yes 160 mg/kg bw/day: one animal showed distinct weight loss from day 6 to 20 p.c.. A treatment related effect is assumed for the weight loss of this animal due to distinct weight loss in individual animals at the 160 mg/kg and 640 mg/kg levels of the main developmental toxicity study (M-011609-01-1). (adverse) Please refer to table 4 in the "any other information on results incl. tables " section.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1:
yes 640 mg/kg bw/day: feed intake of the females which had viable fetuses at cesarean section were decreased (adverse)
yes 40, 160 and 640 mg/kg bw/day: all females which aborted at levels of 40 mg/kg and above and those females which were sacrificed in moribund conditions in the 640 mg/kg group revealed severely decreased feed intakes for measurement periods of 8 or 10 days before abortion or sacrifice. (adverse) Please refer to table 1 in the "any other information on results incl. tables " section.

M-011343:
yes 160 mg/kg bw/day: one female of the 160 mg/kg group, showed severely decreased feed intakes from day 6 to 20 p.c. As individual females of the previous main developmental toxicity study (M-011609-01-1) also showed severely decreased feed intakes at the 160 and 640 mg/kg dose group this finding is considered treatment related. (adverse) Please refer to table 2 in the "any other information on results incl. tables " section.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1:
Yes 40, 160 and 640 mg/kg bw/day: water intakes of the females which aborted or were killed in moribund conditions had been decreased for several consecutive days before abortion or sacrifice at levels of 40 mg/kg bw/day and above (adverse) Please refer to attached PDF (M-011609-01-1 Developmental Toxicity study in rabbits selected tables) in the "attached background material" section.

M-011343: No significant differences in the amount of water consumption were observed. Please refer to attached PDF (M-011343-01-1 Supplementary study for maternal toxicity, selected tables) in the "attached background material" section.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
M-011609-01-1: not examined

M-011343: There was some variation between treatment groups, but there were no meaningful dose dependent effects on either of the parameters of hematology and hemostaseology. In several cases the differences between the groups on days 7, 13 or 20 p.c. reflected differences already evident on day 6 p.c. before start of treatment. Therefore, a treatment related effect on the parameters of hematology and hemostaseology is excluded at levels up to and including 640 mg/kg bw/day. (non adverse)
Please refer to table 5 in the "any other information on results incl. tables " section.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1: not examined

M-011343:
Activity of acetylcholinesterases in blood plasma, erythrocytes and brain:
yes 40, 160 and 640 mg/kg bw/day: acetylcholinesterase activity in plasma was inhibited to a relevant extent (> 20 %) at levels of 40 mg/kg bw/day and above, already after the first test substance administration. (adverse)
yes 160 and 640 mg/kg bw/day: acetylcholinesterase in erythrocytes was inhibited at levels of 160 mg/kg bw/day and above, after first administration. Like in the plasma the inhibition in erythrocytes was more pronounced after 24 h than after 3 h. (adverse)
yes 10, 40, 160 and 640 mg/kg bw/day: after repeated administration the acetylcholinesterase in erythrocytes was inhibited at the 10 mg/kg level. Inhibition of the activity of the acetylcholinesterase was already distinct at the 10 mg/kg level as evident by a 59.5% inhibition in erythrocytes and severe at the 40, 160 and 640 mg/kg levels as evident by a 80.2 to 100 % inhibition in erythrocytes.
Please refer to table 6 in the "any other information on results incl. tables " section.

Clinical chemistry in blood plasma:
yes 160 and 640 mg/kg bw/day: the activity of gamma glutamyltransferase (day 13 and 20 p.c.) and of alanine aminotransferase (day 20 p.c.) were increased, indicative of liver damage. (adverse)
yes 160 and 640 mg/kg bw/day: the mean triglyceride content in the plasma was increased at the 160 mg/kg day 20 p.c. and at the 640 mg/kg level (day 13 and 20 p.c.). Although the value of the 640 mg/kg group of day 20 p.c. was comparable to a previous control value of another study (day 19 p.c.) a treatment related effect is assumed for the increased triglyceride content in the plasma at the 160 mg/kg and 640 mg/kg levels due to the increased triglyceride content in the liver observed at these dose levels. (adverse)
Please refer to table 7 in the "any other information on results incl. tables " section.

Clinical chemistry in the liver:
160 and 640 mg/kg bw/day: the mean triglyceride content in the liver was increased. (adverse)
Please refer to table 8 in the "any other information on results incl. tables " section.
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
Description (incidence and severity):
M-011609-01-1:
not examined

M-011343-01-1:
yes 640 mg/kg bw/day: animals of the 640 mg/kg group revealed a slightly increased absolute and relative liver weight (non-adverse) (adaptive response due to test substance metabolism) . Please refer to table 9 in the "any other information on results incl. tables " section.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1:
yes 640 mg/kg bw/day: one female which was killed in moribund conditions showed extreme filling of the urinary bladder and gaseous contents in the intestines. Another female of this group which aborted showed enlargement of the gallbladder. (adverse)
Please refer to attached PDF (M-011609-01-1 Developmental Toxicity study in rabbits selected tables) in the "attached background material" section.

M-011343-01-1:
yes 160 mg/kg bw/day: one female revealed an uterine anomaly (non-treatment-related, non-adverse)
yes 640 mg/kg bw/day: one female of the 640 mg/kg group had no implantation sites (treatment-related, adverse)
Please refer to attached PDF (M-011343-01-1 Supplementary study for maternal toxicity, selected tables) in the "attached background material" section.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1: not examined

M-011343-01-1:
yes 40, 160 and 640 mg/kg bw/day: hepatocellular hypertrophy was observed in two animals dosed at 40 mg/kg and all animals dosed 160 mg/kg and above; hepatocellular cytoplasmic change consisting of a ground glass
like appearance of the perinuclear cytoplasm found in all animals dosed at 40 mg/kg or above (non-adverse, adaptive response due to test substance metabolism)
yes 640 mg/kg bw/day: in two animals of the high dose group minimal or moderate edema of the gallbladder were observed. In one animal the finding was accompanied by mixed cellular infiltration of the gallbladder wall and mononuclear interstitial infiltration of the liver parenchyma. In the other animal the minimal edema was combined with minimal mononuclear interstitial infiltration and slight multicellular necroses of the liver parenchyma. These findings are considered to be most probably due to infectious origin and therefore are regarded to be unrelated to treatment (non-treatment-related, non-adverse) Please refer to attached PDF (M-011343-01-1 Supplementary study for maternal toxicity, selected tables) in the "attached background material" section.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
no effects observed
Other effects:
not examined
Description (incidence and severity):
no data
Details on results:
Clinical signs
M-011609-01-1:
An increased incidence of females with soft feces was evident at levels of 160 mg/kg bw/day and above. Light color of feces appeared in several animals of the 640 mg/kg dose group (mainly in those which aborted or were killed in moribund
condiditions). Therefore light color of feces was considered an indication of impaired digestion rather than being the consequence of compound excretion.The females which aborted or were killed in moribund conditions showed distinct systemic signs for several days before abortion or sacrifice at levels of 40 mg/kg and above (severely decreased feed intakes, decreased amount of feces, soft feces including diarrhea, decreased water intakes, decreased urination, light-yellow discoloration of urine, body weight loss).

M-011343-01-1: In one female of the 160 mg/kg group reduced amount of feces, reduced water intakes and light-yellow discoloration of urine for which a treatment-related effect is assumed due to the same findings in individual animals at levels of 40 mg/kg body weight/day and above in the previous main developmental toxicity study (M-011609-01-1).

Body weight and weight changes:
M-011609-01-1: Body weight gains of the females which had viable fetuses at cesarean section were decreased at the 640 mg/kg dose level. The females of the 640 mg/kg group lost weight during the first days of treatment (from day 6 to 9 p.c.) which resulted in a decreased weight gain during the treatment period. The two females of this group which were killed in moribund conditions had shown distinct body weight loss from day 6 p.c. until sacrifice. The females which aborted also showed weight loss from day 6 p.c. until abortion which was slight at the 40 mg/kg level (114 g) and distinct at the 160 mg/kg and 640 mg/kg levels.

M-011343: The mean values of body weight gain in the different groups did not reveal a dose related effect. One animal of the 160 mg/kg bw/day group showed distinct weight loss from day 6 to 20 p.c. A treatment related effect is assumed for the weight loss of this animal due to distinct weight loss in individual animals at the 160 mg/kg and 640 mg/kg levels of the main developmental toxicity study (M-011609-01-1).

Food consumption and compound intake:
M-011609-01-1: The feed intakes of the females which had viable fetuses at cesarean section were decreased at the 640 mg/kg dose level. All females which aborted at levels of 40 mg/kg and above and those females which were sacrificed in moribund conditions in the 640 mg/kg group had revealed severely decreased feed intakes for measurement periods of 8 or 10 days before abortion or sacrifice.

M-011343: The mean feed intakes of the different groups did not reveal a dose dependent effect. One female of the 160 mg/kg group, however, showed severely decreased feed intakes from day 6 to 20 p.c. As individual females of the previous main developmental toxicity study (M-011609-01-1) also showed severely decreased feed intakes at the 160 and 640 mg/kg dose group of this study is considered treatment related.

Water consumption:
M-011609-01-1: Water intakes of the females with viable fetuses on day 29 post mating were unaffected by treatment at levels up to and including 640 mg/kg bw/day. Water intakes of the females which aborted or were killed in moribund conditions had been decreased for several consecutive days before abortion or sacrifice at levels of 40 mg/kg bw/day and above.

Haematological findings:
M-011343: There was some variation between treatment groups groups, but there were no meaningful dose dependent effects on either of the parameters of hematology and hemostaseology. In several cases the differences between the groups on days 7, 13 or 20 post coitum (p.c.) reflected differences already evident on day 6 p.c. before start of treatment. Therefore, a treatment related effect on the parameters of hematology and hemostaseology is excluded at levels up to and including 640 mg/kg bw/day.

Clinical biochemistry:
M-011343: Activity of acetylcholinesterases in blood plasma, erythrocytes and brain: acetylcholinesterase activity in plasma was inhibited to a relevant extent (> 20 %) at levels of 40 mg/kg bw/day and above, already after the first test substance administration. The acetylcholinesterase in erythrocytes was inhibited at levels of 160 mg/kg bw/day and above, after first administration. Like in the plasma the inhibition in erythrocytes was more pronounced after 24 h than after 3 h. After repeated administration the acetylcholinesterase in erythrocytes was inhibited at the 10 mg/kg level. Inhibition of the activity of the acetylcholinesterase was already distinct at the 10 mg/kg level as evident by a 59.5% inhibition in erythrocytes and severe at the 40, 160 and 640 mg/kg levels as evident by a 80.2 to 100% inhibition in erythrocytes. The animals of the 2.5 mg/kg group did not reveal any relevant inhibition of the acetylcholinesterase in plasma and erythrocytes.

Clinical chemistry in blood plasma:
The activity of gamma-glutamyltransferase (day 13 and 20 p.c.) and of alanine-aminotransferase (day 20 p.c.) were increased at the 160 and 640 mg/kg dose levels indicative of liver damage. The mean triglyceride content in the plasma was increased at the 160 mg/kg day 20 p.c. and at the 640 mg/kg level (day 13 and 20 p.c.). Although the value of the 640 mg/kg group of day 20 p.c. was comparable to a previous control value of another study (day 19 p.c.) a treatment related effect is assumed for the increased triglyceride content in the plasma at the 160 mg/kg and 640 mg/kg levels due to the increased triglyceride content in the liver observed at these dose levels. Other parameters investigated did not differ significantly in the different dose groups. Therefore, a treatment-related effect on the parameters of clinical chemistry in the plasma except from gamma-glutamyltransferase, alanine-aminotransferase and triglycerides is excluded at levels up to and including 640 mg/kg body weight/day.

Clinical chemistry in the liver: The content of triglycerides in the liver was unaffected by treatment at levels up to and including 40 mg/kg bw/day. The mean values in these groups were lower than or comparable to the control group values. The mean triglyceride content in the liver was increased at the 160 mg/kg and 640mg/kg levels.

Organ weight findings including organ / body weight ratios:
M-011343-01-1: Brain and spleen weights were unaffected by treatment at levels up to and including 640 mg/kg bw/day. The liver weights were unaffected by treatment at levels up to and including 160 mg/kg bw/day as absolute and relative liver weights were comparable to control values. The animals of the 640 mg/kg group revealed a slightly increased absolute and relative liver weight.

Gross pathological findings:
M-011609-01-1: One female of the 640 mg/kg group which was killed in moribund conditions showed extreme filling of the urinary bladder and gaseous contents in the intestines. Another female of this group which aborted showed enlargement of the gallbladder. Extreme filling of the urinary bladder, gas in the intestines and findings in the gallbladder may occur spontaneously in the strain of rabbits used, however, due to the poor general conditions of the affected animals a treatment related effectfor these findings cannot be excluded. Other findings observed at necropsy of the animals are considered incidental.

M-011343-01-1: No meaningful gross pathological findings were ascertained in the dams at levels up to and including 640 mg/kg bw/day. One female of the 160 mg/kg group revealed an uterine anomaly and one female of the 640 mg/kg group had no implantation sites.

Histopathological findings non neoplastic:
M-011343-01-1: Histopathology of brain and spleen did not show treatment related findings at levels up to and including 640 mg/kg bw/day. Hepatocellular hypertrophy was observed in two animals dosed at 40 mg/kg and all animals dosed 160 mg/kg and above. The hepatocellular hypertrophy was predominantly found in centro- and intermedioacinar areas. Additionally hepatocellular cytoplasmic change consisting of a ground glass-like appearance of the perinuclear cytoplasm was found in all animals dosed at 40 mg/kg or above. The severity increased in a dose-related manner. In two animals of the high dose group minimal or moderate edema of the gallbladder was observed. In one animal the finding was accompanied by mixed cellular infiltration of the gallbladder wall and mononuclear interstitial infiltration of the liver parenchyma. In the other animal the minimal edema was combined with minimal mononuclear interstitial infiltration and slight multicellular necroses of the liver parenchyma. These findings are considered to be most probably due to infectious origin and therefore are regarded to be unrelated to treatment.
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1:
yes 40 mg/kg bw/day: one female aborted on day 26 post mating (adverse)
yes 160 mg/kg bw/day: two females aborted on day 28 or 25 post mating (adverse)
yes 640 mg/kg bw/day: three females aborted on day 23 or 24 post mating (adverse) As all animals with abortions revealed signs of maternal toxicity the abortions are not considered an indication of specific developmental toxicity.
Please refer to attached PDF (M-011609-01-1 Developmental Toxicity study in rabbits selected tables) in the "attached background material" section and for historical control data to please refer to attached PDF (M-011609-01-1 Developmental Toxicity study in rabbits historical control data) in the "attached background material" section.

M-011343-01-1: not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
M-011609-01-1: no effects observed
Please refer to table 12 in the "any other information on results incl. tables " section.

M-011343-01-1:
yes 640 mg/kg bw/day: in one animal no implantation sites were observed (non-adverse)
Please refer to attached PDF (M-011343-01-1 Supplementary study for maternal toxicity, selected tables) in the "attached background material" section.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1:
yes 160 and 640 mg/kg bw/day: two single total resorptions appeared in a dose range which was maternally toxic (160 mg/kg: impaired digestion including diarrhea, 640 mg/kg: severely impaired general conditions) (adverse)
Please refer to table 12 in the "any other information on results incl. tables " section.

M-011343-01-1: not examined
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
M-011609-01-1:
yes control and 160 mg/kg bw/day: two early resorptions were observed one in the control group and another one in the 160 mg/kg dose group (non-treatment-related, non-adverse)
yes control, 10, 40, 160 and 640 mg/kg bw/day: late resorptions were observed in all dose groups and in the control group. The number of late reorptions per group were as followed: 12 (control group), 15 (10 mg/kg bw/day), 9 (40 mg/kg bw/day), 9 (160 mg/kg bw/day) and 7 (640 mg/kg bw/day) (non-adverse)
Please refer to table 12 in the "any other information on results incl. tables " section.

M-011343-01-1: not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
M-011609-01-1:
Dead fetuses were not observed in any of the dose groups (non-adverse)
Please refer to table 12 in the "any other information on results incl. tables " section.

M-011343-01-1:
Dead fetuses were not observed in any of the dose groups (non adverse)
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
M-011609-01-1:
yes 40, 160 and 640 mg/kg bw/day: the gestation rate was slightly decreased (93.4%) by one abortion at the 40 mg/kg level, two abortions and one total resorption at the 160 mg/kg bw/day level (81.3%) and due to three abortions and one total resorption at the 640 mg/kg dose level (73.3%). Due to the increased incidence of abortions at the same dose levels a treatment-related effect for these single total resorptions at the 160 mg/kg and 640 mg/kg levels cannot be excluded although single total resorptions may occur spontaneously in the rabbit strain used. (adverse) Please refer to table 11 in the "any other information on results incl. tables " section.

M-011343-1-1: not examined
Other effects:
no effects observed
Description (incidence and severity):
Weight and appearance of placentas The weight and appearance of placentas were unaffected by treatment at levels up to and including 640 mg/kg bw/day.
Details on maternal toxic effects:
Number of abortions:
Single abortions may occur spontaneously in the strain of rabbits used. Nevertheless, a relationship to treatment cannot be totally excluded for the single abortion at the 40 mg/kg level due to the dose-dependent increase in abortions at the 160 mg/kg and 640 mg/kg levels. The animals of the 10 mg/kg group did not reveal any treatment related effects on appearance (including abortions). As all animals with abortions revealed distinct systemic effects for at least eight days before abortion the abortions are considered to be caused by maternal toxicity rather than being an indication of specific developmental toxicity.

Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
2.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed at this dose level
Key result
Dose descriptor:
LOAEL
Remarks:
maternal toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical biochemistry
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: liver, increased activity of gamma glutamyltransferase, alanine aminotransferase and increased triglyceride content
Description (incidence and severity):
liver weights were increased at the 640 mg/kg bw/day level, hepatocellular hypertrophy and hepatocellular cytoplasmic change (ground glass appearance) in centro- and intermedioacinar areas at levels of 40 mg/kg and above due to adaptive responses based on test substance metabolism), increased enzyme activity of gamma glutamyltransferase, alanine aminotransferase and triglyceride content increased at the 160 mg/kg and 640 mg/kg levels
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: inhibition of acetylcholinesterase activity
Description (incidence and severity):
Inhibition of acetylcholinesterase activity levels at 40 mg/kg and above in the plasma and at levels of 10 mg/kg and above in the erythrocytes ) (Statistically significant inhibition by 20% or more in erythrocytes or brain is considered a clear adverse toxicological effect).
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: reduced body weight and food intake
Description (incidence and severity):
At doses of 40, 160 and 640 mg/kg bw/day reduced weight gain and food intake was observed.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: abortions and total resorptions
Description (incidence and severity):
One abortion was observed at 40 mg/kg bw/day dose level, in two females at the 160 mg/kg bw/day dose level and in three females at the 640 mg/kg bw/day dose level. Total litter losses occurred in one female of the 160 mg/kg bw/day dose group and one female of the 640 mg/kg bw/day dose group.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
M-011609-01-1:
no effects observed. Please refer to table 12 in the "other informations on results incl. tables" section.

M-011343-01-1:
not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
M-011609-01-1:
The total number of fetuses was unaffected by treatment at levels up to and including 640 mg/kg body weight/day. Please refer to table 12 in the "other informations on results incl. tables" section.

M-011343-01-1: no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
M-011609-01-1:
no effects observed. Please refer to table 12 in the "other informations on results incl. tables" section.

M-011343-01-1:
not examined
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
M-011609-01-1:
no effects observed. Please refer to table 12 in the "other informations on results incl. tables" section.

M-011343-01-1:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
M-011609-01-1:
no effects observed. Please refer to table 13 in the "any other information on results incl. tables " section.

M-011343-01-1:
yes 640 mg/kg bw/day: acrania was observed in one fetus (non-adverse). Please refer to attached PDF (M-011343-01-1 Supplementary study for maternal toxicity, selected tables) in the "attached background material" section.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
M-011609-01-1:
yes 0 mg/kg bw/day: arthrogryposis was observed in 1 fetus of the control group (non-adverse), supernumerary lumbar vertebra and missing phalanges/metacarpal were observed in 1 fetus of the control group (non-adverse)
yes 10 mg/kg bw/day: arthrogryposis was observed in 5 fetuses (non-adverse), malposition of hind limbs was observed in one fetus (non-adverse), supernumerary lumbar vertebra was observed in one fetus (non-adverse)
yes 40 mg/kg bw/day: arthrogryposis was observed in one 1 fetus (non-adverse), supernumerary lumbar vertebra was observed in one fetus (non adverse)
yes 160 mg/kg bw/day: arthrogryposis was observed in 2 fetuses (non-adverse)
yes 640 mg/kg bw/day: arthrogryposis was observed in 2 fetuses (non-adverse)
Please refer to table 13 in the "any other information on results incl. tables " section. For historical control data, please refer to attached PDF (M-011609-01-1 Developmental Toxicity study in rabbits historical control data) in the "attached background material" section.

M-011343-01-1: no effects observed. Please refer to attached PDF (M-011343-01-1 Supplementary study for maternal toxicity, selected tables) in the "attached background material" section.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
M-011609-01-1:
yes 160 mg/kg bw/day: cardiac septum defect was observed in one fetus (non-adverse)
Please refer to table 13 in the "any other information on results incl. tables " section. For historical control data, please refer to attached PDF (M-011609-01-1 Developmental Toxicity study in rabbits historical control data) in the "attached background material" section.

M-011343-01-1: spina bifida: one fetus (640 mg/kg bw/day dose group) (non-adverse) Please refer to attached PDF (M-011343-01-1 Supplementary study for maternal toxicity, selected tables) in the "attached background material" section.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
640 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed on intrauterine development
Abnormalities:
effects observed, non-treatment-related
Localisation:
visceral/soft tissue: somatic nervous system
Description (incidence and severity):
M-011343-01-1: One fetus of the 640 mg/kg dose group revealed spina bifida and acrania.
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: cranium
Description (incidence and severity):
M-011343-01-1: One fetus of the 640 mg/kg dose group revealed acrania. The same fetus also had spina bifida.
Abnormalities:
effects observed, non-treatment-related
Localisation:
other: arthrogryposis
Description (incidence and severity):
M-011609-01-1: One fetus (control group), 5 fetuses (10 mg/kg bw/day dose group), 1 fetus (40 mg/kg bw/day dose group), 2 fetuses (160 mg/kg bw/day dose group), 2 fetuses (640 mg/kg bw/day dose group)
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: hindlimb
Description (incidence and severity):
M-011609-01-1: malposition of hind limbs was observed in one fetus (10 mg/kg bw/day dose group),
Abnormalities:
effects observed, non-treatment-related
Localisation:
visceral/soft tissue: cardiovascular
Description (incidence and severity):
M-011609-01-1: cardiac septum defect: one fetus (160 mg/kg bw/day dose group)
Abnormalities:
effects observed, non-treatment-related
Localisation:
visceral/soft tissue: central nervous system
Description (incidence and severity):
M-011343-01-1: spina bifida: one fetus (640 mg/kg bw/day dose group).
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: vertebra
Description (incidence and severity):
M-011609-01-1: supernumerary lumbar vertebra: one fetus (10 mg/kg bw/day dose group) and one fetus (40 mg/kg bw/day dose group), supernumerary lumbar vertebra and missing phalanges/metacarpal: one fetus (control group)
Key result
Developmental effects observed:
no

Table 1: Mean feed intakes (g/animal/day) (M-011609-01-1)

Dose(mg/kg bw/day)

0

10

40

160

640

day

0-6

p.c.

84.0

86.6

87.9

84.2

84.2

day

6-10

p.c.

78.4

83.7

85.0

79.1

58.2*

day

10-14

p.c.

67.6

72.6

79.0

74.4

88.9**

day

14-20

p.c.

72.5

72.3

80.4

73.9

76.0

day

20-24

p.c.

85.5

84.8

81.1

82.4

82.3

day

24-29

p.c.

83.8

83.9

81.0

85.6

84.1

*  Statistically significant difference to control p < 0.05

** Statistically significant difference to control p < 0.01

p.c. post coitum

 

Table 2: Mean feed intakes (g/animal/day) (M-011343-01-1)

Dose(mg/kg bw/day)

0 (I)

10

40

160

640

0 (II)

2.5

day

0-6

p.c.

73.3

67.3

80.3

72.4

78.9

80.2

77.9

day

6-11

p.c.

66.7

67.6

79.3

57.7

82.0

66.9

71.6

day

11-16

p.c.

55.8

64.9

72.6

57.1

86.1

59.7

64.3

day

16-20

p.c.

69.2

69.9

75.0

47.9

71.2

76.5

78.8

 

Table 3: Mean body weight gain (g) (M-011609-01-1)

Dose (mg/kg bw/day)

Day 6-19 p.c.

Day 0-29 p.c.

corrected Day 0-29 p.c.

0

48.5

226.5

-137.2

10

59.2

266.3

-123.1

40

42.3

211.3

-137.5

160

29.8

222.8

-157.3

640

5.1

105.8

-131.2

p.c. post coitum

 

Table 4: Mean body weight gain (g) (M-011343-01-1)

Dose (mg/kg bw/day)

Day 6-20 p.c.

0 (I)

7.5

10

19.8

40

50.5

160

-106.0

640

44.3

0 (II)

46.5

2.5

57.8

p.c. post coitum

 

Table 5: Hematology and Hemostaseology Results (M-011343-01-1)

Dose

(mg/kg bw/day)

control

10

40

160

640

Day 6p.c.

 

 

 

 

 

LEUCO (10E9/L)

9.3

9.1

8.3

7.8

6.6

ERY(10E12/L)

6.93

6.98

6.74

6.83

6.88

HB(g/L)

143

147

138

141

139

HCT(L/L)

0.433

0.446

0.425

0.429

0.426

MCV(fl)

62.5

64.0

63.0

63.0

62.0

MCH(pg)

20.6

21.1

20.5

20.6

20.2

MCHC(g/LERY)

329

330

325

328

325

RETI(%0)

22

20

23

22

21

HEINZ (%0)

0

0

0

0

0

THRO(10E9/L)

391

327

348

348

239

TPZ(sec)

7.5

7.4

7.6

7.3

8.7

Day 7 p.c.

 

 

 

 

 

LEUCO (10E9/L)

9.1

8.2

8.2

7.2

7.6

ERY(10E12/L)

6.48

6.43

6.32

6.27

6.58

HB(g/L)

134

136

131

131

134

HCT(L/L)

0.410

0.416

0.398

0.395

0.399

MCV(fl)

63.4

64.8

62.9

63.1

60.6

MCH(pg)

20.7

21.2

20.7

21.0

20.4

MCHC(g/LERY)

327

327

329

332

337

RETI(%0)

18

19

21

19

19

HEINZ (%0)

0

0

0

0

0

THRO(10E9/L)

395

332

344

342

219

TPZ(sec)

7.3

7.3

7.4

7.4

7.6

Day 13 p.c.

 

 

 

 

 

LEUCO (10E9/L)

10.0

7.8

8.5

8.9

8.5

ERY(10E12/L)

7.05

6.77

6.99

6.44

6.63

HB(g/L)

145

144

143

133

133

HCT(L/L)

0.449

0.441

0.444

0.404

0.417

MCV(fl)

63.8

65.2

63.6

62.9

62.9

MCH(pg)

20.6

21.2

20.5

20.7

20.1

MCHC(g/LERY)

323

326

322

330

319

RETI(%0)

26

25

27

27

25

HEINZ (%0)

0

0

0

0

0

THRO(10E9/L)

414

310

406

311

356

TPZ(sec)

7.4

7.8

7.9

7.1

7.5

Day 20 p.c.

 

 

 

 

 

LEUCO (10E9/L)

7.1

6.0

6.1

5.2

5.9

ERY(10E12/L)

6.16

6.23

6.12

5.97

5.98

HB(g/L)

127

132

128

126

123

HCT(L/L)

0.405

0.422

0.404

0.392

0.390

MCV(fl)

65.8

67.8

66.0

65.8

65.2

MCH(pg)

20.7

21.1

21.0

21.2

20.5

MCHC(g/LERY)

314

312

318

323

315

RETI(%0)

57

72

61

67

77

HEINZ (%0)

0

0

0

0

0

THRO(10E9/L)

453

327

410

296

339

TPZ(sec)

7.0

7.1

7.1

7.1

7.3

 

Table 6: Activity of cholinesterase (CHE) in blood plasma, in erythrocytes and in brain (M-011343-01-1)

 

CHE in plasma

CHE in erythrocytes

CHE in brain

Dose mg/kg

Activity kU/l

Inhibition %

Activity kU/l

Inhibition %

Activity kU/l

Inhibition %

Day 6 p.c. (before first administration)

 

 

0 (I)

0.77

 

0.86

 

 

 

10

0.78

 

0.79

8.1

 

 

40

0.79

 

0.83

3.5

 

 

160

0.85

 

0.85

1.2

 

 

640

0.72

6.5

0.73

15.1

 

 

0 (II)

0.83

 

0.51

 

 

 

2.5

0.78

6.0

0.70

 

 

 

Day 6 p.c. (3h after first administration)

 

 

0 (I)

0.72

 

0.76

 

 

 

10

0.63

12.5

0.77

 

 

 

40

0.62

13.9

0.77

 

 

 

160

0.51

29.2

0.77

 

 

 

640

0.38

47.2

0.47

38.2

 

 

0 (II)

0.77

 

0.55

 

 

 

2.5

0.71

7.8

0.66

 

 

 

Day 7 p.c (24 h after first administration)

 

 

0 (I)

0.73

 

0.90

 

 

 

10

0.69

5.5

0.87

3.3

 

 

40

0.57

21.9

0.87

3.3

 

 

160

0.40

45.2

0.41

54.4

 

 

640

0.18

75.3

0.08

91.1

 

 

0 (II)

0.72

 

0.76

 

 

 

2.5

0.65

9.7

0.70

7.9

 

 

Day 13 p.c. (24h after 7th administration)

0 (I)

0.68

 

0.91

 

 

 

10

0.57

16.2

0.61

33.0

 

 

40

0.40

41.2

0.38

58.2

 

 

160

0.31

54.4

0.03

96.7

 

 

640

0.19

72.1

0.00

100.0

 

 

0 (II)

0.73

 

0.70

 

 

 

2.5

0.68

6.9

0.74

 

 

 

Day 20 p.c. (24h after 14th administration)

0 (I)

0.56

 

1.11

 

12.72

 

10

0.49

12.5

0.45

59.5

19.85

 

40

0.36

35.7

0.22

80.2

21.37

 

160

0.26

53.6

0.09

91.9

23.25

 

640

0.18

67.9

0.02

98.2

19.24

 

0 (II)

0.62

 

1.35

 

14.96

 

2.5

0.60

3.2

1.15

14.8

13.67

8.6

 

Table 7 Clinical Chemistry in blood plasma (M-011343-01-1)

Dose

(mg/kg bw/day)

control I

10

40

160

640

Day 6p.c.

ASAT (U/L)

6.6

10.5

8.6

8.7

6.1

ALAT (U/L)

43.2

52.6

49.3

57.9

40.6

APh (U/L)

68

67

64

74

60

GLDH (U/L)

1.8

2.9

2.8

2.0

1.7

GGT (U/L)

7

6

6

6

6

LOH(U/L)

46

54

70

59

59

CK(U/L)

265

347

476

428

280

CHOL (mmol/L)

0.91

1.29

1.02

0.82

0.83

TRIGL (mmol/L)

0.36

0.46

0.43

0.43

0.47

CREA (mcmol/L)

126

124

125

122

105

UREA (mmol/L)

8.91

7.42

8.16

8.39

7.53

BILI-t (mcmol/L)

1.1

1.2

1.1

0.8

0.8

PROT (g/L)

70.2

67.7

67.7

70.1

65.9

ALBUMIN (g/L)

45.2

43.2

42.3

44.7

41.5

Day 7p.c.

ASAT (U/L)

6.3

10.0

8.6

8.5

6.6

ALAT (U/L)

44.1

53.1

50.9

63.9

48.9

APh (U/L)

63

61

62

68

61

GLDH (U/L)

1.4

2.2

2.0

1.7

1.0

GGT (U/L)

7

6

6

7

7

LOH(U/L)

41

50

81

47

34

CK(U/L)

399

596

1058

760

221

CHOL (mmol/L)

0.95

1.30

1.08

0.83

0.89

TRIGL (mmol/L)

0.40

0.44

0.46

0.46

0.51

CREA (mcmol/L)

127

127

127

125

110

UREA (mmol/L)

8.07

6.75

7.75

7.31

6.49

BILI-t (mcmol/L)

1.1

1.2

1.1

1.1

1.0

PROT (g/L)

69.2

67.4

65.4

67.1

65.4

ALBUMIN (g/L)

44.0

42.4

42.0

43.7

40.9

Day 13 p.c.

ASAT (U/L)

6.7

10.7

7.7

7.6

6.2

ALAT (U/L)

39.3

50.9

50.1

54.2

37.8

APh (U/L)

57

59

60

59

51

GLDH (U/L)

2.2

2.6

2.3

1.7

1.3

GGT (U/L)

6

6

6

10

10

LOH(U/L)

36

58

44

37

52

CK(U/L)

67

117

115

180

310

CHOL (mmol/L)

0.93

1.20

1.21

0.95

1.10

TRIGL (mmol/L)

0.35

0.50

0.48

0.64

0.90

CREA (mcmol/L)

137

130

142

119

100

UREA (mmol/L)

8.16

7.24

8.01

7.33

6.29

BILI-t (mcmol/L)

1.2

1.3

1.3

0.9

1.2

PROT (g/L)

66.1

67.1

65.2

65.9

61.9

ALBUMIN (g/L)

42.0

41.4

40.4

42.1

39.3

Day 20 p.c.

ASAT (U/L)

8.6

16.1

8.4

17.3

10.9

ALAT (U/L)

34.1

53.4

39.9

112.5

74.1

APh (U/L)

52

49

53

72

65

GLDH (U/L)

2.6

4.3

2.1

7.8

2.3

GGT (U/L)

5

5

6

20

11

LOH(U/L)

48

50

45

46

50

CK(U/L)

66

82

89

167

194

CHOL (mmol/L)

0.25

0.33

0.42

0.47

0.33

TRIGL (mmol/L)

0.61

0.72

0.84

1.28

0.97

CREA (mcmol/L)

96

96

95

97

77

UREA (mmol/L)

6.72

5.71

6.03

5.40

4.44

BILI-t (mcmol/L)

1.2

1.2

1.2

1.3

1.1

PROT (g/L)

62.2

65.3

62.6

60.8

58.1

ALBUMIN (g/L)

41.2

42.5

42.2

40.2

39.1

 

Table 8 Clinical chemistry results (liver) (M.011343-01-1)

Dose (mg/kg bw/day)

TRIGL (mcmol/g)

0 (I)

7.97

10

11.01

40

11.83

160

22.94

640

21.68

0 (II)

11.32

2.5

5.77

 

Table 9: Organ weights (M-011343-01-1)

Dose

(mg/kg bw/day)

brain weight

absolute [g]

liver weight

absolute [g]

liver weight

relative [g/g]

spleen weight

absolute [g]

spleen weight

relative [g/g]

0 (I)

7.91

53.13

6.80

0.94

0.12

10

7.58

57.36

7.61

0.80

0.11

40

8.32

67.53

8.12

0.90

0.11

160

8.33

70.71

8.59

0.72

0.09

640

7.77

75.30

10.16

0.68

0.09

0 (II)

6.93

64.53

9.29

0.70

0.10

2.5

8.07

61.02

7.62

0.74

0.09

 

Table 10: General reproduction data (M-011609-01-1)

Dose

(mg/kg bw/day)

0

10

40

160

640

mated females

16+

16+

16

16

18+#

mated females

evaluated

15

15

16

16

15

females with

implantations

15

15

15

16

15

in % of those

mated

100

100

93.8

100

100

mean values per female with implantation sites

corpora lutea

8.3

9.0

7.8

8.3

7.4

preimplantation loss

0.3

0.3

0.3

0.4

0.9

implantations

8.0

8.7

7.5

7.9

6.5

+ one female with uterine anomaly

# two females killed in moribund conditions

 

Table 11: Gestation rate (M-011609-01-1)

Dose

animals with viable fetuses

mg/kg bw/day

total

% of animals with implantations

0

15

100

10

15

100

40

14

93.3

160

13

81.3

640

11

73.3

 

Table 12: Mean values intrauterine development (M-011609-01-1)

Dose (mg/kg bw/day)

0

10

40

160

640

number of dams with implantations (a)

15

15

14

14

12

number of dams with viable fetuses (b)

15

15

14

13

11

females with implantations (a)/viable fetuses (b)

mean placental weight [g] per female (b)

4.12

4.00

4.23

4.12

4.13

mean number of fetuses values per female (b)

7.1

7.7

6.9

7.7

6.5

dead fetuses per group (a)

0

0

0

0

0

dead fetuses per group (b)

0

0

0

0

0

mean postimplantation loss values per female (a)

0.9

1.0

0.6

0.7

0.6

mean postimplantation loss values per female (b)

0.9

1.0

0.6

0.5

0.2

preimplantation (a)

5

5

4

6

11

preimplantation (b)

5

5

4

6

11

early resorptions (a)

1

0

0

1

0

early resorptions (b)

1

0

0

1

0

late resorptions (a)

12

15

9

9

7

late resorptions (b)

12

15

9

6

2

mean litter [%] males (b)

49.9

40.5

49.7

41.5

50.5

mean litter [%] females (b)

50.1

59.5

50.3

58.5

49.5

males [%]

49.9

40.5

49.7

41.5

50.5

mean fetal weight [g]

35.9

35.5

36.8

34.9

37.3

 

Table 13: Malformations (M-011609-01-1)

Dose (mg/kg bw/day)

0

10

40

160

640

multiple malformation

 

 

 

1

 

arthrogryposis

1

5(4)

1

2(2)

2(2)

malposition of hind limbs

 

1

 

 

 

supernumerary lumbar

vertebra

 

1

1

 

 

supernumerary lumbar

vertebra and missing phalanges/

metacarpal

1

 

 

 

 

cardiac septum defect

 

 

 

1

 

number of fetuses/group

107

115

96

100

71

number of fetuses with malformations

1

7

2

4

2

malformed fetuses/group [%]

0.9

6.1

2.1

4.0

2.8

number of litters/group

15

15

14

13

11

number of litters with malformations

1

5

2

4

2

malformed litters/group [%]

6.7

33.3

14.3

30.8

18.2

() number of litters affected

Conclusions:
CLP: not classified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
21.4 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable studies (Klimisch score 1), and are thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Additional information:

Developmental toxicity was tested in a GLP compliant study in Wistar rats according to OECD 414 (1997q). In this study 28 inseminated rats per dose level were treated daily by gavage with 100, 300 and 1000 mg test substance /kg bw/day suspended in 0.5% tylose suspension from day 6 to day 15 post coitum. The animals of the control group received vehicle only (0.5% tylose suspension). At day 20 of gestation fetuses were delivered by caesarian section. Examinations of maternal animals included general tolerance of the test substance, appearance, behavior feed and water consumption, excretory products, mortality, body weight and gross pathological examinations. The following parameters were determined at caesarian section: number of corpora lutea, number of implantations, uterus weight, individual weight and appearance of the placentas, number of resorptions, number of live fetuses, sex of live fetuses, individual weights of fetuses, external malformations and other findings deviating from normal, visceral malformations and other findings deviating from normal, findings in abdominal, pelvic and thoracic organs as well as skeletal findings.

 

In the 1000 mg/kg bw/day dose group red stains around the nose were detected in two animals, this finding was not considered treatment-related, since it was also observed in control animals of previous studies. In addition, 7 dams showed light discoloration of the feces which was attributed to the elimination of test material, a white powder. This finding is not considered to be of toxicological significance. No further general toxicity findings (clinical findings, behavior, appearance mortalities, body weight, gross pathology findings, food and water consumption) were observed. In the 1000 mg/kg bw/day dose group a slightly elevated pre-implantation loss was observed, but the rate was within the normal range of historical control values and therefore not considered related to treatment. At the same dose level total resorption of conceptus was seen in 2 animals. Historical control data indicated that single total resorptions occur by chance. Furthermore a preceding developmental toxicity screening study did not reveal any total resorptions in 20 treated animals even at dose levels of 2000 mg/kg bw/day (1994b). These findings were therefore considered incidental. In the highest dose group significantly elevated placental weights were observed, but only when calculated on an individual basis. Furthermore, a slightly increased number of engorged placentas was determined. However, without a clear dose- response when elevated on a per litter basis and considering that similar incidence rates were also observed in control groups of previous studies, the findings are not considered treatment-related. Except the before mentioned findings, no further maternal effects were observed. Parameters like gestation rate, resorption rate, number of corpora lutea, implantations and preimplantation loss were comparable among the groups.

Analysis of fetuses revealed a slightly, but statistically significantly elevation of fetal weights at 1000 mg/kg bw/day. Since this finding was only observed on an individual but not per litter basis, it was considered incidental. The rate of external, skeletal and visceral malformations was slightly elevated in the 300 mg/kg bw/day group, but not at the next higher dose level of 1000 mg/kg bw/day. Because of missing dose-dependency and the fact that similar malformation rates were seen in control animals of previously performed studies in the same rat strain, it was concluded that neither the type nor the incidence rate and types of malformations indicate a treatment- related, adverse effect on intrauterine development. Also observed skeletal system deviations and retardation at a dose of 1000 mg/kg bw/day were not considered test substance related, since most were only significant on an individual, but not per litter basis, have either also been reported at similar incidence levels in control fetuses of previous studies or did not display a clear dose-response. No effects were observed on other fetal parameters such as number of live offspring, sex ratio, and litter weights. Thus, no effects indicative for maternal toxicity and/or adverse effects on intrauterine development were observed in the rat. The NOAEL for maternal and developmental toxicity was determined to be 1000 mg/kg bw/day. 

 

Further, developmental toxicity was tested in a GLP compliant study in Himalayan rabbits according to OECD 414 (1997r). The test substance formulated in 0.5% tylose was administered to 16 rabbits per dose group orally by gavage at doses of 10, 40, 160 and 640 mg/kg bw/day. The dose levels used in this study were based on a dose range finding study (100, 200, 400, 600 or 1000 mg/kg body weight/day) which was showing severe maternal toxicity at dose levels of 800 and 1000 mg/kg bw/day (1995h). An additional study at a further dose level was performed especially for the evaluation of maternal toxicity in 4 rabbits per dose level (1997s). Doses used in this supporting study were 2.5, 10, 40, 160 and 640 mg/kg bw/day. Since results of both studies were consulted for determination of the no-observed-adverse-effect level in the main developmental toxicity study (1997s), the studies are provided as one study entry and are also discussed together. In both the main developmental toxicity study and the additional study for maternal toxicity animals were treated daily between Day 9 and 19 post mating. At Day 29 of gestation in the main study and Day 20 of gestation in the additional study, the fetuses were delivered by caesarean section.

In the main study examinations included general tolerance of the treatment, appearance, behavior, food and water intake, excretory products, mortality, body weight development and gross pathological investigations. At caesarian section the following endpoints were investigated: number of corpora lutea and implantations, uterus weight, individual weights and appearance of placentas, number of live and dead fetuses or embryos, sex of all live fetuses, individual weights of live fetuses, occurrence of external malformations and other findings deviating from normal, occurrence of findings in abdominal, pelvic and thoracic organs as well as skeletal findings.

In the additional study on maternal toxicity the following parameters were investigated: general tolerance of the test substance, appearance, behavior, feed and water intakes, excretory products, mortality, body weight development, hematology and hemostaseology, clinical chemistry of blood plasma, erythrocytes, liver and brain, gross pathological investigations, organ weights and histopathology.

General toxicity to maternal animals was observed in both the main and the additional study for maternal toxicity. In the main study two females dosed at 640 mg/kg bw/day were killed in moribund conditions. Clinical signs included an increased incidence of females with soft feces at 160 mg/kg bw/day and above in both the main and the additional study on maternal toxicity. Furthermore, light color of the feces was observed in several animals of the 640 mg/kg bw/day dose group in the main study. This finding was considered an indication of impaired digestion rather than being the consequence of test substance excretion. In both the main and the additional study, reduced weight gain was observed at 160 and 640 mg/kg bw/day. In animals that aborted a slight reduction in body weight was even visible at 40 mg/kg bw/day dose group. In line with reduced body weights, also food consumption was decreased in animals of the 40, 160 and 640 mg/kg bw/day dose level that aborted or were killed in moribund conditions. At the highest dose level also females which had viable fetuses, showed a reduced food uptake. In accordance with findings of the main study, one female of the 160 mg/kg bw/day dose group showed severely decreased food intake in the additional study. Beside a decrease in food consumption, decreased water intake was observed in animals that aborted (40, 160 and 640 mg/kg bw/day). Gross pathology showed extreme filling of the urinary bladder and gaseous contents in the intestine only in one female of the 640 mg/kg bw/day which was killed in moribund conditions. Another female of this dose group which aborted showed enlargement of the gallbladder (main study). In the additional study on maternal toxicity, one female of the 160 mg/kg bw/day dose group had a uterine anomaly. These isolated findings were considered to be the consequence of the bad general condition of the animals rather than being an indication of a specific

target toxicity.

 

Parameters additionally investigated for maternal toxicity (1997s) did not reveal treatment-related effects on hematology and hemostaseology at levels up to and including 640 mg/kg bw/day. Analysis of clinical biochemistry revealed, a reduction of acetylcholinesterase activity at levels of 10 mg/kg bw/day and above in erythrocytes and in plasma at levels of 40 mg/kg bw/day and above. Only in the brain no significant inhibition was observed. In the plasma, the activity of gamma-glutamyltransferase (day 13 and 20 p.c.) and alanine-aminotransferase (day 20 p.c.) were increased. In addition, both in the plasma and the liver, the mean triglyceride content was increased in the 160 and 640 mg/kg bw/day dose groups. In line with these signs of liver toxicity, organ weight analysis revealed slightly increased absolute and relative liver weight in animals of the highest dose group. Furthermore, histopathology revealed hepatocellular hypertrophy in two animals dosed at 40 mg/kg and all animals dosed 160 mg/kg and above. Furthermore, hepatocellular cytoplasmic change consisting of a ground glass-like appearance of the perinuclear cytoplasm was found in all animals dosed at 40 mg/kg or above. At the 640 mg/kg bw/day dose level minimal or moderate edema of the gall bladder were observed. In one animal the finding was accompanied by mixed cellular infiltration of the gall bladder wall and mononuclear interstitial infiltration of the liver parenchyma. In the other animal the minimal edema was combined with minimal mononuclear interstitial infiltration and slight multicellular necroses of the liver parenchyma. These findings were considered to be most probably due to infectious origin and were therefore regarded to be unrelated to treatment.

 

In the main study different signs of maternal developmental toxicity occurred. One abortion was observed at 40 mg/kg bw/day dose level, in two females at the 160 mg/kg bw/day dose level and in three females at the 640 mg/kg bw/day dose level. Although single abortions may occur in this strain of rabbits, the observed dose dependent increase in abortions indicates a potential treatment-related effect at dose levels of 40 mg/kg bw/day and above. The animals of the 10 mg/kg group did not reveal any treatment related effects and no abortions occured. As all animals with abortions revealed distinct systemic effects for at least eight days before abortion the abortions are considered to be caused by maternal toxicity rather than being an indication of specific developmental toxicity. Total litter losses occurred in one female of the 160 mg/kg bw/day dose group and one female of the 640 mg/kg bw/day dose group. In both animals maternal toxicity, impaired digestion including diarrhea or severely impaired general conditions were observed. Therefore, a specific developmental effect is not assumed for these two isolated cases of total resorptions. Due to the abortions and the total resorptions, the gestation rate was decreased to 93.4%, 81.3% and 73.3% in the 40, 160 and 640 mg/kg bw/day dose group, respectively. No pre- and post-implantation loss was observed in the main study. In the additional study one female without implantation sites was observed at the 640 mg/kg be/day dose level. However, since this finding was only limited to one animal and similar findings were not observed in the main study at the same dose, the effect was considered non-treatment-related. Two early resorptions were observed in the main study, one in the control group and another one in the 160 mg/kg dose group. Late resorptions were observed in all dose groups and in the control group. Since dose dependency was missing and early and late resorptions were also observed in the control group, the findings were not considered treatment-related. In both the main and the additional study, dead fetuses were not observed. Further, a reduction in number of dams with live offspring was observed in the 160 and 640 mg/kg bw/day dose group.

External malformations, acrania was only observed in one fetus of the 640 mg/kg bw/day dose group in the additional study on maternal toxicity and was therefore considered incidentally. Skeletal malformations, arthrogryposis occurred in all dose and the control groups in the main study, since dose-dependency was missing the findings were not considered treatment-related. Isolated findings in the 10 mg/kg bw/day dose group were malposition of hind limb in one fetus and supernumerary lumbar in another fetus. Supernumerary lumbar vertebra was observed in one fetus of the 40 mg/kg bw/day dose group. Supernumerary lumbar vertebra and missing phalanges/metacarpal were observed in 1 fetus of the control group. Since skeletal effects were not observed in the additional study and observed findings were isolated and not dose dependent, a treatment-related effect was not considered. Isolated visceral malformations were a cardiac septum defect in one fetus of the 160 mg/kg bw/day dose group in the main study and spina bifida in one fetus of the 640 mg/kg bw/day dose group in the additional study. Since findings were isolated, they were not considered related to test substance administration. Other parameters, the number of fetuses, fetal body weight, changes in sex ratio, litter size and weights and external appearance of placentas were unaffected by the treatment at levels up to and including 640 mg/kg body weight/day.

 

Based on maternal findings of both the main developmental and the additional study on maternal toxicity, only at the lowest dose level of 2.5 mg/kg bw/day no signs of maternal toxicity were observed, while doses of 10 mg/kg bw/day and above were maternally toxic. Signs of maternal toxicity included inhibition of acetylcholinesterase at 10 mg/kg bw/day and at higher dose levels also (160 and 640 mg/kg bw/day) liver toxicity, effects on body weight, feed and water intakes. Based on fetal analysis a NOAEL of 640 mg/kg bw/day was determined and a teratogenic potential was not evident.

 

Taken together results of the main developmental toxicity study and the additional study on maternal toxicity, signs of general maternal toxicity were observed at dose levels of 10 mg/kg bw/day and above. Developmental effects, abortions and total litter loss were observed at a dose of 40 mg/kg bw/day and above. Since systemic signs of toxicity occurred in all animals with abortions and total resorptions, these findings were considered to be caused by maternal toxicity rather than being an indication of specific developmental toxicity.

 

Further, as supporting data, a teratology study similar to OECD 414 was performed in pregnant Sprague-Dawley rats (1994b) which was designed as a preliminary study for the main teratology study (1997q). The test substance (suspended in 2% Cremophor ELsolution) was administered orally by gavage at a dose of 500, 1000 and 2000 mg/kg bw/day once daily from day 6 to day 15 of gestation. Control animals were administered 2% Cremophor EL solution, only. The number of animals differed in the treatment and control groups and was as follows: 10 control animals, 5 animals dosed at 500 mg/kg bw/day and 16 animals dosed at 1000 mg/kg bw/day, in an additional examination 5 contol animals and 20 animals dosed at 2000 mg/kg bw/day were used.

Examinations in dams included clinical findings, body weight and food consumption. At day 20 of gestation, caesarean section was carried out for analyses of corpora lutea, number of implantations, number of embryonic deaths, number of live fetuses, sex ratio, live fetal weights and placental weights. Fetuses were examined for external, visceral and skeletal changes.

In maternal animals no clinical signs, behavior, mortalities, changes in body weight, food consumption or organ weights were observed. Gross pathology revealed a rough surface of the liver and a yellowish kidney in one animal of the 2000 mg/kg bw/day dose group at caesarian section day 20 of gestation, but this finding was considered incidental as only 1 animal was affected. All animals were pregnant and abortions, post implantation loss, dead fetuses, abnormal number of live fetuses or sex ratio were not observed at any dose level. Numbers of corpora lutea, pre-implantaion loss and early or late resorptions did not differ significantly between dose and control groups.

At 2000 mg/kg bw/day an increase in placental weights was noticed. Furthermore a significant increase in fetal weight was observed in females at 2000 mg/kg bw/day. In other dose groups no alterations of fetal weights were observed. External examinations of live fetuses revealed absence of tail in one fetus of the control group and one fetus with a club foot at the 2000 mg/kg bw/day dose group. Since the incidence of these findings was very low, and a statistically significant increase was not observed, these effects were considered incidental.

Skeletal malformations were not observed in any dose group. However, a significant increase of skeletal variations was observed at the 1000 mg/kg bw/day dose level, but not at the 2000 mg/kg bw/day dose level. The incidence of fetuses with variations was 57.2% (60/104 fetuses) at 1000 mg/kg bw/day. Dumb-bell shaped thoracic vertebral body was observed at high frequency and the incidence was significantly increased at the dose level of 1000 mg/kg bw/day. Furthermore, retarded ossification of hyoid, dumb-bell shaped sternebrae body, asymmetrical sternebrae body and retarded ossification of pups, was observed at relatively high frequency at this dose. In the 2000 mg/kg bw/day dose group cervical rib, lumber rib and shortening of 13th ribs were sporadically observed, but the incidence of these variations was statistically not significant. Changes suggesting developmental retardation, the number of ossified sternebrae body, metacarpal, metatar sals and sacro-caudal vertebral body as indicators of fetal development were not observed.

At a dose level of 1000 mg/kg bw/day, visceral malformations, microphthalmia, abnormal aorta accompanied with right aortic arch and vascular ring and the right subclavian artery were observed in 3/117 (11.7%) fetuses. However these findings were not statistically significant and visceral malformations, microphthalmia and dilatation of the ventricles of the brain were also observed in one fetus of the control group. At the next higher dose level of 2000 mg/kg bw/day such findings were not observed. Therefore, these findings were considered to be incidental and not test substance-related. Visceral variations, a persistence of the thymus in the lateral side of the thyroid, dysplasia of liver and lung, dilatation of the renal pelvis and existence of the umbilical artery in the left side of the bladder were sporadically observed in some fetuses of all test groups. These findings were statistically not significant and the incidence was not clearly dose-related. Thus, these findings were not attributed to the test substance administration.

In conclusion, a remarkable effect of test substance on maternal animals was not observed up to a dose of 2000 mg/kg bw/day. External malformations and visceral findings were not considered treatment-related. Skeletal malformations were not detectable; however a possibility of an increase of skeletal variations could not be excluded.

 

Overall conclusion on developmental toxicity

 

Prenatal developmental toxicity was evaluated in rats and rabbits. Based on the available data, no effects indicative for maternal toxicity and/or adverse effects on prenatal development were observed in the rat up to and including 1000 mg/kg bw/day. In contrast, maternal toxicity was evident in rabbits starting at 10 mg/kg bw/day. Signs of maternal toxicity included inhibition of acetylcholinesterase at 10 mg/kg bw/day and liver toxicity, effects on body weight, feed and water intakes at higher dose levels. Developmental effects, abortions and total litter loss were observed starting at 40 mg/kg bw/day. Since systemic signs of toxicity occurred in all rabbits with abortions and total resorptions, these findings were considered to be caused by maternal toxicity rather than being an indication of specific developmental toxicity. Thus, in prenatal developmental toxicity studies, no concern for in utero development arose.

In the One- and Two-Generation studies performed in rats, reduced live birth and reduced viability indices and/or reduced lactation index and reduced mean litter size were observed at the highest dose level (1800 ppm and/or 3200 ppm in the Two-Generation sudy and One-Generation study, respectively). Moreover, depressed body weight and weight gain of the offspring was evident during the lactation periods At the same dose levels, an inhibition of cholinesterase activity and adaptive liver alterations were observed in the maternal animals. Thus, adverse effects indicating developmental and/or lactational effects were observed at dose levels which in parallel induced adverse effects in maternal animals. Taken together, signs of general maternal toxicity indicated by cholinesterase inhibition and developmental effects occurred at the same dose levels.Thus, effects observed in the offspring of treated maternal animals were considered to be caused by maternal toxicity rather than being an indication of specific developmental toxicity.

In conclusion, the test substance does not meet the classification criteria for reproductive/developmental toxicity according to Regulation EC No 1272/2008.

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Justification for classification or non-classification

No adverse effects on fertility have been observed in the available studies on reproduction. Moreover, effects observed in the offspring are considered to be caused by maternal toxicity indicated by a tremendous inhibition of cholinesterase activation.

In conclusion, the test substance does not meet the classification criteria for reproductive/developmental toxicity according to Regulation EC No 1272/2008.

 

Additional information