4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1985

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

EC Number:

605-140-1

Cas Number:

158237-07-1

Molecular formula:

C16H20ClN5O2

IUPAC Name:

4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-1,2,3,4-tetrazole-1-carboxamide

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

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)

Doses / concentrations 2

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)

Doses / concentrations 3

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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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".

Details on results (P0)

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.

Effect levels (P0)

1

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

2

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

3

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

4

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

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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".

Effect levels (P1)

1

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

2

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

3

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

4

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

5

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

Target system / organ toxicity (P1)

1

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

2

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

3

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

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

2

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

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

No significant clinical findings were made in F2 pups during the four week lactation period at levels of up to 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.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Description (incidence and severity):

no data

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Description (incidence and severity):

no data

Effect levels (F2)

1

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

2

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

Overall reproductive toxicity

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

Any other information on results incl. tables

   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 Observations^1)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.

Applicant's summary and conclusion

Conclusions:

CLP: not classified