Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Nov 1987 - 17 Feb 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
other: Amendment
Title:
Unnamed
Year:
1990
Report date:
1990
Reference Type:
other: Amendment
Title:
Unnamed
Year:
1992
Report date:
1992
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted 2001
Deviations:
yes
Remarks:
Please refer to "Principles of method if other then guideline"
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted 1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Version / remarks:
adopted 1984
Deviations:
no
Principles of method if other than guideline:
The study differs from the actual version of the OECD test guideline in the following points: oestrus cycle as well as sperm parameters were not evaluated, pairs without progeny were not evaluated to determine cause of infertility, age of vaginal opening and preputial separation for F1 weanings selected for mating was not recorded, data on physical landmarks in pups and other postnatal developmental data were not recorded, uteri of primiparous females were not examined for presence of implantation sites, brains of parental animals and offspring were not weighed, spleen and thymus of offspring were not weighed.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
428-040-8
EC Name:
-
Cas Number:
138261-41-3
Molecular formula:
C9H10ClN5O2
IUPAC Name:
2-chloro-5-{[2-(nitroimino)imidazolidin-1-yl]methyl}pyridine

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Common strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: KFM, Kleintierfarm Madoerin AG, Fuellinsdorf, Switzerland
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5 weeks; (F1) 3 weeks
- Weight at study initiation: (P) Males: 123-169 g; Females: 81-137 g; (F1) Males: 176-298 g; Females: 132-213 g
- Fasting period before study: not applicable
- Housing: individually in Makrolon type-3 cages with wire mesh tops and standard granulated, softwood bedding (Lignocel, Schill AG, Muttenz, Switzerland), cohoused during pairing
- Diet: pelleted standard Kliba 343, rat/mouse maintenance diet (Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 Oct 1987 To: 17 Feb 1989

Administration / exposure

Route of administration:
oral: feed
Mass median aerodynamic diameter (MMAD):
other: not applicable
Remarks on MMAD:
not applicable
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: at least every 2 weeks
- Mixing appropriate amounts with granulated food: The test article was mixed to granulated food in a Buehler Mixer, type DDMA-0.5 and pelleted in a Buehler pelleting machine type DFPL (Buehler Ltd., Uzwil, Switzerland). Water was added to each feed preparation at an approximately 1:10 volume/weight ratio to ensure pelleting, after which the pellets were dried with warm air for 48-96 hours before storage.
- Storage temperature of food: room temperatue in the dark
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 22 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 post coitum
- After 22 days of unsuccessful pairing replacement of first male by another male.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the test article concentration, its homogeneity and stability in the food pellets was determined before starting the study with the dose finding study using HPLC.
The content and homogeneity of the test article in the food pellets was determined at start of the prepairing periods, mating and at the end of gestation/start of lactation periods. The test substance was stable for 3 weeks in the diet. The overall mean concentrations were 98.1 %, 97.1 % and 97.7 % of the nominal concentration for the 100 ppm, 250 ppm and 700 ppm dose group, respectively. The homogeneity varied in the range of -10 % to +9 % of the analyzed feed batches.
Duration of treatment / exposure:
P generation
during a 84-day prepairing period and also during the pairing-, gestation- and lactation periods for breeding the F1A and F1B litters.

F1 animals
starting at 7 or 8 weeks of age, during their growth into adulthood (105-days prepairing period) and also during the pairing-, gestation- and lactation periods for breeding the F2A and F2B litters
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
- F1 parental animals not mated until 18 weeks after selected from the F1 litters (after 105 days of feeding with test substance)
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 17 weeks (P generation for F1A litters), 28 weeks (P generaion for F1B litters), 22 weeks (F1 parents for F2A litters), ca 28-31 weeks (F1 parents for F2B litters)
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm (nominal)
Remarks:
corresponding to actual dose ingested:
5-13 mg/kg bw/day for P generation males (P0),
6-13 mg/kg bw/day for P generation females (P0) during prepairing and gestation,
10-18 mg/kg bw/day for P generation females (P0) during lactation,
5-9 mg/kg bw/day for F1 parental males (P1),
6-9 mg/kg bw/day for F1 parental females (P1) during prepairing and gestation,
11-18 mg/kg bw/day for F1 parental females (P1) during lactation
Dose / conc.:
250 ppm (nominal)
Remarks:
corresponding to actual dose ingested:
12-32 mg/kg bw/day for P generation males (P0),
16-32 mg/kg bw/day for P generation females (P0) during prepairing and gestation,
25-46 mg/kg bw/day for P generation females (P0) during lactation,
12-23 mg/kg bw/day for F1 parental males (P1),
16-24 mg/kg bw/day for F1 parental females (P1) during prepairing and gestation,
26-42 mg/kg bw/day for F1 parental females (P1) during lactation
Dose / conc.:
700 ppm (nominal)
Remarks:
corresponding to actual dose ingested:
35-89 mg/kg bw/day for P generation males (P0),
44-96 mg/kg bw/day for P generation females (P0) during prepairing and gestation,
73-121 mg/kg bw/day for P generation females (P0) during lactation,
36-68 mg/kg bw/day for F1 parental males (P1),
41-68 mg/kg bw/day for F1 parental females (P1) during prepairing and gestation,
78-120 mg/kg bw/day for F1 parental females (P1) during lactation
No. of animals per sex per dose:
30 (P generation (P0))
26 (F1 parental animals (P1))
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected based on a previous range finding experiment (M-027080-01-2). There, 10 male and 10 female rats were fed with 0, 20, 100 or 500 ppm of the test substance in pelleted diet. Administration was continued for a three-week prepairing period and throughout the pairing, gestation and lactation periods. Pups of the F1 generation were reared for a further week on the respective test diet after weaning (from post partum Day 21). It was shown that at 500 ppm, P females had a reduced body weight gain during the preparing period and consequently decreased mean body weights during the following gestation and lactation periods, they also consumed less food than control animals during the lactation period. In the F1 generation, decreased mean body weight on day 0 post partum and throughout the lactation and rearing period were observed in the 500 ppm group as well as reduced food consumption during the rearing period (days 21 - 28 post partum). Therefore, the NOEL of the test substance was set to 100 ppm and the dose rationale for the main study was set to 100, 250 and 700 ppm.
- Fasting period before blood sampling for clinical biochemistry: no
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
Animals were examined for clinical signs of reaction related to the treatment, affecting behaviour and general appearance.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (with exception of pairing periods), after mating on days 0, 7, 14, and 21 and on days 0, 4, 7, 14 and 21 post partum

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
- Time schedule: weekly (with exception of pairing periods). During the lactation periods, food consumption was recorded only till day 14 post partum


OTHER:
Blood samples for hematology and clinical biochemistry were collected from 10 randomly selected animals per group and sex of the F1 generation prior to necropsy. Parameters checked were erythrocyte count (RBC), hemoglobin (HB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, reticulocyte count (retic.), nucleated erythrocytes normoblasts (NEN), total leucocyte count (WBC), differential leukocyte count (diif. WBC count, analyzed for band neutrophils, segmented neutrophils, eosinophils, basophils, lymphocytes, monocytes, plasma cells, blast cells), red cell morphology, thromboplastin time (PT), partial thromboplastin time (PTT), glucose, urea, creatinine, bilirubin total (Bili-T), total lipids, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (ALP), gamma-gutamyl-transferase (GGT), calcium, phosphorus, sodium, potassium, chloride, total protein, protein electrophoresis

Samples of liver were taken from all animals mentioned above at necropsy for the assay of triglycerides, cytochrome P-450 and N-and 0-demethylase activity.
Oestrous cyclicity (parental animals):
Mating data were considered for detection of anomalies affecting the estrus cycle of the females.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
- males that failed to induce pregnancy: testis weight, epididymides weight, histopathological examination of testes, epididymides, prostate and seminal vesicles
- all animals of control and high dose group: histopathological examination of testes, epididymides, prostate and seminal vesicles

F1 parent animals:
- males that failed to induce pregnancy: testis weight, epididymides weight, histopathological examination of testes, epididymides, prostate and seminal vesicles
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities, weight gain (F1A and F1B pups were weighed individually on days 0, 4, 7, 14 and 21 of lactation. F2A and F2B pups were weighed individually on days 0 (if possible) and/or 1, 4, 7, 14 and 21 of lactation)

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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed, exact time point is not reported
- Maternal animals: All surviving animals where sacrificed after the weaning of the F1B litter

GROSS NECROPSY
- Gross necropsy consisted of all gross lesions, liver, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, thyroid gland, testes with epididymides, uterus and cervix, vagina

HISTOPATHOLOGY / ORGAN WEIGHTS
- tissues prepared for microscopic examination and weighed, respectively: liver, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, thyroid gland, testes with epididymides, uterus and cervix, vagina
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations as follows: all pups were examined macroscopically, from one male and one female per litter specified organs were weighed and tissues preserved for histopathological examination

GROSS NECROPSY
- Gross necropsy consisted of all gross lesions, liver, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, thyroid gland, testes with epididymides, uterus and cervix, vagina

HISTOPATHOLOGY / ORGAN WEIGHTS
- tissues prepared for microscopic examination and weighed, respectively: liver, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, thyroid gland, testes with epididymides, uterus and cervix, vagina
Statistics:
If the variable could be assumed to follow a normal distribution: Univariate one-way analysis of variance was used.
If significance was noted, Dunnett many-one t-test was used for inter-group comparison.
For reproduction parameters, An one-way univariate analysis of variance based on Wilcoxon ranks together with the Kruskall-Wallis test was applied.
For the spontaneous mortality of pups data, Fisher's Exact test for 2x2 tables was used.
Reproductive indices:
mean precoital time, fertility index, conception rate and gestation index
Offspring viability indices:
not reported

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
incidental findings were observed, mainly hair loss and wounds but no dose-relationship was noticed and findings were occasional. Therefore, they were considered incidental.
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- Control: 1 female was found dead on Day 38 of the preparing period and 1 female was killed for humane reasons on day 21 post coitum breeding for F1B litters because of poor condition.
- 100 ppm: 1 female was found dead on Day 36 of the preparing period.

Since no dose-response was observable, these deaths were considered incidental.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males
- 700 ppm: reduced mean body weight in the preparing period compared to controls (from Day 8 on, from -5.2 to -9.1%), the body weight gain was similar to controls during post-paring period but the significantly lower mean body weights remained.

Females
Prepairing period for breeding F1A litters
- 100 ppm: statistically significant increased body weight to controls on Day 1 (+4.5%), considered incidental
- 250 ppm: statistically significant increased body weight to controls on Day 1 (+4.5%), considered incidental
- 700 ppm: reduced body weight to controls throughout the prepairing period with statistical significance on days 29, 36, 43, 57, 71 and 78 (from -5.5 to -7.0%)

Gestation period for breeding F1A litters
- 700 ppm: body weight remained lower than controls (statistically significant up to Day 14, from -6.9 to -7.0%) but body weight gain was similar to control animals, therefore this was not considered related to treatment.

Lactation period for breeding F1A litters
- 700 ppm: statistically significant reduced body weight compared to controls until Day 7 (from -6.9 to -7.2%)

Gestation period for breeding F1B litters
- 700 ppm: statistically significant reduced body weight compared to controls throughout gestation period (from -4.9 to -5.8%) but body weight gain similar to controls (42.5 vs 42.9% in controls)

Lactation period for breeding F1B litters
- 700 ppm: statistically significant lower mean body weight at Day 0 (-5.5%), increased body weight gain compared to controls

Summarized data can be found in Table 1 (Attachment 1) in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
- 700 ppm: reduced food consumption during preparing period for F1A litters (statistically significant compared to controls for days days 8-15 (-5.7%), 22-57 (from -6.3 to -9.5%) and 64 - 78 (from -5.7 to -6.0%)

Females
Prepairing period for breeding F1A litters
- 700 ppm: reduced compared to controls from Day 8 to Day 84 (statistically significant different to controls from Days 22-43 and 64-71 with reduction from -7.7 to -8.8 and -7.1, respectively)

Gestation period for breeding F1A litters
- 700 ppm: statistically significant reduced food consumption throughout the gestation period (between -7.5 and -8.0%) compared to controls

Lactation period for breeding F1A litters
- 700 ppm: reduced food consumption throughout the lactation period (statistically significant from Day 4 to Day 14 with reduction from -10.4 and -11.5%) compared to controls

Gestation period for breeding F1B litters
- 700 ppm: statistically significant reduced food consumption throughout the gestation period (between -7.7 and -12.1%) compared to controls

Lactation period for breeding F1B litters
- 700 ppm: reduced food consumption throughout the lactation period (statistically significant from Day 7 to Day 14 with reduction from -9.3%)

Summarized data can be found in Table 2 (Attachment 2) within the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test article related changes were noted at the histological examination of the coagulating glands, epididymides, liver, ovaries, pituitary glands, prostate gland, seminal vesicles, testes, thyroid glands, uterus and vagina in control and high dose animals.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
not applicable
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
not applicable
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance, fertility, duration of gestation, litter size at first litter check, postnatal loss from Day 0 to Day 4 post partum and breeding loss from Day 4 to Day 21 post partum were similar throughout all groups including the control.

Non treatment related findings were
- 700 ppm: statistical significant more pubs per dam in F1B litter but within the historical control range, one female lost its entire F1B litter between days 6 and 12 of the lactation period (considered incidental)

There was no evidence of an adverse effect of treatment with on any of the reproduction indices: mean precoital time, fertility index, conception rate and gestation index.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this concentration
Remarks on result:
other: corresponding to 20 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
700 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: corresponding to 50 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 700 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest concentration tested
Remarks on result:
other: corresponding to 40 mg/kg bw/day

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of reaction related to treatment.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- 100 ppm: one male died after blood sampling at the end of the study (no abnormal findings were noted at necropsy).
- 250 ppm: one male was killed for human reasons on day 14 of the post-pairing period for breeding F2B litters because of poor condition.

These deaths were considered incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males
- 700 ppm: since body weight was reduced in pups of this group at weaning, mean body weight was reduced between Days 1 and 22 of the prepairing period but increased thereafter. Body weight gain was slightly higher increased compared to controls during the prepairing period or similar during the post-pairing period compared to controls.

Therefore, the findings were considered incidental.

Females
Prepairing period for breeding F2A litters
- 700 ppm: body weight was reduced throughout the prepairing period compared to controls (from -7.5 to -9.3%)

Gestation period for breeding F2A litters
- 700 ppm: reduced mean body weight was continued and statistically significant throughout the gestation period compared to controls (from -10.0 to -10.2%) but body weight change was similar to control group.

Lactation period for breeding F2A litters
- 700 ppm: reduced mean body weight was continued and statistically significant throughout the gestation period compared to controls (from -6.8 to -11.2%) but body weight change was similar to control group.

Gestation period for breeding F2B litters
- 700 ppm: reduced mean body weight was continued and statistically significant throughout the gestation period compared to controls (from -7.9 to -9.6%) but body weight change was similar to control group.

Lactation period for breeding F2B litters
- 700 ppm: reduced mean body weight was continued and statistically significant throughout the gestation period (from -5.7 to -10.9%) but body weight change was similar to control group.

The reduced body weight of females was considered a secondary effect of the lower body weights noted from the F1B pups compared to controls at weaning and therefore not directly related to treatment with the test article. Summarized Data can be found in Table 3 (Attachment 3) within the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males
-100 ppm: food consumption was higher than in controls, statistical significance was found for the period between Day 1-8 of the preparing period (+6.7%) but considered incidental
- 700 ppm: slightly increased relative food consumption compared to control group during some weeks of the prepairing period but considered incidental.

Females
Prepairing period for breeding F2A litters
- 700 ppm: reduced statistically significant compared to controls from Days 15-29 (from -7.7 to -11.8%) and 57-105 (from -5.4 to -16.0%)

Gestation period for breeding F2A litters
- 700 ppm: statistically significant reduced food consumption compared to controls throughout the gestation period (between -11.0 and -13.8%)

Lactation period for breeding F2A litters
- 100 ppm: slightly increased food consumption (between +4.3 and +7.0%) but not statistically significant compared to controls
- 700 ppm: slightly reduced food consumption from Day 1 to 14 (from -2.6 to -6.6%) but without statistical significance compared to controls

Gestation period for breeding F2B litters
- 250 ppm: reduced food consumption compared to controls from Day 0-7 (-7.8%) and Day 7-14 (-6.8%)
- 700 ppm: statistically significant reduced food consumption compared to controls throughout the gestation period (between -10.7 and -13.7%)

Lactation period for breeding F2B litters
- 250 ppm: reduced food consumption but without statistical significance compared to controls (from -4.0 to -8.4%)
- 700 ppm: reduced food consumption but statistical significance compared to controls only from Day 7-14 (-9.5%)

The findings for 250 ppm were considered incidental because they were probably mostly caused by the slightly reduced mean number of pups noted in this group.

Summarized data can be found in Table 4 (Attachment 4) within the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
no effects observed
Description (incidence and severity):
All statistical differences in the results of the haematology parameters were considered to be incidental and of normal variation for rats of this strain and age.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 250 ppm: slight but statistical significant increase in O-demethylase activity compared to controls (23% in females, seen as adaptive proicess as described below)
- 700 ppm: slight but statistical significant increase in hepatic Cytochrome P-450 content (26%, males) and N-demethylase activity (18%, males), slight increase in O-demethylase activity (36% males/37%, females), all compared to controls

These findings may indicate a slight stimulation of mixed-function oxidase activities. However, other markers for liver toxicity were not observed (namely liver-derived plasma enzymes, plasma bilirubin, liver triglycerides or any effect on liver morphology).

Summarized data can be found in Table 5 in Attachment 5.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- 700 ppm: statistically reduced absolute ovary weights (-13.5%) compared to controls. However, the ovaries did not show any other abnormal macroscopical or histopathological changes and the relative ovary weights were similar to those of the control females. Thus, this finding was considered to be a secondary effect of the reduced mean body weight and not considered directly treatment related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item related macroscopic findings were observed in any of the treatment groups.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test article related changes were noted at the histological examination of the coagulating glands, epididymides, liver, ovaries, pituitary glands, prostate gland, seminal vesicles, testes, thyroid glands, uterus and vagina in control and high dose animals.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
not applicable
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
not applicable
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating performance, fertility, duration of gestation, litter size at first litter check, postnatal loss from Day 0 to Day 4 post partum and breeding loss from Day 4 to Day 21 post partum were similar throughout all groups including the control.

Non treatment related findings were
- 250 ppm: breeding loss in F2A litters from day 4 to day 21 post partum was statistically increased (4.7% vs 0.7% in controls). This was considered incidental and within the normal range for this strain of rat. In F2B litters, the litter size was slightly reduced (9.2 vs 10.8 in controls) but no dose relationship was found.

There was no evidence of an adverse effect of treatment on any of the following reproduction indices: mean precoital time, fertility index, conception rate and gestation index.

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
250 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to this concentration
Remarks on result:
other: corresponding to 20 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
700 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: corresponding to 50 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 700 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest concentration tested
Remarks on result:
other: corresponding to 40 mg/kg bw/day

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
not applicable
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- Control: one F1A pub was found dead after parturition and showed cheiloschisis.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 700 ppm: in F1A litters, the body weight gain was reduced during the lactation period compared to controls, this was statistically significant from Day 4, resulting in reduced mean body body weight at Day 21(-13.4%). In F2A litters, the body weight gain was reduced during the lactation period, this was statistically significant compared to controls from Day 7, resulting in reduced mean body weight at Day 21(-9.6%) compared to controls.

Summarized data can be found in Attachmend 6.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable because only evaluated in F1 parental animals (=P1)
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable because only evaluated in F1 parental animals (=P1)
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Sexual maturation:
not examined
Description (incidence and severity):
not applicable
Anogenital distance (AGD):
not examined
Description (incidence and severity):
not applicable
Nipple retention in male pups:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- 700 ppm: absolute weights of the liver was reduced in F1A (-17.3% males, -14.6% females) and F1B (12.9% males, -8.6% females) pups compared to controls, absolute weights of testes or ovaries were reduced in F1A pups (-12% males, -25% females), testes/body weight ratio was increased in male F1B pups (+4.5%) compared to controls.

These findings were considered incidental because they were not seen in the other generation and showed no correlation with the reduced mean body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item related macroscopic findings were observed in any of the treatment groups.
Histopathological findings:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined
Description (incidence and severity):
not applicable

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest concentration tested
Remarks on result:
other: corresponding to ca. 40 mg/kg bw/day (during lactation)
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
700 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: corresponding to 100 mg/kg bw/day (during lactation)

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- 250 ppm: one female pup showed a stumped tail at external examination at first litter check.
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
not applicable
Mortality / viability:
no mortality observed
Description (incidence and severity):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 700 ppm: in F2A litters, the body weight gain was reduced compared to controls during the lactation period, this was statistically significant from Day 7, resulting in reduced mean body body weight at Day 21(-9%). In F2B litters, the body weight gain was reduced throughout the lactation period, this was statistically significant compared to controls from Day 7, resulting in reduced mean body body weight at Day 21(-13.4%).

Summarized data can be found in Attachment 7.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Sexual maturation:
not examined
Description (incidence and severity):
not applicable
Anogenital distance (AGD):
not examined
Description (incidence and severity):
not applicable
Nipple retention in male pups:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- 250 ppm: increase of the absolute and relative weight of the ovaries in F2A litter (23.5% and 23.0%, respectively) as compared to controls.
- 700 ppm: decreased liver weight in F2B males (-10.3%) but similar liver/body weight ratio as in control groups


The increased ovary weight was considered incidental since no dose-relationship was observed and the finding was not seen in the F2B litter. The decreased liver weight is considered a result of the reduced body weight and therefore not directly treatment related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test article realted findings were observed. In the pups that were killed 4 days post partum, occasional findings included reduced kidney weight, increased kidney size, agnesia of the left testis and worms in the intestine. These findings were spred through all treatment and control groups, no dose-relationship was found and they were therefore considered incidental.
Histopathological findings:
no effects observed
Description (incidence and severity):
No test article related changes were noted at the histological examination of the coagulating glands, epididymides, liver, ovaries, pituitary glands, prostate gland, seminal vesicles, testes, thyroid glands, uterus and vagina in control and high dose animals.
Other effects:
not examined
Description (incidence and severity):
not applicable

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined
Description (incidence and severity):
not applicable

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
250 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this concentration
Remarks on result:
other: corresponding to 40 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Generation:
F2
Effect level:
700 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: corresponding to 100 mg/kg bw/day

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
To assess reproductive toxicity, a 2-generation study was conducted according to OECD TG 416 with male and female rats. The test item was offered to the animals via feed, at doses of 100, 250 and 700 ppm. No treatment-related mortality occurred. As compared to the untreated controls, reduced food consumption was observed for P0 and P1 animals that received 700 ppm of the test substance during the prepairing period. For females, reduced food consumption was also observed at 700 ppm during all gestation and lactation periods (P0 and P1 animals). Consequently, animals of the 700 ppm group had reduced body weight. The body weight gain was especially reduced during the prepairing period of P0 animals (male and female), after that, it was similar to controls but body weight remained low because of the established weight difference. In all offspring generations of the 700 ppm dose group (F1A, F1B, F2A and F2B), reduced body weight gain was observed. In the P1 generation, increased O-demethylase activity in the liver was observed for females receiving 250 ppm test substance and both sexes of the 700 ppm group. Also, hepatic N-demethylase activity and hepatic cytochrome P-450 content was increased in males at 700 ppm. This indicates increased xenobiotic metabolism but no further evidence of liver toxicity was found. External examination of pups revealed no obvious abnormalities indicative of teratogenicity in any group of either generation. Therefore, the NOAEL for parental toxicity can be set at 250 ppm for male and female rats under the conditions of the study. The NAOEL for fertility was >= 700 ppm, since no alteration of reproduction parameters was observed and the NOAEL for developmental toxicity was set at 250 ppm.

The study was conducted according to GLP and in accordance with the OECD Guideline 416 in the version of 1981.