2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridine carboxylate; imazapyr (ISO)

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-09-03 to 1986-09-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

estrous cycle was not evaluated; Sperm parameters were not examined; Organ weights were not determined for P0 and P1 parental animals

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: AC 4866-062

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in an airtight container at room temperature and in a dark, cool and dry place
- Stability under test conditions: stable (please refer to analytical verification)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CD-CRL:COBS CD (SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) ca. 6 wks; (F1) ca. 14 wks
- Weight at study initiation: (P) Males: 187 - 240 g; Females: 128 - 166 g; (F1b) Males: 118 - 127 g; Females 105 - 111 g
- Fasting period before study: No
- Housing: individually in stainless steel cages (except mating period)
- Diet: ad libitum (Certified Purina Rodent Chow (No. 5002))
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: basal diet
- Storage temperature of food: at room temperature

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 6 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: Two litters were produced for each parental generation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 20 g portions of each rodent meal sample were assayed by HPLC Method M-1465 described in another report. This method is validated in rodent meal for concentrations from 50 to 20000 ppm.
Test substance concentrations in diet after 7 and 14 days in feeders exposed in the rat room were 991 SD 47 ppm and 902 SD 18 ppm (99.1 % CV 4.7 % and 90.2 % CV 2.0 % of nominal) for the 1000 ppm batch. Corresponding values at 10000 ppm were 10153 SD 194 ppm and 9122 SD 249 ppm (101.5 % CV 1.9 % and 91.2 % CV 2.7 % of nominal). Adequate stability was concluded.

Duration of treatment / exposure:

P0 generation: treated for 64 days prior to placement for mating, throughout the two mating periods and until necropsy, approximately 3 weeks after the end of the second mating period (males). The females were also treated throughout gestation and lactation.

P1b generation: For at least 78 days prior to mating. The treatment of the P1b generation adult females was continued throughout the mating, gestation and lactation periods. The P1b generation adult males were treated until necropsy, approximately 4 weeks after the end of the second mating period.

Frequency of treatment:

daily

Details on study schedule:

- P1 parental animals not mated until ca. 11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

84.25 mg/kg bw/day (nominal)

Remarks:

P0 generation: corresponding to 1000 ppm

Doses / concentrations 2

Dose / conc.:

425.85 mg/kg bw/day (nominal)

Remarks:

P0 generation: corresponding to 5000 ppm

Doses / concentrations 3

Dose / conc.:

835.65 mg/kg bw/day (nominal)

Remarks:

P0 generation: corresponding to 10000 ppm

Doses / concentrations 4

Dose / conc.:

92.9 mg/kg bw/day (nominal)

Remarks:

P1b generation: corresponding to 1000 ppm

Doses / concentrations 5

Dose / conc.:

466.85 mg/kg bw/day (nominal)

Remarks:

P1b generation: corresponding to 5000 ppm

Doses / concentrations 6

Dose / conc.:

938.15 mg/kg bw/day (nominal)

Remarks:

P1b generation: corresponding to 10000 ppm

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: mortality, overt signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed weekly except for mated females which were weighed on days 0, 7, 14 and 21 of gestation and on days 0, 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

Oestrous cyclicity (parental animals):

Not examined

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 F1a/F2a/F2b offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (approx. 3 weeks for the F0 generation and approx. 4 weeks for the F1b generation, after the end of the second mating phase)
- Maternal animals:
Those females which failed to mate were killed 26 days after the end of the second mating phase and were given a detailed macroscopic examination. Females which mated but did not litter after the second mating phase were sacrificed on day 26 or 27 post coitum.
The dams were killed on day 21, 22 or 23 post partum of the second lactation period and given a macroscopic examination.

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

HISTOPATHOLOGY
The tissues indicated were prepared for microscopic examination. Epididymides, seminal vesicle, testes, liver, mammary glands (Thoracic and inguinal), uterus, vagina, ovaries and prostate were examined for control and high dose rats. Any other eventual abnormalities were examined for all groups.

Postmortem examinations (offspring):

SACRIFICE
- The F1a offspring and all F2 (a,b) offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Pups born dead or malformed and pups dying on or before day 7 post partum were fixed in Bouin' s fluid and, subsequently given a detailed external and internal examination with a dissecting microscope using a modified Barrow and Taylor technique. Pups found dead between days 8 and 21 post partum were examined in a detailed gross necropsy.
One male and one female weanling (between 21 and 28 days post partum) from each litter of the F1b and F2b generations were randomly selected where possible, killed by carbon dioxide -asphyxiation followed by exsanguination from the abdominal aorta and given a gross necropsy examination. Weanling rats not selected for either breeding or pathological examination were given an external examination and those with external abnormalities were subjected to a gross necropsy examination. Pups externally normal were killed on day 21, 22 or 23 post partum using carbon dioxide asphyxiation.
Abnormal tissues from pups found dead between days 8 and 21 post partum, F1a, F1b and F2a generation weanlings necropsied because of external abnormalities and F1b generation weanlings selected for necropsy were preserved in 10 % neutral buffered formalin.

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

HISTOPATHOLOGY
On completion of the gross pathology examination of the F2b generation weanlings, the following tissues and organs were retained in 10% neutral buffered formalin for fixation and preservation:
adrenals lungs (samples of 2 lobes), spinal cord (thoracic and lumbar), aorta (thoracic), bone and marrow (sternum), lymph nodes (mandibular and mesenteric), spleen, stomach, brain (3 levels), mammary gland (inguinal), testes, optic nerve, thymus, cecum, colon, duodenum, ovaries, thyroid (and parathyroids), pancreas, epididymides, pituitary tongue esophagus, prostate, trachea, eyes, salivary gland (sub-maxillary), urinary bladder, heart, uterus (corpus and cervix), ileum, sciatic nerve jejunum, seminal vesicles, vagina, kidneys, skeletal muscle, liver (sample of 2 lobes), skin, any other tissue(s) with gross lesions

Statistics:

Please refer to any other information on materials and methods incl. tables

Reproductive indices:

The mating, fertility and gestation indeces were calculated for the parental animals. Furthermore the conception rate has been determined.

Offspring viability indices:

For the offspring the viability, survival and lactation index has been calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (P0)"

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (P0)"

Food consumption and compound intake (if feeding study):

no effects observed

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (P0)"

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (P0)"

Details on results (P0)

Clinical signs
There were no clinical signs related to treatment with the test substance. Occasional incidental findings such as areas of alopecia/scabbing and ocular discharge were seen among males and females (control and/or treated groups). Two females, No. 357 (5000 ppm) and 451 (10000 ppm) displayed, respectively, a mass and a swelling at the axillary region. However, there were no gross pathological findings for No. 451 and the mass of No. 357 was histologically found to be active mammary tissue. During weeks 18 and 19, (the period between the first and second reproductive phases), most animals displayed clinical findings indicative of an infection with sialodacryoadenitis virus (S.D.A.V.). These findings included ventral cervical swelling, ocular redness, ocular swelling, ocular discharge, ocular opacity, and respiratory abnormalities such as nasal discharge and sneezing. As a result of this infection, the second mating phase was delayed by approximately one week in order for the clinical signs to subside. (The reproductive parameters for the second littering phase were either similar to the first littering phase or showed only the changes expected with their age and did not appear to be affected by the S.D.A.V. infection).

Body weight and weight gain
The body weights of males in the treated groups were not significantly different from those of the control group. There were, however, occasional statistically significant intergroup differences for body weight gains which were not attributed to treatment. These included significantly lower values relative to the control, during week 2 for the 5000 and 10000 ppm groups (P < 0.05) and again during week 7 for the 10000 ppm group (P < 0.01) and significantly (P < 0.01) greater values relative to the control, during week 15 for the 1000 and 5000 ppm groups. All groups showed a weight loss during week 18, the time of the S.D.A.V. infection.
For females neither the body weights nor the body weight gains of the treated groups differed significantly from values of the control group for the pre-mating period or gestation and lactation periods of both reproductive phases.

Gross pathological and histopathological findings
Few gross or microscopic findings were observed in the P0 generation male and female rats.
In the males, slight to severe degeneration of the testicular seminiferous epithelium was observed in 3/25 rats in Group 1 (Control) and in 4/25 rats in Group 4 (10000 ppm). The lesion was usually unilateral and associated, in some cases, with a variable degree of aspermatogenesis. In addition, in 2 rats (no. 109 and 119) in Group 1 and in 1 rat (no. 401) in Group 4, the corresponding epididymis showed a complete absence of sperm (recorded as empty). In the prostate, there was a purulent exudate in the acini in 14/25 animals in Group 1, and 16/25 animals in Group 4. This finding was accompanied, in many cases, by a focal chronic prostatitis. These prostatic changes were usually slight. In one female (no. 464) in Group 4, there was an embryonic resorption in the right uterine horn, characterized by the presence of an embryo in the lumen, focal ulceration of the endometrium, and neutrophil infiltration. Female 357 in Group 3 (5000 ppm) had a pale, firm, lobulated mass in the left axilla. However, microscopic examination of the mass revealed that it was physiologically active mammary tissue.
In conclusion, in the P0 generation male and female rats, there were no observable lesions which were treatment-related. The gross and microscopic findings were considered to be incidental or background changes observed in rats of this age group.

Reproductive performance
The conception rate of the 10000 ppm group at the first mating phase was significantly (P < 0.05) less than the control value, while in the second mating phase the conception rate was markedly higher than the control value. Consideration of the data for the numbers of fertile males and females, derived from combining the data for the two mating phases, indicates no effect of treatment. The day of mating in the control and treated groups was similar. The gestation index, length of gestation, length of parturition, number of live pups at birth and the sex ratio of both littering periods were unaffected by treatment with the test substance.
The incidence of dead pups for the second littering phase was found to be significantly (P < 0.05) higher for the 10000 ppm group as compared to the control. However, as this difference was primarily a result of a large litter loss in one litter (No. 455), the numbers of litters affected were comparable to the control and thus it does not appear to be of toxicological significance.
One pup from litter No. 455 of the F1b generation was sacrificed on day 13 post partum because it had been bitten in the head by its mother.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (P1)"

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Please refer to "details on results (P1)"

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (P1)"

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (P1)"

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (P1)"

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (P1)"

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P1)

Mortality
One male (No. 3118) in the 5000 ppm treated group was found dead during week 21 with lymphoid leukemia and multifocal erosions of the gastric mucosa. No other animals died and none were sacrificed preterminally.

Clinical findings
Occasional incidental findings were observed among males and females of the control and/or treated groups. Examples of these were areas of alopecia, deviating or broken teeth and discharge and/or staining in the periorbital region. Two males (No. 1110-Control and 4104-10000 ppm) displayed swellings at the urogenital and ventral cervical regions, respectively. (The former was found to be the result of an inflammation in the preputial gland and the latter was not seen at necropsy).

Body weights and weight gain
The body weight of male animals of the 5000 ppm group was significantly (P < 0.05) higher than the control following weaning, week 0. The body weights of the 1000 ppm group were significantly (P< 0.01) greater than the control group for weeks 7, 9 to 15, and 18 and 19. The body weight gains of all the treated groups were frequently significantly different, either higher or lower, than the control values. These differences were believed to represent intergroup variation.
The body weights and body weight gains of the females were not affected by treatment. There were a number of assessment occasions at which there were statistical differences between the control and treated groups. These included differences in body weight gains during the premating period and higher weights in the 1000 ppm group seen frequently during the reproductive phases.

Food consumption
Food intake levels for the control and treated groups of male animals were comparable exrept during week 19 in the 10000 ppm group where there was a statistically significantly (P < 0.05) lower intake value relative to the control group. There were no significant differences for food consumption between control and treated groups in the female animals.

Gross pathological and histopathological findings
Miscellaneous gross or microscopic findings were observed in the P1b generation male and female rats. In the male reproductive tract, moderate to severe degeneration of the testicular seminiferous epithelium was noted in 3/25, 3/8, 2/5 and 2/25 rats in Group 1 (control), Group 2 (1000 ppm), Group 3 (5000 ppm) and Group 4 (10000 ppm), respectively. This change was unilateral or bilateral and associated, in all cases, with a severe aspermatogenesis/hypospermatogenesis. Additionally, in many of these rats, the corresponding epididymis showed a complete absence of sperm (recorded as empty). One rat (no. 2110) in Group 2 also had a spermatic granuloma with mineralization in one testis. In the prostate, there was a purulent exudate in the acini in 15/25 animals in Group 1, 1/7 in Group 2, 3/4 in Group 3 and 11/25 in Group 4. In many cases, this finding was accompanied by a focal, slight or mild, chronic prostatitis. The most frequent gross changes in the males were observed in the lungs, and mainly included mottled lungs and single or multiple, dark or pale, raised or depressed areas of variable consistency. These pulmonary changes were characterized microscopically by various combinations of the following: perivascular mononuclear cell infiltration, histiocytosis and intra-alveolar hemorrhage. These findings varied from slight to moderate and were observed in 11/11 males in Group 1, 7/7 in Group 2, 12/13 in Group 3 and 8/8 in Group 4. One rat (no. 3118) in Group 3, which died during the course of the study, had a lymphoid leukemia in the liver and multifocal erosions of the gastric mucosa. In rat no. 1110 in Group 1, a pale, firm mass was found in the right urogenital region. The microscopic examination revealed a mixed cell infiltration in the preputial glands, associated with ductular dilatation and accumulation of secretion. One female (no. 3172) in Group 3, had an ovarian cyst. No other microscopic observations were observed in the female reproductive tract. Only female no. 4157 in Group 4 had a finding in the lungs; this was a slight histiocytosis. In conclusion, in the P1b generation (male and female rats), there were no lesions which were treatment-related. The gross and histological findings were considered to be incidental or background changes found in rats.

Maternal performance
The gestation index, length of gestation, duration of parturition, number of live pups at birth and sex ratio were unaffected by treatment for both littering phases. There was marked intergroup variation in the incidence of dead groups at birth. In the second littering phase, the incidence of dead pups in the 1000 and 5000 ppm groups was statistically significantly (P < 0.05 and P < 0.01, respectively) lower than control values. The incidence of litters with dead pups, however, did not achieve statistical significance.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (F1)

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (F1)

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (F1)

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (F1)

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Clinical findings
The clinical condition of the pups was unaffected by treatment with the test substance. Occasional incidental findings included subcutaneous hematomas and small, weak and dehydrated pups. One male pup in the control group of the F1b generation (156/2) had a domed skull. (Gross pathological findings of dilatation of the lateral ventricles confirmed this to be internal hydrocephalus). Another pup of the same generation from litter No. 253 had a missing digit on day 4 post partum, presumably as a result of maternal cannibalism.

Body weight
The total group mean litter mean pup body weight of the F1a generation pups in the 10000 ppm group was significantly (P < 0.05) elevated on day 0 post partum. Male, female and total pup weights of treated groups were similar to values of the control group for all other assessments.

Gross pathological and histopathological findings
Some F1a generation pups were born dead or died during the course of the study. The majority of these pups were found dead on the day of parturition. With the exception of pup 160/B in Group 1 (control) which showed findings consistent with cannibalism, no other gross findings were observed in the remaining pups. Three F1a generation pups were sacrificed during the course of the study. Pup 266/A in Group 2 (1000 ppm) had a scab and a clot on the cranium, and a dark discoloration of the rostral portion of the brain. Additionally, the liver was pale and the left cornea was dry. In pup 353/1 and pup 353/2 in Group 3 (5000 ppm), there were no gross findings.
Few F1b generation pups were born dead or died during the course of the study. The majority of these pups were found dead on the day of parturition. There was evidence of cannibalism in 2 pups (no. 162/A and 162/B) in Group 1 (control) and in 1 pup (no. 362/A) in Group 3 (5000 ppm). Pup 455/A in Group 4 (10000 ppm) had a severe reduction of renal papillae. This kidney finding is often associated with delayed renal development and is not an uncommon observation in rats. Infrequent gross findings were observed in the F1b generation pups sacrificed between days 25 and 27 post partum. One pup (no. 459/8) in Group 4 had a small eye (microphthalmia). In pup 156/2 (control group), there was a dilatation of the lateral ventricles of the brain (hydrocephalus) and a clot in the cranial cavity. These abnormalities, found in these two pups, were considered to be spontaneous. One pup (no. 154/7) in control group and 1 pup (no. 264/3) in Group 2 (1000 ppm) had dilated renal pelves. Multiple cysts were observed in the kidneys of pups 168/3 and 465/1. These renal findings are seen with regularity in rats and were not considered significant. In pup 455/N, sacrificed on day 13 post partum, there was a clot in the cranial cavity and a skin laceration on the head.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

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):

Please refer to "details on results (F2)"

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (F2)"

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "details on results (F2)"

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

Clinical signs
There were few clinical signs among pups in the control and treated groups. The most common incidental finding was area(s) of subcutaneous hematomas.

Pathological and histopathological findings
In F2a generation groups there were pups born dead or pups which died during the course of the study. Most of these pups were found dead on the day of parturition. Autolysis was observed in pups no. 2153/A in Group 2 (1000 ppm) and 4167/B in Group 4 (10000 ppm), and evidence of cannibalism was noted in pup no. 2174/A in Group 2. No other gross findings were noted in the remaining pups found dead. One pup (no. 1173/A) in Group 1 (control) was sacrificed on day 13 post partum. There was a perforation of the calvarium associated with a dark discoloration of the brain and a clot in the cranial cavity.
In all groups, some F2b generation pups were born dead or died during the course of the study. The majority of these pups were found dead on the day of parturition. In pups no. 1163/A and 1173/A in Group 1 (control), no. 2168/A in Group 2 (1000 ppm) and no. 4164/A in Group 4 (10000 ppm), there was a moderate reduction of the renal papilla(e). This renal finding is seen with some regularity in neonatal rats and is often associated with delayed renal development. Autolysis was noted in both control and test groups. In all pups of female no. 4154 which drowned, the lungs were uncollapsed. In pups no. 1163/G, 2153/A and 4158/A, the lungs were uncollapsed and/or mottled. No other gross findings were observed in the remaining pups.
Few gross or microscopic findings were observed in the F2b generation male and female pups sacrificed at the end of the study. A focal, chronic prostatitis was observed in 1 male (no. 2731) in Group 2 (1000 ppm), 1 male (no. 3603) in Group 3 (5000 ppm) and 1 male (no. 4662) in Group 4 (10000 ppm). One female (no. 1755) in Group 1 (control) and 1 female (no. 3617) in Group 3 were hydrocephalic. In one female (no. 1707) in Group 1, there was a bilateral anophthalmia. In the kidneys, unilateral or bilateral hydronephrosis and/or cysts were found in some males and females in the control and test groups. These renal findings are seen with some regularity in rats of this strain. Pulmonary changes which included mainly intra-alveolar, pleural and/or perivascular hemorrhage or edema were observed in some pups of both sexes in all groups. These lung findings were considered to be agonal and related to the method of euthanasia. Other findings in various organs and tissues were infrequent and appeared to be incidental.
In conclusion, in the F2b generation male and female pups, there were no lesions which were treatment-related.

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1 First mating phase P0 Generation

	No. of males	No. of females	No. of females failing to mate	Mean number of days to mating (SD)	No. of pregnant females	Mating index (%)	Fertility index (%)	Conception rate (%)
Group 1 (control)	25	25	2	2.7 (2.120)	22	92	88	95.7
Group 2 (1000 ppm)	25	25	0	3.4 (2.739)	21	100	84	84
Group 3 (5000 ppm)	25	25	0	3.61 (2.904)	21	100	84	84
Group 4 (10000 ppm)	25	25	0	2.44 (1.044)	18	100	72	72A

A = P < 0.05

Table 2 First littering phase P0-Generation

	Gestation index (%)	Length of gestation (days)	Length of parturition (hours)	No. of pups at birth		Incidence of dead pups at birth		Sex ratio (%M)
				live	dead	pups	litters
Group 1 (control)	100	21.6	2.21	13.5	0.27	6/304	4/22	45.3
Group 2 (1000 ppm)	100	21.8	2.52	14.8	0.25	5/300	3/20	46.9
Group 3 (5000 ppm)	100	21.7	2.4	13.2	0.16	3/254	3/19	52.2
Group 4 (10000 ppm)	100	21.7	2.87	13	0.22	4/238	4/18	51.6

 

Table 3 Second mating phase P0 Generation

	No. of males	No. Of females	No. of females failing to mate	Mean number of days to mating (SD)	No. of pregnant females	Mating index (%)	Fertility index (%)	Conception rate (%)
Group 1 (control)	25	25	2	2.86 (2.587)	17	92	68	73.9
Group 2 (1000 ppm)	25	25	2	2.78 (1.704)	21	92	84	91.3
Group 3 (5000 ppm)	25	25	1	2.04 (0.955)	18	96	72	75
Group 4 (10000 ppm)	25	25	1	2.54 (1.141)	21	96	84	87.5

 

Table 4 Second littering phase P0 Generation

	Gestation index (%)	Length of gestation (days)	Length of parturition (hours)	No. Of pups at birth		Incidence of dead pups at birth		Sex ratio (%M)
				live	dead	pups	litters
Group 1 (control)	100	21.7	1.92	13.5	0.19	3/219	2/16	45.1
Group 2 (1000 ppm)	100	21.8	1.93	13.2	0.24	5/282	3/21	50
Group 3 (5000 ppm)	100	21.8	2.46	13.4	0.22	4/246	4/18	48.6
Group 4 (10000 ppm)	95.2	21.8	1.81	13.7	0.75	15/288A	4/20	50.1

A = P < 0.05

Table 5 First mating phase F1b Generation

	No. of males	No. Of females	No. of females failing to mate	Mean number of days to mating (SD)	No. of pregnant females	Mating index (%)	Fertility index (%)	Conception rate (%)
Group 1 (control)	25	25	3	2.38 (1.284)	22	88	88	100
Group 2 (1000 ppm)	25	25	1	2.39 (2.039)	22	96	88	91.7
Group 3 (5000 ppm)	25	25	0	2.38 (1.377)	21	100	84	84
Group 4 (10000 ppm)	25	25	1	2.71 (1.301)	20	96	80	83.3

 

Tabel 6 First littering phase F1b Generation

	Gestation index (%)	Length of gestation (days)	Length of parturition (hours)	No. Of pups at birth		Incidence of dead pups at birth		Sex ratio (%M)
				live	dead	pups	litters
Group 1 (control)	100	21.6	1.66	12.8	0.36	8/290	3/22	52.2
Group 2 (1000 ppm)	100	21.7	2.38	13.7	0.18	4/305	4/22	49.1
Group 3 (5000 ppm)	100	21.7	2.15	12	0.24	5/257	5/21	52.7
Group 4 (10000 ppm)	100	21.6	1.77	13	0.1	2/262	1/20	53.9

 

Table 7 Second mating phase F1b Generation

	No. of males	No. Of females	No. of females failing to mate	Mean number of days to mating (SD)	No. of pregnant females	Mating index (%)	Fertility index (%)	Conception rate (%)
Group 1 (control)	25	25	3	2.91 (1.231)	20	88	80	90.9
Group 2 (1000 ppm)	25	25	6	2.47 (1.020)	18	76	72	94.7
Group 3 (5000 ppm)	25	25	0	2.72 (1.487)	23	100	92	92
Group 4 (10000 ppm)	25	25	2	2.96 (1.551)	19	92	76	82.6

 

Table 8 Second littering phase F1b Generation

	Gestation index (%)	Length of gestation (days)	Length of parturition (hours)	No. Of pups at birth		Incidence of dead pups at birth		Sex ratio (%M)
				live	dead	pups	litters
Group 1 (control)	100	21.8	1.75	13	0.65	13/273	5/20	45.3
Group 2 (1000 ppm)	100	21.9	1.81	15	0.22	4/274A	4/18	44.8
Group 3 (5000 ppm)	100	21.9	1.97	13.1	0.09	2/303	2/23	53.5
Group 4 (10000 ppm)	100	21.8	1.81	13.3	0.42	8/260	6/19	53.2

A = P < 0.05

Table 9 Viability Data F1a generation

	Day 0-4 post partum Viability index (%)	Day 4-7 post partum Survival index (%)	Day 4-14 post partum Survival index (%)	Day 4-21 post partum Lactation index (%)
Group 1 (control)	99.7	100	100	100
Group 2 (1000 ppm)	98.4	100	100	99.4
Group 3 (5000 ppm)	97.4	100	100	100
Group 4 (10000 ppm)	98.6	100	100	100

 

Table 10 Viability Data F1b generation

	Day 0-4 post partum Viability index (%)	Day 4-7 post partum Survival index (%)	Day 4-14 post partum Survival index (%)	Day 4-21 post partum Lactation index (%)
Group 1 (control)	98.8	100	100	100
Group 2 (1000 ppm)	99.2	100	100	100
Group 3 (5000 ppm)	100	98.6	100	100
Group 4 (10000 ppm)	94.5	100	95	95

 

Table 11 Viability Data F2a generation

	Day 0-4 post partum Viability index (%)	Day 4-7 post partum Survival index (%)	Day 4-14 post partum Survival index (%)	Day 4-21 post partum Lactation index (%)
Group 1 (control)	98.4	100	99.4	99.4
Group 2 (1000 ppm)	99.1	100	100	100
Group 3 (5000 ppm)	98.9	100	100	100
Group 4 (10000 ppm)	99.2	100	100	100

 

Table 12 Viability Data F2b generation

	Day 0-4 post partum Viability index (%)	Day 4-7 post partum Survival index (%)	Day 4-14 post partum Survival index (%)	Day 4-21 post partum Lactation index (%)
Group 1 (control)	99.7	98.7	98.7	98.7
Group 2 (1000 ppm)	96.7	100	100	100
Group 3 (5000 ppm)	98	99.5	98.8	98.8
Group 4 (10000 ppm)	98.5	100	99.3	99.3