2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 November 2001 to 16 June 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: European Economic Community. Methods for the Determination of Toxicity. Official Journal of the European Communities, Vol. 31, No. L 133, May 30, 1988. ISSN 0378-6978, 1988.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: The Japan Ministry of Agriculture, Forestry and Fisheries, Notification of 12- Nohsan-8147, Guideline 2-1-17, Reproduction Study, November 24, 2000.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

EC Number:

604-721-7

Cas Number:

150114-71-9

Molecular formula:

C6H4Cl2N2O2

IUPAC Name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: tan powder

Specific details on test material used for the study:

Purity: 94.5%

Test animals

Species:

rat

Strain:

other: Crl:CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: CD (Crl: CD (SD) IGS BR)
- Age at study initiation: ca. 6 weeks
- Weight at study initiation (parental generation group means on day -2): 193.7 - 193.9 g (males); 147.6 - 147.8 g (females)
- Housing: Animals were housed individually in stainless steel cages with wire-mesh floors suspended above catch pans. Cages contained feed containers and pressure activated nipple-type watering systems. Dams were housed one per cage (with their litter) in plastic cages provided with corn cob nesting material from approximately day 19 of gestation and throughout the lactation phase of the study.
- Diet: ad libitum
- Water: municipal water, ad libitum
- Acclimation period: at least two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 40 - 70 % (relative)
- Air changes: 12 - 15 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIETARY EXPOSURE
Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Pre-mixes were prepared periodically throughout the study based on stability data. Diets were prepared weekly for approximately ten weeks prior to breeding of the P1 adults. The concentrations of the test material in the diets were calculated from the most recent body weight and feed consumption data. Initial concentrations of test material in the diet were calculated from pre-exposure body weights and feed consumption data. To avoid potential overdosing during the breeding period, animals co-housed were provided with either the male or female diet, whichever is of lower concentration. During gestation, females from each dose group were provided with the appropriate dietary concentration of test material given during breeding. Dietary concentrations supplied during lactation were adjusted using historical control feed consumption data for lactating females to account for the large and rapid increase in feed consumption (2-3x increase) typical for rats in late lactation (Carney et al., 1998). Until all litters were weaned, weanlings received a diet containing the same concentration of test material that was given to the P1 females during the third week of lactation. Dams awaiting necropsy received a diet containing the same concentration of test material that was given during the breeding period until all litters had finished the lactation phase. Dietary concentrations for the P2 generation were calculated as described for the P1 animals.

Details on mating procedure:

Breeding for the P1 and P2 adults commenced after approximately ten weeks of treatment.
- M/F ratio per cage: 1 male: 1 female.
- Length of cohabitation: until mating occurred or two weeks elapsed.
- Proof of pregnancy: the presence of a vaginal plug, or sperm in vaginal smear, was referred to as day 0 of pregnancy.
The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. If available, one rat/sex/litter was randomly selected for the P2 mating to produce the F2 generation. More than one weanling may have been selected from the litters, if necessary, to achieve 30 breeding pairs/dose level for the second generation. Cohabitation of F1 male and female littermates was avoided. In cases where a mating partner had died or was otherwise not available, the animal was paired with the next available partner. A second breeding of the first or second -generation adults was not conducted.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity
Representative samples from the test diets were evaluated to ensure homogeneous distribution of the test material at the lowest and highest concentrations in the feed at least once during the study.
Analyses confirmed that the test material was homogeneously distributed in the diets.

- Stability
A previous four-week toxicity study in mice demonstrated the test material to be stable for at least 21 days in rodent chow at concentrations ranging from 0.005 to 3 %. A two-year dietary chronic toxicity/oncogenicity and chronic neurotoxicity study has demonstrated the test material to be stable for at least 55 days at a 7 % concentration. An 18 month chronic toxicity/oncogenicity study has demonstrated the test material to be stable for at least 35 days at 0.0258 %. Test diets for the current study were prepared and used within these stability limits.

- Concentration Verification
Analyses of all test diets to determine concentration of the test material were conducted at least three times (at the start, midway, and near the end of the study). The analyses were conducted using a solvent extraction method followed by liquid chromatography-mass spectrometry (LC-MS) and solvent standards incorporating an internal standard.
One of the analyses was done on a breeding diet which is common to both sexes. The mean concentrations of test material in the test diets over the entire study period were 94.1, 100, and 102 % of target for the male 50, 250, and 1000 mg/kg/day dose levels, and 104, 108, and 106 % of target for the female 50, 250, and 1000 mg/kg/day dose levels, respectively.

Duration of treatment / exposure:

Animals were dosed for approximately ten weeks prior to breeding, and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations.

Frequency of treatment:

Continuous (in diet)

Details on study schedule:

If available, one rat/sex/litter was randomly selected for the P2 mating to produce the F2 generation. More than one weanling may have been selected from the litters, if necessary, to achieve 30 breeding pairs/dose level for the second generation. Cohabitation of F1 male and female littermates was avoided. In cases where a mating partner had died or was otherwise not available, the animal was paired with the next available partner. A second breeding of the second-generation adults was not conducted.

- Culling and Weaning
To minimise variation in pup growth due to differences in litter size, F1 and F2 litters were standardised to eight pups per litter on postnatal day 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were then randomly selected using a computer generated selection procedure, so that four males and four females (whenever possible) remained in each litter. Litters with fewer than eight pups were not culled. All litters were weaned on postnatal day 21.
One male and one female per litter were randomly selected as P2 animals to produce the second generation. If there were fewer than 30 litters from which to select P2 animals, additional animals were randomly selected from available litters, as needed, in order to obtain the required number of animals/dose level. When possible, one pup/sex/litter was selected for a gross examination with the collection of organ weights and two additional pups/sex/litter were selected for a gross examination only. Any weanlings either not held for the next generation of adult animals or not selected for necropsy were euthanised.

- Physical Maturational Landmarks
All F1 weanlings selected for mating were observed daily for vaginal opening beginning on postnatal day 28 or preputial separation beginning on day 35. The age and body weights at the time of landmark acquisition were recorded. Because there was not a treatment-related effect on the F1 sex ratio, age at vaginal opening or age at preputial separation and anogenital distance were not measured in the F2 pups.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 males and 30 females per dose.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected based on the previous 13-week dietary probe study in CD rats. Administration of test material at the high-dose (1000 mg/kg/day) was expected to produce increases in cecum weights with possible epithelial hyperplasia of the cecum (based on existing data from the reproductive toxicity probe study). The top dose level of 1000 mg/kg/day for this study was a limit dose as defined by the relevant regulatory guidelines. The middle- and low-doses were expected to provide dose response data for any treatment-related effects observed in the high-dose group and to establish a no-observed-effect level (NOEL).
- Rationale for animal assignment: Prior to test material administration, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice each day a cage-side examination was conducted. To the extent possible the following parameters were evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were conducted on all males pre-study and weekly thereafter. Clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm- or plug-positive) females received clinical examinations on gestation day (GD) 0, 7, 14, and 21. In addition, mated females were observed for signs of parturition beginning on or about day 20 of gestation. Females that delivered litters were subsequently evaluated on lactation day (LD) 0, 1, 4, 7, 14, and 21. Females that failed to mate or failed to deliver litters were examined weekly. Examinations included a careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), swellings, masses, and animal behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period and weekly during the pre-breeding treatment period. Body weights for males were recorded weekly throughout the course of the study. Sperm- or plug-positive females were weighed on days 0, 7, 14, and 21 of gestation. Females that delivered litters were weighed on days 1, 4, 7, 14, and 21 of lactation. Females that failed to mate were not weighed during the subsequent gestation and lactation segments of the study. Females that failed to deliver a litter were not weighed during the lactation phase of the study.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Feed consumption was determined pre-exposure and weekly during the ten-week pre-breeding period for all animals by weighing feed crooks at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption was measured weekly in males and dietary concentrations were adjusted accordingly. During gestation, feed consumption was measured on GD 0, 7, 14, and 21. During lactation, feed consumption was measured on days 1, 4, 7, 11, 14, 17, 19, and 21. Feed consumption was not measured in females that failed to mate or deliver a litter. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feeder- final weight of feeder) / number of days in measurement cycle

Oestrous cyclicity (parental animals):

Vaginal lavage samples were collected daily for all P1 and P2 females for three weeks prior to mating and during cohabitation until each female was sperm- or plug-positive or until the two-week mating period elapsed. Lavage samples were collected by gently irrigating the vagina with water and transferring loosely adherent vaginal cells to a slide with a pipette. Vaginal lavage slides were examined to determine oestrous cycle length and pattern. Additionally, on the day of scheduled necropsy, the stage within the oestrous cycle was determined for all P1 and P2 female rats.

Sperm parameters (parental animals):

Parameters examined in P1 and P2 male parental generations: testis weight, epididymis weight and seminal vesicles with coagulating glands (and seminal fluid) weight

SPERM ANALYSIS
Unless circumstances dictated otherwise, the left and right epididymides and testes were allocated as follows: right epididymis – motility and histopathology; left epididymis – counts; right testis – histopathology; left testis – counts.
- Motility
Sperm motility was evaluated in all surviving P1 and P2 males at termination. Immediately after euthanasia of males and isolation of their epididymides, a small sample of sperm from the right cauda epididymis was expressed into a dish containing ca. 5 mL of SpermPrep Medium and was incubated at room temperature for approximately 2-3 minutes. An aliquot of the incubated sperm suspension was placed in a chamber of the HTM Integrated Visual Optical System (IVOS) for the determination of total percent motile (showing any motion) and percent progressively motile (showing net forward motion) sperm. After sperm were released, the epididymis was placed in Bouin’s fixative and, when deemed appropriate, subjected to histological examination.
- Counts
The left testis and cauda epididymis were weighed and frozen at -80 °C for subsequent determination of the number of homogenisation-resistant spermatids and cauda epididymal sperm per testis/epididymis and per gram of testicular/epididymal tissue. Thawed testis or epididymis was minced, diluted and stained with a fluorescent DNA-binding dye and spermatid or sperm count were determined from an aliquot loaded into the IVOS analyser as described by Stradler et al., (1996). Samples from the high-dose and control animals were evaluated.
- Morphology
An aliquot of sperm suspension was also taken, placed on a slide, and a smear prepared and then air dried for subsequent evaluation of sperm morphology. At least 200 sperm per male were evaluated and classified as normal or abnormal as described by Filler (1993). Samples with less than 200 sperm were excluded from analysis. Morphological evaluation of sperm from control and high-dose males was conducted. Sperm morphology was scored blind with respect to treatment group.

Litter observations:

LITTER DATA
Litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, 4, 7, 14, and 21 postpartum, and the sex and the weight of each pup on LD 1, 4 (before and after culling), 7, 14, and 21, and any visible physical abnormalities or demeanour changes in the neonates. Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, if possible, for external and visceral defects. These pups were preserved in neutral, phosphate-buffered 10 % formalin.

CULLING AND WEANING
To minimise variation in pup growth due to differences in litter size, F1 and F2 litters were standardised to eight pups per litter on postnatal day 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were then randomly selected using a computer generated selection procedure, so that four males and four females (whenever possible) remained in each litter. Litters with fewer than eight pups were not culled. Culled pups were euthanised by administration of Socumb euthanasia solution into the buccal cavity, and then discarded. All litters were weaned on postnatal day 21.
One male and one female per litter were randomly selected as P2 animals to produce the second generation. If there were fewer than 30 litters from which to select P2 animals, additional animals were randomly selected from available litters, as needed, in order to obtain the required number of animals/dose level. When possible, one pup/sex/litter was selected for a gross examination with the collection of organ weights and two additional pups/sex/litter were selected for a gross examination only. Any weanlings either not held for the next generation of adult animals or not selected for necropsy were euthanised by CO₂ inhalation and discarded.

PHYSICAL MATURATIONAL LANDMARKS
All F1 weanlings selected for mating were observed daily for vaginal opening beginning on postnatal day 28 (Cooper et al., 1989) or preputial separation beginning on day 35 (Korenbrot et al., 1977). The age and body weights at the time of landmark acquisition were recorded. As there was not a treatment-related effect on the F1 sex ratio, age at vaginal opening or age at preputial separation and anogenital distance were not measured in the F2 pups.

Postmortem examinations (parental animals):

SACRIFICE
A complete necropsy of all P1 and P2 adults was performed as close as possible to the weaning of the last litter. Fasted adult rats submitted alive for a necropsy were weighed and anaesthetised by the inhalation of carbon dioxide. Vaginal lavage slides were prepared from all P1 and P2 females for later determination of oestrous cycle stage. While anaesthetised, their tracheas were exposed and clamped, and they were euthanised by decapitation.

GROSS NECROPSY
A complete necropsy was conducted on all animals. The necropsy included an examination of the external tissues, and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10 % formalin using a hand-held syringe and blunt needle. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The uteri of all females were stained with an aqueous solution of 10 % sodium sulfide stain (Kopf et al., 1964) for approximately 2 minutes and were examined for the presence and number of implantation sites. Uteri from females that did not deliver a litter and had no visible implantation sites were examined for evidence of early resorption in order to verify pregnancy status. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10 % formalin.

ORGAN WEIGHTS
Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides (total weights for both epididymides and cauda weight for the left side only), seminal vesicles with coagulating glands (and seminal fluid), prostate, brain, pituitary, liver, kidneys, adrenal glands, spleen, cecum (empty and full) and thyroid (after fixation) were recorded. Organ-to-body weight ratios were calculated for all weighed organs with the exception of the left testis and left cauda epidymidis.

HISTOPATHOLOGY
Representative samples of the following tissues were collected and preserved in neutral, phosphate-buffered 10 % formalin, except that the ovaries, right testis and right epididymis were preserved by immersion in Bouin’s fixative. Similar necropsy procedures were followed for animals found dead or moribund (paired testes and epididymides were preserved for dead males).
- adrenals, aorta, auditory sebaceous glands, bone (including joint), bone marrow, brain (cerebrum, brainstem, cerebellum), cecum, cervix, coagulating glands, colon, cranial nerve - optic, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal/ Harderian glands, larynx, liver, lungs, mammary gland (females only), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues/pharynx, oesophagus, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina.
Histologic examination of the adult rats included the reproductive tissues, target organs, and relevant gross lesions of all control and high-dose rats as listed above (including thyroid gland due to weight increases). Reproductive organs of low-and mid-dose animals with signs of reduced fertility also were examined histologically. A qualitative evaluation of the testis included examination for retained spermatids, missing germ cell layers or types, multinucleated giant cells, and sloughing of spermatogenic cells into the lumen. The right epididymis, from which sperm were obtained for motility assessment, also was examined including evaluation of the caput, corpus and cauda. Examination of the ovaries included enumeration of primordial follicles of the P2 females using a method similar to Bucci et al. (1977). A subset of 15 randomly selected control and high-dose P2 females were chosen for primordial follicle enumeration, based on a power analysis. As no treatment-related effects were observed at the high dose, primordial follicle enumeration from the lower dose levels was not conducted. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with haematoxylin and eosin and examined using a light microscope. Examinations of tissues from the remaining groups were limited to those tissues that demonstrated treatment-related histologic effects at the high-dose and relevant gross lesions. Rats found dead or moribund were histologically examined in a similar manner.

Postmortem examinations (offspring):

SACRIFICE
When litter size permitted, three pups/sex/litter from the F1 and F2 litters were randomly selected at the time of weaning for a complete necropsy. In order to control for variations in body and organ weights, pups selected for a complete necropsy were euthanised at the same age (postnatal day 22). The pups were anaesthetised with carbon dioxide, weighed and euthanised by decapitation.

GROSS NECROPSY
For the F1 and F2 pups that were examined macroscopically, one pup/sex/litter was randomly selected for the collection of brain, spleen, uterus and thymus weights. Organ-to-body weight ratios were calculated. Gross pathological examination was performed as described above for adults, except that the weanlings were not fasted overnight. Representative samples of grossly abnormal tissues were collected from all weanlings at the scheduled necropsy. In addition, the brain, spleen, thymus, cecum and ileum were saved for the weanlings selected for organ weight measurements in the event that an effect on organ weight was observed and/or future evaluation was desirable. Tissues were fixed in neutral phosphate-buffered 10 % formalin. There were no treatment-related gross lesions in the F1 or F2 weanlings; therefore, histological examination was not conducted.

Statistics:

See "Any other information on materials and methods incl. tables" for information.

Reproductive indices:

Reproductive indices were calculated for all dose level groups as follows:
- Female mating index = (No. females with evidence of mating / No. paired) x 100
- Male mating index = (No. males with evidence of mating / No. paired) x 100
- Female conception index = (No. females with evidence of delivering a litter / No. mated) x 100
- Male conception index = (No. males siring a litter / No. mated) x 100
- Female fertility index = (No. females with evidence of delivering a litter / No. paired) x 100
- Male fertility index = (No. males siring a litter / No. paired) x 100
- Gestation index = (No. females delivering a viable litter / No. females delivering a litter) x 100
- Gestation survival index = percentage of delivered pups alive at birth
- Post-implantation loss = (No. implants – No. viable offspring / No. implants) x 100

Offspring viability indices:

- Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4 / No. born live) x 100
- Day 7, 14, or 21 pup survival index = (No. viable pups on day 7, 14 or 21 / No. live after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Overall, no treatment-related effects on behaviour or demeanour were observed in any phase of the study at any dose level in the P1-generation animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the first generation two males and one female died prior to scheduled termination, however on investigation these were not attributed to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Aside from one isolated value, there were no statistically identified differences in the body weights of the P1 or P2 males or the body weights and body weight gains of the P1 and P2 females during their respective pre-mating, mating, gestation, or lactation periods. The exception was an isolated finding of decreased body weight gain in the low-dose P1 females over the entire lactation period (LD 1-21), which was considered spurious due to the lack of dose-response and lack of repeatability in the P2 generation. Low-dose P2 females had the largest gain over the entire lactation period in the P2 generation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Several statistically identified increases and decreases in feed consumption were noted at different periods throughout the study. However these were slight and there was no consistent pattern of change nor any corroborating findings on paternal or maternal body weight or body weight gains during these time periods. These findings were not considered to be biologically significant.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic changes due to exposure to the test material in any of the tissues examined. The observations present were considered to be spontaneous alterations, unassociated with exposure to the test material. The cecal organ weight and gross pathologic changes were unassociated with microscopic changes indicative of a significant toxicologic effect. The normally formed faeces in the descending colon and rectum further supports the conclusion of a physiologic effect localised to the cecum and of no toxicological significance. The absence of microscopic changes in the cecum, as well as the other tissues examined, demonstrates the test material and/or its metabolite(s) were not significantly toxic, even at the highest dose tested.
Histopathologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effects considered due to test material administration. There were no treatment related or statistically-identified differences in the mean number of small and growing ovarian follicles in control females as compared to females given 1000 mg/kg/day.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no evidence of an effect on oestrous cyclicity at any dose level of test material.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no effects of the test material on any sperm analysis parameter at any dose level.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, post-implantation loss, time to mating, gestation length, pup survival or pup sex ratio in either generation. The only parameter that was significantly altered was pup survival, which was statistically increased on LD 4 in the 1000 mg/kg/day dose group of the P1 generation. This finding is likely spurious and is not considered adverse.

Details on results (P0)

MORTALITY AND CLINICAL SIGNS
Overall, no treatment-related effects on behaviour or demeanour were observed in any phase of the study at any dose level in either the P1- or P2-generation animals.
In the first generation two males and one female died prior to scheduled termination, however on investigation these were not attributed to treatment.
In the second generation one adult male died prior to the start of the pre-breeding phase, other incidental observations were noted in other P2 animals. None of these were attributed to treatment with the test material.

FEED CONSUMPTION
Several statistically identified increases and decreases in feed consumption were noted at different periods throughout the study. However these were slight and there was no consistent pattern of change nor any corroborating findings on paternal or maternal body weight or body weight gains during these time periods. These findings were not considered to be biologically significant.

BODY WEIGHTS
Aside from one isolated value, there were no statistically identified differences in the body weights of the P1 or P2 males or the body weights and body weight gains of the P1 and P2 females during their respective pre-mating, mating, gestation, or lactation periods. The exception was an isolated finding of decreased body weight gain in the low-dose P1 females over the entire lactation period (LD 1-21), which was considered spurious due to the lack of dose-response and lack of repeatability in the P2 generation. Low-dose P2 females had the largest gain over the entire lactation period in the P2 generation.

ANATOMIC PATHOLOGY
- Organ Weights: There were statistically identified differences in cecal weights of middle- and high-dose P1 males and females when compared to their respective controls. Other organ weights and terminal body weights were not altered by treatment with the test material in either sex of P1 adult rats. Similar to the P1 males, there were statistically identified differences in cecal weights of middle- and high-dose P2 males. However, the low-dose P2 males also had significantly increased relative empty cecal weights, contrary to the P1 low-dose males. In the P2 females, cecum weights were altered only at the highest dose level of test material. Other organ weights and terminal body weights were not altered by treatment with the test material in either sex of P2 adult rats.
- Gross Pathology: All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material, except those in the cecum of male and female rats. In the 250 mg/kg/day dose group, a few animals (P1 and P2 females, P2 males) had grossly observed increases in cecum size, but this cecal observation was noted most frequently in high-dose males and females in both the P1 and P2 generations, where the incidence of animals affected was 33-70 %. The cecum of P1 and P2 adults of both sexes in the 50 mg/kg/day group were grossly unaffected by treatment.
- Histopathology: There were no treatment-related microscopic changes due to exposure to the test material in any of the tissues examined. The observations present were considered to be spontaneous alterations, unassociated with exposure to the test material. The cecal organ weight and gross pathologic changes were unassociated with microscopic changes indicative of a significant toxicologic effect. The normally formed faeces in the descending colon and rectum further supports the conclusion of a physiologic effect localised to the cecum and of no toxicological significance. The absence of microscopic changes in the cecum, as well as the other tissues examined, demonstrates the test material and/or its metabolite(s) were not significantly toxic, even at the highest dose tested.
Histopathologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effects considered due to test material administration. There were no treatment related or statistically-identified differences in the mean number of small and growing ovarian follicles in control females as compared to females given 1000 mg/kg/day.

SPERM PARAMETERS
There were no effects of the test material on any sperm analysis parameter at any dose level.

OESTROUS CYCLICITY
There was no evidence of an effect on oestrous cyclicity at any dose level of test material.

REPRODUCTIVE INDICES, PUP SURVIVAL AND SEX RATIO
There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, post-implantation loss, time to mating, gestation length, pup survival or pup sex ratio in either generation. The only parameter that was significantly altered was pup survival, which was statistically increased on LD 4 in the 1000 mg/kg/day dose group of the P1 generation. This finding is likely spurious and is not considered adverse.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(parental toxicity)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicologically adverse effects at highest dose tested.

2

Key result

Dose descriptor:

NOAEL

Remarks:

(reproductive toxicity)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no reproductive effects at the highest dose tested

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

Overall, no treatment-related effects on behaviour or demeanour were observed in any phase of the study at any dose level in the P2-generation animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the second generation one adult male died prior to the start of the pre-breeding phase, other incidental observations were noted in other P2 animals. None of these were attributed to treatment with the test material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Aside from one isolated value, there were no statistically identified differences in the body weights of the P1 or P2 males or the body weights and body weight gains of the P1 and P2 females during their respective pre-mating, mating, gestation, or lactation periods. The exception was an isolated finding of decreased body weight gain in the low-dose P1 females over the entire lactation period (LD 1-21), which was considered spurious due to the lack of dose-response and lack of repeatability in the P2 generation. Low-dose P2 females had the largest gain over the entire lactation period in the P2 generation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Several statistically identified increases and decreases in feed consumption were noted at different periods throughout the study. However these were slight and there was no consistent pattern of change nor any corroborating findings on paternal or maternal body weight or body weight gains during these time periods. These findings were not considered to be biologically significant.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Similar to the P1 males, there were statistically identified differences in cecal weights of middle- and high-dose P2 males. However, the low-dose P2 males also had significantly increased relative empty cecal weights, contrary to the P1 low-dose males. In the P2 females, cecum weights were altered only at the highest dose level of test material. Other organ weights and terminal body weights were not altered by treatment with the test material in either sex of P2 adult rats.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material, except those in the cecum of male and female rats. In the 250 mg/kg/day dose group, a few animals (P1 and P2 females, P2 males) had grossly observed increases in cecum size, but this cecal observation was noted most frequently in high-dose males and females in both the P1 and P2 generations, where the incidence of animals affected was 33-70 %. The cecum of P1 and P2 adults of both sexes in the 50 mg/kg/day group were grossly unaffected by treatment.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic changes due to exposure to the test material in any of the tissues examined. The observations present were considered to be spontaneous alterations, unassociated with exposure to the test material. The cecal organ weight and gross pathologic changes were unassociated with microscopic changes indicative of a significant toxicologic effect. The normally formed faeces in the descending colon and rectum further supports the conclusion of a physiologic effect localised to the cecum and of no toxicological significance. The absence of microscopic changes in the cecum, as well as the other tissues examined, demonstrates the test material and/or its metabolite(s) were not significantly toxic, even at the highest dose tested.
Histopathologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effects considered due to test material administration. There were no treatment related or statistically-identified differences in the mean number of small and growing ovarian follicles in control females as compared to females given 1000 mg/kg/day.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no evidence of an effect on oestrous cyclicity at any dose level of test material.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no effects of the test material on any sperm analysis parameter at any dose level.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, post-implantation loss, time to mating, gestation length, pup survival or pup sex ratio in either generation.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicologically adverse systemic effects at the highest dose tested

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on reproduction at the highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pup survival was statistically increased on LD 4 in the 1000 mg/kg/day dose group of the P1 generation. This finding is likely spurious and is not considered adverse. There were no effects of treatment on the number of pups born live, number of pups born dead, or on litter size at any time in any dose group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on pup body weights at any time in any dose group. Pup weights were significantly higher than the controls on LD 14 in the F1 litters of the 250 and 1000 mg/kg/day dose groups. This increases were considered spurious because this response lacked of a dose-response relationship, occurred at isolated time points, and was not accompanied by other reproductive or developmental effects.

Sexual maturation:

no effects observed

Description (incidence and severity):

Age at vaginal opening or preputial separation was similar in all groups, indicating no influence of treatment.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on terminal body weights or organ weights for the male and female F1 weanlings. F1 female weanling in the 250 mg/kg/day dose group exhibited a significant decrease in absolute uterine weight, but this effect was considered spurious based on the absence of a dose-response relationship and the lack of reproducibility in the F2 weanlings.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material.

Histopathological findings:

not examined

Details on results (F1)

Observations made on the F1 and F2 pups during their respective lactation periods revealed no effects related to treatment. Incidental findings were noted. However, no malformed pups were observed in either generation at any dose level tested.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects at the highest dose tested

Results: F2 generation

General toxicity (F2)

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no effects of treatment on the number of pups born live, number of pups born dead, or on litter size at any time in any dose group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on pup body weights at any time in any dose group. Pup weights were significantly higher than the controls on LD 7 in the F2 litters of the 1000 mg/kg/day dose group. These increases were considered spurious because this response lacked of a dose-response relationship, occurred at isolated time points, and was not accompanied by other reproductive or developmental effects.

Sexual maturation:

no effects observed

Description (incidence and severity):

Age at vaginal opening or preputial separation was similar in all groups, indicating no influence of treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The final body weight and organ weight data for the male and female F2 weanlings were unaffected by treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to the test material.

Details on results (F2)

Observations made on the F1 and F2 pups during their respective lactation periods revealed no effects related to treatment. Incidental findings were noted. However, no malformed pups were observed in either generation at any dose level tested.

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at the highest dose tested

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Significant Organ Weight Effects

P1 Male rats	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Final body weight (g)	525.2	518.6	523.2	520.8
Cecum full (g)	5.223	4.951	6.493*	11.534*
Cecum full (g/100)	1.001	0.961	1.251*	2.230*
Cecum empty (g)	2.206	2.172	2.418	3.022#
Cecum empty (g/100)	0.420	0.420	0.462#	0.580#
P1 Female rats	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Final body weight (g)	278.0	275.0	277.1	268.6
Cecum full (g)	3.913	3.701	4.723*	6.653*
Cecum full (g/100)	1.405	1.345	1.694*	2.469*
Cecum empty (g)	1.874	1.876	2.036	2.412*
Cecum empty (g/100)	0.675	0.682	0.739*	0.898*
P2 Male rats	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Final body weight (g)	580.8	584.5	587.9	564.7
Cecum full (g)	4.614	5.211	6.065*	8.700*
Cecum full (g/100)	0.795	0.899	1.036*	1.546*
Cecum empty (g)	1.994	2.283	2.561*	2.878*
Cecum empty (g/100)	0.343	0.390*	0.439*	0.513*
P2 Female rats	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Final body weight (g)	296.3	308.2	307.5	293.3
Cecum full (g)	3.493	3.753	4.093	5.050#
Cecum full (g/100)	1.183	1.234	1.344	1.757#
Cecum empty (g)	1.590	1.580	1.744	2.059*
Cecum empty (g/100)	0.546	0.517	0.572	0.720*

*Statistically different from control mean by Wilcoxon's test, alpha = 0.05

#Statistically different from control mean by Dunnett's test, alpha = 0.05

Bold indicates effects considered treatment-related

Table 2: Incidence of Gross Pathologic Findings

P1 Male rats	Dose level (mg/kg/day)
Target organ/Lesion	0	50	250	1000
Cecum - increased size	0/30	0/30	0/30	21/30
P1 Female rats	Dose level (mg/kg/day)
Target organ/Lesion	0	50	250	1000
Cecum - increased size	0/30	0/30	2/30	20/30
P2 Male rats	Dose level (mg/kg/day)
Target organ/Lesion	0	50	250	1000
Cecum - increased size	0/30	0/30	2/30	17/30
P2 Female rats	Dose level (mg/kg/day)
Target organ/Lesion	0	50	250	1000
Cecum - increased size	0/30	0/30	4/30	10/30

Table 3: Mean Pup Weights

F1 litters	Dose level (mg/kg/day)
Mean pup weights (g)	0	50	250	1000
14 days after culling - Female	30.6	31.4	32.8#	32.3
14 days after culling - Male	31.1	32.1	33.6#	33.3#
21 days after culling - Female	49.6	50.1	49.4	50.9
21 days after culling - Male	50.8	51.2	50.7	53.0
F2 litters	Dose level (mg/kg/day)
Mean pup weights (g)	0	50	250	1000
7 days after culling - Female	15.3	16.1	16.0	16.5#
7 days after culling - Male	16.0	16.9	16.6	17.4
14 days after culling - Female	32.2	32.0	33.0	33.2
14 days after culling - Male	33.6	33.4	33.9	34.7

#Statistically different from control mean by Dunnett's test, alpha = 0.05

Table 4: F1 Weanling Uterine Weights

F1 weanlings	Dose level (mg/kg/day)
Parameter	0	50	250	1000
Uterus (g)	0.047	0.046	0.039*	0.043
Uterus (g/100 g)	0.091	0.091	0.079	0.084

* Statistically different from control mean by Wilcoxon's test, alpha = 0.05

Applicant's summary and conclusion

Conclusions:

There were no adverse effects of the test material on any parameter of reproductive function, pup survival, growth or development. Based upon the absence of any significant adverse effects, the No-Observed-Adverse-Effect-Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day, while 1000 mg/kg/day (the highest dose level tested) was considered a No-Observed-Effect-Level (NOEL) for reproductive toxicity.

Executive summary:

The toxicity of the test material to reproduction was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 870.3800, OECD 416, EEC 0378 -6978 and JMAFF 2 -1 -17.

The purpose of the two-generation dietary reproduction toxicity study was to evaluate the potential effects of the test material on male and female reproductive function, as well as the survival, growth and development of the offspring.

During the study groups of 30 male and 30 female rats were fed diets supplying 0, 50, 250, and 1000 mg test material/kg/day for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations. In-life parameters included clinical observations, feed consumption, body weights, oestrous cyclicity, reproductive performance, puberty onset, pup survival, and pup body weights. In addition, post-mortem evaluations included gross and histopathology, organ weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

Body weights of both adult P1 and P2 animals and weanling F1 and F2 pups were unaffected by treatment. Furthermore, there were no treatment-related effects on organ weights with the exception of the cecum. Statistically significant increases in absolute and relative full and empty cecal weights were identified in the 1000 mg/kg/day P1 and P2 adults of both sexes. Similar effects were seen at 250 mg/kg/day, although of lesser severity. At 50 mg/kg/day, the only significant parental effect was a statistically significant increase in relative empty cecal weights in P2 males. Increased cecal size also was noted at gross necropsy of the 250 and 1000 mg/kg/day adults. There were no treatment-related histopathologic changes in any tissue examined, including the cecum. Thus, cecal enlargement was interpreted to represent an adaptive process to a physiological effect localised to the cecum and was not considered toxicologically significant. There were no adverse effects of the test material on any parameter of reproductive function, pup survival, growth or development.

Based upon the absence of any significant adverse effects, the No-Observed-Adverse-Effect-Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day, while 1000 mg/kg/day (the highest dose level tested) was considered a No-Observed-Effect-Level (NOEL) for reproductive toxicity.