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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2001 to 13 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental 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 Japanese Ministry of Agriculture, Forestry and Fisheries, Notification of 12-Nohsan-8147, Guideline 2-1-18, Teratogenicity 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:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Sexually mature adults, 5 - 6 months of age
- Weight at study initiation: 2.5 - 3.5 kg
- Housing: Animals were housed individually in suspended stainless steel cages with flattened tube grid floors. Cages contained a stainless steel hanging feeder and a pressure-activated nipple-type watering system.
- Diet: ad libitum
- Water: municipal water, ad libitum
- Acclimation period: ca. 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 40 - 60 % (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: gavage

Vehicle:

other: 0.5 % METHOCEL® A4M

Details on exposure:

DOSE PREPARATION
The test material was administered as a suspension in an aqueous vehicle of 0.5 % METHOCEL® A4M such that a dose volume of 4 mL/kg body weight yielded the targeted dose. Dose volumes were adjusted daily based on individual body weights. Due to the length of the dosing period, dose suspensions were prepared periodically throughout the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity
The low- and high-dose suspensions from the first mix were analysed prior to the start of dosing to verify homogeneous distribution of the test material in vehicle.
Phase I: Analysis of aliquots from the low- and high-dose suspensions indicated that the test material was homogeneously distributed.
Phase II: These suspensions were also found to be homogeneously distributed.

- Concentration Verification
Analysis of all dosing suspensions from the first mix for both Phase I and Phase II were initiated prior to the start of dosing using HPLC with ultraviolet detection and external standards to determine concentrations.
Phase I: Analyses of all dosing suspensions from the first mix revealed mean concentrations of test material ranging from 103 to 106 % of the targeted concentrations.
Phase II: The mean concentrations for both dosing suspensions (500 mg/kg/day and 750 mg/kg/day) from the first mix were found to be 102 % of the targeted concentrations.

- Stability
The test material was previously found to be stable in 0.5% METHOCEL® A4M at concentrations ranging from 62.5 mg/mL to 250 mg/mL for at least 32 days. Since the low concentration in the present study was 6.25 mg/mL, stability of the test material in the vehicle for the low-and high-dose levels was confirmed for the period of use (i.e., 14 days). Stability analyses were conducted concurrent with the conduct of the study.
Phase I: Reanalysis of the high- and low-dose suspensions after 14 days of use revealed mean dose concentrations for the low- and high-dose suspensions were 90.1 and 106.4 % of the initial values, respectively, indicating that the solutions were stable during this time. Thereafter, dosing suspensions were mixed periodically throughout the study based upon these stability data.
Phase II: Subsequent dose suspensions were mixed periodically based upon stability data generated during Phase I of this study.

Details on mating procedure:

Sexually mature, adult virgin females, weighing approximately 2500-3500 g were naturally mated with males of the same strain at the test animal supply laboratory. The observed day of breeding was considered day 0 of gestation. Day 0 body weights and records of mating pairs were provided by the animal supplier and maintained in the study record. Rabbits were shipped on day 0 or 1 of gestation and arrived in the test laboratory on the same day.

Duration of treatment / exposure:

Animals were administered on days 7-27 of gestation.

Frequency of treatment:

Daily

Duration of test:

Animals were necropsied on gestation day 28.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Phase I

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Phase I

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Phase I

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Phase I

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Phase II

Doses / concentrations 6

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Phase II

Doses / concentrations 7

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Phase II

No. of animals per sex per dose:

26 females per dose.

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels for Phase I were selected based on the preliminary results of the probe study. The high-dose (250 mg/kg/day) was expected to induce overt signs of maternal toxicity (i.e., decreased maternal weight gain). The lower dose levels were selected to provide dose response data for any toxicity that may be observed among the high-dose group rabbits. However, rabbits at 250 mg/kg/day in the full study did not exhibit the significant body weight gain reductions that were observed at 250 mg/kg/day in the probe study. Consequently, a second dosing phase was added to this study. Dose levels for Phase II were chosen based upon body weight gain decreases and decreased food consumption observed at the next highest doses (500 and 750 mg/kg/day) in the probe study.
- Rationale for animal assignment: Randomisation of time-mated rabbits into dose groups was performed using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice each day cage-side examinations were conducted and to the extent possible, the following 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. Morning cage-side examinations were conducted approximately one hour after the completion of dosing on gestation days 7-27.
Moribund animals that were not expected to survive until the next observation period, and any animals found dead, were necropsied on that day. However, animals found dead after routine working hours or on weekends, and holidays were refrigerated until the next scheduled workday, at which time they were necropsied. For animals showing indications of premature delivery, the delivered foetuses were counted and examined to the extent possible. Dosing was discontinued, and animals were euthanised and necropsied on that day or, if found after regular working hours, the next scheduled workday.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were conducted daily throughout the study period. This examination included a careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), unusual swelling or masses and animal behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on day 0 by the supplier, day 4, and daily during the dosing period, and at necropsy (day 28 of gestation). Statistical analyses of body weights and body weight gains were performed using data collected on gestation days 0, 4, 7, 10, 13, 16, 20, 24, and 28.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Feed consumption was recorded for all animals, daily beginning on day 4 by weighing feed containers at the start and end of a measurement cycle. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed container - final weight of feed container) / number of days in measurement cycle

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- All surviving females were euthanised by an I.V. injection of a euthanasia solution (Beuthanasia-D Special) and a limited gross pathologic examination (necropsy) was performed. Given the rapid growth and ossification of the late gestation foetuses, the timing of the maternal sacrifice was counterbalanced across groups (e.g., control, high, middle and low).
The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. The stomach, liver, and kidneys were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded, and the weight of the liver, kidneys, and gravid uterus were recorded. The ratios of liver and kidney weights to day 28 (terminal) body weight were calculated. Representative sections of liver with gallbladder, kidneys, and gross lesions were preserved in neutral, phosphate buffered 10 % formalin. Microscopic examination of tissues was not conducted.

Any animal that died, appeared moribund or showed indications of premature delivery was submitted for a complete necropsy. This necropsy was performed as described above with the following exceptions: 1) animals submitted alive for necropsy were euthanised by the inhalation of CO₂ and subsequent decapitation, 2) the head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined, 3) all viscera were dissected from the carcass and re-examined, 4) liver, kidney and gravid uterine weights were not recorded, 5) the number of corpora lutea and the sex and body weight of foetuses from these animals were not recorded. Development of the conceptuses was determined to the extent possible by external examination (as appropriate for gestational age). The conceptuses were not examined for visceral or skeletal alterations. Following external examination, these conceptuses were discarded. Near term foetuses were euthanised.

Ovaries and uterine content:

The uterine horns were exteriorised through an abdominal incision and the following data were recorded:
1) The number and position of live foetuses in utero.
2) The number and position of dead foetuses.
3) The number, position and classification (early, late) of resorption sites - resorptions were classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognisable embryonic/foetal form.
4) The number of ovarian corpora lutea.
5) The sex and body weight of each foetus.
6) Any external gross alterations in the foetus.
Corpora lutea were not counted for non-pregnant females or for females with totally resorbed litters. The uteri of females without grossly visible evidence of pregnancy were stained with a 10 % aqueous solution of sodium sulphide (Kopf et al., 1964) and examined for evidence of early resorptions to determine pregnancy status.

Fetal examinations:

An external examination of all live and dead foetuses was conducted. This examination included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum, and tail. All live foetuses were euthanised by oral administration of sodium pentobarbital solution. All foetuses were examined by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984).
The visceral examination included observations of the thymus, trachea, oesophagus, lungs, great vessels, heart (external and internal), liver, gallbladder, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. At least one-half of the foetuses in each litter, chosen randomly via computer, were designated for examination of the internal structures of the head. The heads of these selected foetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965).
All foetuses were preserved in alcohol, eviscerated, cleared, stained with alizarin red-S (Dawson, 1926) and examined for skeletal alterations. All foetal alterations were classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development, or function and/or which occurs at a relatively low incidence in the specific species/strain.

Statistics:

Maternal body weights, maternal body weight gains, organ weights (absolute and relative), foetal body weights and feed consumption were evaluated by Bartlett’s test (alpha=0,01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha=0.05, analysis by Dunnett's test (alpha=0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha=0.05; Hollander and Wolfe, 1973) with Bonferroni's correction (Miller, 1966) was performed, respectively.
Frequency of pre-implantation loss, post-implantation loss, resorptions per litter and resorptions per foetal population and foetal alterations were analysed using a censored Wilcoxon test with Bonferroni’s correction (Haseman and Hoel, 1974). The number of corpora lutea and implantations and litter size was evaluated using a nonparametric ANOVA (alpha=0.05) followed by the Wilcoxon Rank-Sum test (alpha=0.05) with Bonferroni's correction. Pregnancy rates were analysed using the Fisher exact probability test (alpha=0.05; Siegel, 1956) with Bonferroni’s correction. Foetal sex ratios were analysed using a binomial distribution test. Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method (alpha=0.02; Grubbs, 1969), and excluded if justified.
As numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data took into consideration statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Indices:

- Pre-implantation loss = [(No. corpora lutea - implantations) / No. corpora lutea] x 100
- Post-implantation loss = [(No. implantations - live born pups) / No. implantations] x 100
Percent pre- and post-implantation loss was determined for each litter, followed by calculation of the mean and standard deviation of these litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details on maternal effects below

Mortality:

mortality observed, treatment-related

Description (incidence):

see details on maternal effects below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Phase I: There were no statistically identified differences in the body weights or body weight gains for any treated groups when compared to their respective controls.
Phase II: The body weight of rabbits administered 750 mg/kg/day was significantly decreased by 6 % relative to controls on gestation day 10. Mean body weights of the 500 mg/kg/day rabbits were comparable to controls throughout the study. Body weight gains of rabbits administered 500 or 750 mg/kg/day were significantly decreased relative to controls on gestation days 7-10. In the controls (0 mg/kg/day), 4 out of 24 rabbits lost weight with a mean weight gain of 25 g reported for the group during gestation days 7-10. At 500 and 750 mg/kg/day, weight losses were seen during this interval in 12 out of 25 and 18 out of 25 rabbits, respectively. Mean weight losses of 11.8 g for the 500 mg/kg/day group and 70.0 g for the 750 mg/kg/day group were reported. During subsequent periods (gestation days 10-28), body weight gain at 500 mg/kg/day were comparable to or higher than controls. At 750 mg/kg/day weight gain remained slightly decreased on gestation days 13-16. The decreases in weight gain observed in the 750 mg/kg/day rabbits were accompanied by significant decreases in feed consumption during the same period. Due to the magnitude of the body weight gain decrease seen on gestation days 7-10 and the severity of clinical signs observed in several 750 mg/kg/day rabbits, this entire dose group was removed from study early without further data collection.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Phase I: There were no significant differences in the amount of feed consumed by any treated groups when compared to their respective controls.
Phase II: Feed consumption of rabbits administered 750 mg/kg/day was decreased for several days (gestation days 7-17) following the initiation of dosing. Statistically identified decreases were noted on 8 of 10 days and the magnitude of the decreased feed consumption ranged from 20 to 45 % of the control values. The 750 mg/kg/day group was terminated early as previously mentioned due to the severity of maternal toxicity observed. Rabbits administered 500 mg/kg/day had an 18 % decrease in feed consumption relative to controls on GD 7-8 that was statistically identified.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Phase I: There were no statistically identified differences in any of the measured parameters for any treated groups when compared to their respective controls.
Phase II: There were no statistically identified differences in any of the measured parameters for the 500 mg/kg/day group compared to the controls. Organ weights were not evaluated for the 750 mg/kg/day group which was terminated early due to excessive toxicity.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Phase I: 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.
Phase II: Treatment-related gross pathologic observations were noted in three rabbits given 750 mg/kg/day which were terminated early due to excessive toxicity. These observations included, pale kidneys (cortex) and an increased incidence of alterations in the glandular mucosa of the stomach (erosion-ulcer, hyperaemia or mottled appearance). A single rabbit given 500 mg/kg/day had ulcers/erosions in the glandular mucosa of the stomach which may have been treatment-related. All other gross pathologic observations in rabbits given 500 mg/kg/day were considered to be spontaneous alterations, unassociated with exposure to the test material.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Mean percent pre-implantation loss was significantly increased at 25 mg/kg/day; however, this result was considered spurious based on the lack of a dose-response and because treatment is initiated when implantation is complete.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
> Phase I: Examinations performed on all animals prior to the study and at termination revealed no treatment-related findings. Health evaluations performed on all animals upon arrival revealed various lacerations and scratches which were likely inflicted during breeding at the supplier’s facility.
During the dosing period, soft faeces and absent or decreased amounts of faeces were noted in various rabbits in the treated and untreated (control) groups. In the absence of any significant decreases in body weight or body weight gain, these incidences were considered within the normal range for rabbits. One rabbit (2085) was removed from study on gestation day 3 (prior to initiation of treatment). This rabbit had been assigned to the 250 mg/kg/day group, and was apparently not eating after arrival and had decreased activity. In addition, this doe had soft/watery faeces with an abnormal odour which contained a mucoid matter. A single 250 mg/kg/day rabbit (2088) had blood in her cage and aborted one resorbing foetus on gestation day 19 with no prior clinical observations. This rabbit was subsequently euthanized due to the abortion and examined grossly at necropsy. Gross findings for this rabbit included pale kidneys (bilateral) with evidence of recent abortion in the uterus. The cause of this abortion was not determined. Another 250 mg/kg/day rabbit (2095) was found dead on gestation day 15. This rabbit had exhibited a reflux of the test material at the time of dosing (gestation day 15) and subsequently had noisy and laboured respiration along with bluish skin and mucous membranes. At necropsy, this rabbit was found to be pregnant with 14 normally developed foetuses and one early resorption within the uterus. This rabbit also had mottled lungs with oedema, froth in the trachea and bronchi, and external soiling (blood tinged froth) on the nares and muzzle. All of these finding supported a cause of death due to a gavage complication. Finally, one 25 mg/kg/day rabbit (2037) had blood in her cage and aborted four foetuses on gestation day 26. In the days preceding (gestation day 14-26), this doe was noted as having decreased and mucoid faeces in her cage pan. Upon gross examination following euthanasia, this rabbit was found to have seven normally developed foetuses and one late resorption still within the uterus. Other observations for this rabbit included perineal soiling and dark ingesta/watery contents in the cecum. The cause of the abortion was not determined. All other Phase I rabbits survived to the scheduled necropsy.
> Phase II: As in Phase I, health evaluations performed on all animals upon arrival revealed several lacerations, scratches and bruises which were likely inflicted during breeding at the supplier’s facility. Two 750 mg/kg/day rabbits were sacrificed early on gestation day 17 (moribund) with prior clinical signs that included inco-ordinated gait, perineal urine soiling, decreased faeces, and decreased activity. Additionally, these two does showed significant decreases in body weight gains and feed consumption (gestation days 7-16). At gross necropsy examination, both rabbits were pregnant and observed to have pale kidneys (cortex), watery, dark cecal contents, erosions/ulcers in the glandular mucosa of the stomach and hairballs. The moribund condition of these rabbits was considered due to treatment. Another 750 mg/kg/day rabbit died on gestation day 8. Just prior to death, this rabbit was observed to have perineal urine and faecal soiling, laboured respiration, and an absence of faeces in its cage pan. Gross findings at necropsy included perineal soiling, congestion in the kidneys and liver, watery contents in the cecum, lung consolidation (anterior ventral portions), abnormal lung distension (dorsal portions), exudate in the bronchus, mucoid exudate and froth in the trachea, and petechia in the stomach. The cause of death was attributed to a gavage complication. One doe in the 500 mg/kg/day dose group died on gestation day 27. Observations at gross necropsy included a frothy red discharge around the external nares and muzzle, froth in the trachea and bronchi (bilateral), and dark consolidation in both lungs with accumulation of serosanguinous fluid and abnormal lung distension (bilateral and diffuse). Death was attributed to the diffuse, oedematous state of the lungs, which suggests a gavage complication. All other rabbits survived their respective test periods. Treatment-related clinical signs were noted in rabbits administered 750 mg/kg/day and included increased incidences of decreased amounts of faeces (associated with significant reductions in body weight and body weight gain), and inco-ordinated movement shortly following dosing.
The incoordination observed at 750 mg/kg/day was characterised by a reluctance to move unless manually stimulated by the observer. Significant stiffening or dragging of the limbs was evident when movement was attempted and on various occasions several rabbits tipped onto their sides. In most cases, the inco-ordination was transient, with complete resolution of the inco-ordination within two hours post-dosing. Also, the degree of inco-ordination observed did not appear to progressively worsen on subsequent days. A similar incidence of inco-ordination was also observed among the rabbits in the 500 mg/kg/day dose group. However, the degree of inco-ordination observed was much less pronounced than that observed in the 750 mg/kg/day rabbits. In general, when stimulated to move, the 500 mg/kg/day rabbits exhibited cautious placement of their paws with a slight stiffening or dragging of the forelimbs. Unlike the 750 mg/kg/day rabbits, none of the 500 mg/kg/day rabbits tipped over when moving. The inco-ordination observed at 500 mg/kg/day resolved within two hours of dosing. Due to the severity of clinical signs and corresponding significant decreases in body weight gain on gestation days 7-10 observed in the majority of the 750 mg/kg/day rabbits, this entire dose group was removed from study on October 8, 2001.

BODY WEIGHTS
> Phase I: There were no statistically identified differences in the body weights or body weight gains for any treated groups when compared to their respective controls.
> Phase II: The body weight of rabbits administered 750 mg/kg/day was significantly decreased by 6 % relative to controls on gestation day 10. Mean body weights of the 500 mg/kg/day rabbits were comparable to controls throughout the study. Body weight gains of rabbits administered 500 or 750 mg/kg/day were significantly decreased relative to controls on gestation days 7-10. In the controls (0 mg/kg/day), 4 out of 24 rabbits lost weight with a mean weight gain of 25 g reported for the group during gestation days 7-10. At 500 and 750 mg/kg/day, weight losses were seen during this interval in 12 out of 25 and 18 out of 25 rabbits, respectively. Mean weight losses of 11.8 g for the 500 mg/kg/day group and 70.0 g for the 750 mg/kg/day group were reported. During subsequent periods (gestation days 10-28), body weight gain at 500 mg/kg/day were comparable to or higher than controls. At 750 mg/kg/day weight gain remained slightly decreased on gestation days 13-16. The decreases in weight gain observed in the 750 mg/kg/day rabbits were accompanied by significant decreases in feed consumption during the same period. Due to the magnitude of the body weight gain decrease seen on gestation days 7-10 and the severity of clinical signs observed in several 750 mg/kg/day rabbits, this entire dose group was removed from study early without further data collection.

FEED CONSUMPTION
> Phase I: There were no significant differences in the amount of feed consumed by any treated groups when compared to their respective controls.
> Phase II: Feed consumption of rabbits administered 750 mg/kg/day was decreased for several days (gestation days 7-17) following the initiation of dosing. Statistically identified decreases were noted on 8 of 10 days and the magnitude of the decreased feed consumption ranged from 20 to 45 % of the control values. The 750 mg/kg/day group was terminated early as previously mentioned due to the severity of maternal toxicity observed. Rabbits administered 500 mg/kg/day had an 18 % decrease in feed consumption relative to controls on GD 7-8 that was statistically identified.

ANATOMIC PATHOLOGY
Organ Weights
> Phase I: There were no statistically identified differences in any of the measured parameters for any treated groups when compared to their respective controls.
> Phase II: There were no statistically identified differences in any of the measured parameters for the 500 mg/kg/day group compared to the controls. Organ weights were not evaluated for the 750 mg/kg/day group which was terminated early due to excessive toxicity.

Gross Pathology
> Phase I: 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.
> Phase II: Treatment-related gross pathologic observations were noted in three rabbits given 750 mg/kg/day which were terminated early due to excessive toxicity. These observations included, pale kidneys (cortex) and an increased incidence of alterations in the glandular mucosa of the stomach (erosion-ulcer, hyperaemia or mottled appearance). A single rabbit given 500 mg/kg/day had ulcers/erosions in the glandular mucosa of the stomach which may have been treatment-related. All other gross pathologic observations in rabbits given 500 mg/kg/day were considered to be spontaneous alterations, unassociated with exposure to the test material.

REPRODUCTIVE PARAMETERS
> Phase I: Mean percent pre-implantation loss was significantly increased at 25 mg/kg/day; however, this result was considered spurious based on the lack of a dose-response and because treatment is initiated when implantation is complete. Thus, there were no significant treatment related effects on pregnancy rates, numbers of corpora lutea, implantations, or viable foetuses per litter, percent pre-implantation loss, percent post-implantation loss, resorption rates, foetal body weights, foetal sex ratios or gravid uterine weights at any dose level.
> Phase II: There were no significant treatment related effects on pregnancy rates, numbers of corpora lutea, implantations, or viable foetuses per litter, percent pre-implantation loss, percent post-implantation loss, resorption rates, foetal body weights, foetal sex ratios or gravid uterine weights at 500 mg/kg/day.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

other: maternal toxicity

2

Key result

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse test substance related effects at the highest dose level with fetuses

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see details on fetal effects below

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see details on fetal effects below

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FOETAL ALTERATIONS
> Phase I: There were no treatment-related differences in the incidence of any foetal malformations in any of the dose groups. There were two malformed control foetuses from two separate litters. One control foetus had a bifurcated renal artery, while the other exhibited skeletal malformations in the thoracic region which included fused vertebrae and centra coupled with a hemivertebra. A total of four 25 mg/kg/day foetuses from three litters had malformations. Three foetuses presented with bifurcated renal arteries. A fourth foetus had a missing gallbladder. Among the eight 100 mg/kg/day foetuses (six affected litters) that were malformed, a single foetus had a bifurcated renal artery. A second foetus had a persistent truncus arteriosus. Two other foetuses had hemivertebra in the cervical region (atlas) with one also having a missing gallbladder. Another foetus had visceral malformations which included multiple missing lung lobes in association with a diaphragmatic hernia and a missing gallbladder. The sixth foetus had malformed ribs (fused and forked) with a hemivertebra. Another foetus had multiple skeletal malformations which included misshapen short and long bones of the fore- and hind-limbs along with class II wavy ribs. The final 100 mg/kg/day foetus had fused ribs and centra along with a hemivertebra in the thoracic region. Finally, three foetuses from three separate 250 mg/kg/day litters were malformed. One foetus had a bifurcated renal artery. Another had a displaced aortic arch (right-sided) and missing innominate artery. The third foetus had persistent truncus arteriosus. With regard to foetal variations, the only statistically identified difference was a decrease in the incidence of fused sternebrae at 100 mg/kg/day when compared to controls. There were no fused sternebrae at either 25 or 250 mg/kg/day. These decreases in fused sternebrae were considered spurious as a decrease rather than an increase in incidence was identified. Other foetal variations observed during Phase I of this study occurred at low frequencies and/or did not follow a dose-response relationship in litter incidence. Therefore, there were no differences in the incidence of foetal alterations related to treatment with the test material.
> Phase II: There were no treatment-related differences in the incidences of any foetal malformations or variations at 500 mg/kg/day when compared to controls. In total, there were ten malformed foetuses from 5 litters in the control group. Of these, six foetuses had bifurcated renal arteries. One foetus had a cardiac malformation consisting of transposition of the great vessels, a misshapen aortic semilunar valve and right ventricle, and a ventricular septal defect. Other control foetuses had dilated cerebral ventricles, missing cervical vertebrae, and fused ribs. At 500 mg/kg/day, three foetuses from a single litter were malformed. One foetus had a missing testis, one had fused thoracic centra, and another had fused thoracic centra associated with a hemivertebra.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse test substance related effects at the highest dose level

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Phase II Clinical Observations

Clinical observations*	Dose level (mg/kg/day)
	0	500	750
Faeces, abnormal quantity, decreased	1	3	6
Gait, incoordinated	0	23	23

*n = 26 animals per treatment group

Table 2: Phase II Select Mean Body Weights and Body Weight Gains

Endpoint	Dose level (mg/kg/day)
	0	500	750
Body weight on GD 10 (g)	3265.0	3246.7	3068.7*
Body weight gain on GD 7-10 (g)	25.0	-11.8#	-70.0#
Body weight gain on GD 13-16 (g)	97.3	91.8	33.2
Body weight gain on GD 0-28 (g)	359.7	374.5	no data

*Statistically difference from control mean by Dunnett's test, alpha = 0.05

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

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the no-observed-effect-levels (NOEL) for maternal and developmental toxicity were 250 mg/kg/day and 500 mg/kg/day, respectively.

Executive summary:

The developmental toxicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 414, EPA OPPTS 870.3700, EEC 0378-6978 and JMAFF 2-1-18.

During the study groups of 26 time-mated female New Zealand White rabbits were administered test material, via gavage, at targeted dose levels of 0, 25, 100, or 250 mg/kg/day (Phase I) or 0, 500 or 750 mg/kg/day (Phase II) on gestation days 7 through 27. In-life maternal parameters included clinical observations, body weight, body weight gain, and feed consumption. On gestation day 28, all surviving rabbits were euthanised and examined for gross pathologic alterations and changes in liver, kidney and gravid uterine weights. The numbers of corpora lutea, uterine implantations, resorptions and live/dead foetuses were determined. All foetuses were weighed, sexed and examined for external, visceral and skeletal alterations. Also, the internal structures of the head were examined by serial sectioning for approximately one-half of the foetuses in each litter.

Phase I rabbits given ≤ 250 mg/kg/day test material did not exhibit any treatment-related maternal or developmental toxicity, thus prompting the evaluation of higher doses in Phase II. At 500 mg/kg/day, maternal toxicity was evidenced by transient uncoordinated gait (23/26 does) and decreased body weight gains (gestation days 7-10), but there was no evidence of embryonal/foetal effects. At 750 mg/kg/day, uncoordinated gait was accompanied by decreased amounts of faeces, significant reductions in body weight, body weight losses, decreased body weight gains, and decreased feed consumption. Also, two 750 mg/kg/day does were euthanised in moribund condition (gestation day 17). At necropsy, these does had pale kidneys, watery, dark cecal contents, erosions/ulcers of the stomach (glandular mucosa) and hairballs. As 750 mg/kg/day exceeded the maximum tolerated dose, all surviving rabbits in the dose group were removed from study.

Based upon these results, the no-observed- effect-levels (NOEL) for maternal and developmental toxicity were 250 mg/kg/day and 500 mg/kg/day, respectively.