Nitrapyrin

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 1993 to 14 April 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines, and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Methods for the Determination of Toxicity (EEC 1988)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF Toxicity Tests Guidelines (JMAFF 1985)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nitrapyrin

EC Number:

217-682-2

EC Name:

Nitrapyrin

Cas Number:

1929-82-4

Molecular formula:

C6H3Cl4N

IUPAC Name:

2-chloro-6-(trichloromethyl)pyridine

Test material form:

solid: crystalline

Details on test material:

- Appearance: white crystalline solid
- Storage conditions of test material: room temperature

Test animals

Species:

rat

Strain:

other: CD [SD] BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 50 days on arrival, 66 days on initiation of mating (females); 49 days on arrival (males)
- Weight at study initiation: 200 - 309 g (females)
- Housing: Individually, except during mating, in stainless steel suspended cages with wire mesh floors and fronts.
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 16 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 40 - 66 %
- Photoperiod (hrs dark / hrs light): 12 hours of darkness / 12 hours of light (07:00 to 19:00)

IN-LIFE DATES: From: 26 April 1993 To: 16 June 1993

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of test material were dissolved in the vehicle. Two batches of solutions were prepared at each concentration (dose) level and used for dosing in the study. When not being dosed, these solutions and vehicle were stored at ambient temperature. A correction for the purity of the test material was made in the preparation of dosing solutions.

DOSE VOLUME
5 mL/kg bw/day; initial dose volumes administered to individual animals were derived from Day 6 gestation body weights. The dose volumes were adjusted on Days 9 and 12 of gestation to changes in the individual animal's body weights. Dosing of the animals initiated at approximately the same time each day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity Samples/ Analyses: Homogeneity of 400 mL batches of solutions prepared at concentration levels of 10 and 40 mg/mL was confirmed in the range-finding study. Since the low concentration for this study (3 mg/mL) was out of this range, a mock batch of solution at this concentration was prepared pre-test and evaluated for homogeneity and stability. Additionally, since the batch size of solutions prepared for this study was increased from that used in the range-finding study, an additional homogeneity assessment was performed at the high-concentration level (24 mg/mL). These evaluations at the high-concentration were performed on the first batch of solution used for dosing. To assess homogeneity three samples were collected from each of the top, middle and bottom portions of these solutions while stirring, and analysed.

- Stability Samples/ Analyses: Stability of the dosing solutions at concentration levels of 10 to 40 mg/mL were established for 22 days post-preparation at ambient storage during the previous study. Since the high concentration level for this study (24 mg/mL) was within this range the only additional stability assessments performed for this study were for the low-concentration level (3 mg/mL). The mock batch of solution prepared at this concentration level to assess homogeneity was held at ambient temperature. This solution was sampled for analysis on Days 7, 14 and 21 post-preparation to determine stability.

- Analyses to Confirm Concentrations of Dosing Formulations: The homogeneity analyses at the high-concentration served as confirmation for the use of this batch of dosing solution on study. At the low- and mid-concentration levels, samples of solutions from the first batch were taken on the day of preparation and analysed in duplicate. For the second batch of dosing solutions prepared for use on study, one sample was taken from each concentration level and analysed in duplicate.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male: 1 female
- Females selected for mating were placed with male rats nightly in a 1:1 ratio. Vaginal smears were taken early in the morning following intervals of nightly cohabitation and females were considered to have mated if sperm was noted microscopically in the vaginal rinse and/or a plug was observed in the vaginal opening. The day on which evidence of mating was observed was defined as Day 0 of gestation.
The evenings for co-habitation of males with females were scheduled to provide females at Day 20 gestation sacrifice during the Monday-to-Friday work week. The number of females caged nightly with males was also controlled to limit the number of mated females available for sorting into groups on a particular day. In this study, the maximum number of mated females sorted into groups on a particular day was 19. When the number of females mated after an evening exceeded the number to be sorted among the groups that day, females excluded from the sorting procedure were determined using a random numbers table and the female's temporary cage card number.

Duration of treatment / exposure:

Treatment for the Day 6 - 15 gestation interval

Frequency of treatment:

Daily

Duration of test:

Animals were sacrificed on Day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

120 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

28 mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose selection was based on findings from a rang-finding study.
- Rationale for animal assignment: A sufficient number of female rats was received so as to discard unhealthy females prior to initiation of mating.
The population of animals received for study was evaluated for suitability to use on test by the staff veterinarian prior to the initiation of mating. Females which mated were assigned to the groups daily in such a way as to provide an equal distribution of mated females among groups and equalise, as best possible, the Day 0 gestation mean body weights between groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (morning and afternoon)
- Cage side observations included checks for signs of pharmacologic or toxicologic effects and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each female was given a detailed physical examination on Days 0, 6 - 16 and 20 of gestation. On Days 6 - 15 of gestation, the detailed physical examinations were conducted approximately 1 hour after all animals had been dosed.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded on Days 0, 6, 9, 12, 16 and 20 of gestation. Day 20 gestation body weights are presented as actual and corrected (the actual Day 20 gestation body weight minus the weight of the gravid uterus).

FOOD CONSUMPTION: Yes
- Recorded for the following intervals during gestation: Days 0 - 6, 6 - 9, 9 - 12, 12 - 16 and 16 - 20.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 by exsanguination following anaesthesia with carbon dioxide.
- Complete gross postmortem examinations were performed on all test females. External gross examinations of all surfaces, all orifices, the cranial cavity, carcass, the external surface of the spinal cord and sectioned surfaces of the brain, nasal cavity and paranasal sinuses, the thoracic, abdominal and pelvic cavities and their viscera and the cervical tissues and organs were performed for all animals. The carcass of each female was discarded at completion of the gross postmortem evaluation.

ORGAN WEIGHTS: Yes
- The liver and kidneys were weighed for all females, and liver/body weight and kidney/body weight ratios were calculated using the corrected Day 20 gestation body weights.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

The intact uterus (ovaries attached) was removed from the abdominal cavity, weighed and the number and location of the following were recorded for each uterine horn: live foetuses (movement in response to touch); dead foetuses (lack of movement in response to touch but no evidence of tissue degeneration); late resorptions (recognisable dead foetus undergoing degeneration regardless of size); early resorptions (evidence of implantation but no recognisable foetus); and implantation sites.
The ovaries were dissected free from the uterus and evaluated for the presence and number of corpora lutea. When no uterine implants were grossly apparent, the uterus was stained with ammonium sulphide. When no foci were visualised post-staining, the female was considered not pregnant.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Each foetus was individually identified, weighed, sexed externally (ano-genital distance) and given a gross examination for external malformations/variations to include observation for palatal defects.

- Visceral Evaluations
Approximately one-half of the foetuses in each litter (alternating foetuses within the litter) were evaluated for visceral malformations/variations using a microdissection procedure. Evaluations were performed on the fresh foetal specimens shortly after removal from the uterus. Foetuses designated for visceral evaluation were decapitated (head placed in appropriately labelled tissue bags and fixed in Bouin's solution for later evaluation). The foetal specimens were then secured beneath a dissecting microscope and dissected so as to permit evaluation of tissues in the thoracic and abdominal cavities. At the completion of the foetal examination, the foetuses were eviscerated (viscera discarded) and placed in individual plastic cassettes and stored in a 70 % ethanol solution. Following a period of fixation, the foetal heads were sectioned using a razor blade. The serial, transverse sections generated during this procedure were evaluated for malformations of the palate, eyes and brain under a dissecting microscope. Following evaluation, head sections were placed in plastic cassettes for storage (one litter/jar) in a 70b% ethanol solution.

- Skeletal Evaluations
The remaining foetuses in each litter were killed via an overdose of inhaled carbon dioxide. The intact foetuses were eviscerated (internally sexed by inspection of the gonads) and processed for staining of the ossified skeletal structures using a modified Alizarin Red S staining procedure. Foetal skeletal specimens were evaluated under a dissecting microscope for ossification variations and malformations.

- Late Resorptions
Late resorptions were weighed, examined grossly for external malformations and discarded.

Statistics:

Data were analysed between control (Group I) and treated groups (Groups II-IV).

- Interval Data
Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. First, Bartlett's test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a non-parametric procedure for testing equality of means was needed, the Kruskal-Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control.
The Bartlett's test was conducted at the 1 %, two-sided risk level. All other statistical tests were conducted at the 5 and 1 %, two-sided risk level. All ratios were transformed via the arc sine transformation prior to analysis.

- Incidence Data
Statistical analysis of incidence data was performed using contingency tables. First, a standard chi-square analysis was performed to determine if the proportion of incidences differed between the groups tested. Next, each treatment group was compared to the control group using a 2x2 Fisher Exact Test; the significance level was corrected via the Bonferroni inequality to assure an overall test of the stated significance level. Thirdly, Armitage's test for linear trend of the stated significance level. As standard, if any one cell had an expected value less than 5, the chi square and test was not reported. When this occurred, only the Fisher Exact test (corrected via Bonferroni inequality) was performed and reported.
All tests were reported at the 5 and 1 % level of significance.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY
No mortality occurred among the control or treated groups as all animals survived to scheduled sacrifice.

PREGNANCY RATES
Pregnancy rates for the low- and mid-dose groups were comparable to control data. In the high-dose group (120 mg/kg/day), the pregnancy rate was 82.1 % (23/28 females). This value was lower than the pregnancy rate of the control group (89.3 %); however, this slight difference was not statistically significant and was not considered indicative of an adverse effect of treatment with test material.

BODY WEIGHTS
Mean body weights during gestation (Days 0, 6, 9, 12, 16 and 20) and mean body weight gain during the pre-treatment (Days 0-6), treatment (Days 6 - 9, 9 - 12, 12 - 16 and 6 - 16) and post-treatment (Days 16 - 20) intervals for the low-dose group (15 mg/kg/day) were comparable to control. Likewise, the mean corrected Day 20 gestation body weight and mean body weight gain over the Day 6 - 20 gestation interval using these corrected weights, for the low-dose group, were comparable to control.
In the mid-dose group (50 mg/kg/day), mean body weights during gestation to include the corrected Day 20 gestation weights were comparable to control. Likewise, mean body weight gains for the mid-dose group during the pre- and post-treatment periods and for Day 6 - 9 and 9 - 12 intervals of the treatment period were comparable to control. During the Day 12 - 16 gestation interval and over the entire treatment period (Days 6 - 16), mean body weight gains for the mid-dose group were lower than control and these differences were statistically significant and considered indicative of a treatment-related response. Mean body weight gain for the mid-dose group over the Day 6 - 20 gestation interval using the corrected Day 20 gestation body weights, was comparable to control.
In the high-dose group, mean body weights on Gestation Days 9, 12, 16 and 20 (actual and corrected) were lower than control and while these differences were slight (<10 %) many were statistically significant. During the Day 6 - 9 gestation interval representing the first three days of treatment, the high-dose group experienced a mean weight loss of 5 g compared to a mean weight gain of 12 g for the control group; this difference was statistically significant. Thirteen of the 23 pregnant high-dose females (56.5 %) lost weight during the Day 6 - 9 gestation interval compared to one of 25 control females (4.0 %). Mean body weight gain for the high-dose group during the Gestation Day 9 - 12 treatment interval (11 g) was lower than control data (21 g); however, this difference was not statistically significant. Four high-dose females (17.4 %) lost weight during the Day 9 - 12 gestation interval while none of the control females lost weight over this same interval. Mean body weight gains for the high-dose group over the Day 12 - 16 and 16 - 20 gestation intervals were comparable to control. Mean body weight gains for the high-dose group over the entire treatment interval (Days 6 - 16) and for the Day 6 - 20 gestation interval using the corrected Day 20 gestation body weights, were lower than control and these differences were statistically significant. The weight loss seen in the high-dose group for the Day 6 - 9 gestation interval and reductions in body weight gains for the Day 9 - 12, 6 - 16 and 6 - 20 gestation intervals were considered indicative of treatment-related responses.
Thus, no adverse effect of treatment with test material at a dose level of 15 mg/kg/day was indicated from maternal body weight or body weight change data during gestation. At the higher dose levels evaluated, a maternal toxic response to treatment was indicated from reduced body weight gain during the treatment period. These changes were most severe at the 120 mg/kg/day dose level.

FOOD CONSUMPTION
The only adverse effect of treatment seen in feed consumption data occurred at the high-dose level. Mean feed consumption data for the low- and mid-dose groups during the pre-treatment (Days 0 - 6), treatment (Days 6 - 9, 9 - 12 and 12 - 16) and post-treatment (Days 16 - 20) intervals of gestation were comparable to control. In the high-dose group, mean feed consumption data during the Days 6 - 9 and 9 - 12 gestation intervals were lower than control; however, only for the Day 6 - 9 interval was this difference of -19.2 % statistically significant. During the Day 12 - 16 and 16 - 20 gestation intervals, mean feed consumption data for the high-dose group were higher than control and these differences were statistically significant. The latter was considered to represent compensatory feeding in the high-dose animals related to the reduced level of feeding seen earlier in the treatment period.
Thus, no adverse effect of treatment with test material at a dose level up to and including 50 mg/kg/day was indicated from maternal feed consumption data during gestation. At the 120 mg/kg/day dose level, feed consumption was reduced early in the treatment period (Days 6 - 9 and 9 - 12) and elevated at later intervals (Days 12 - 16 and 16 - 20).

PHYSICAL OBSERVATIONS
Alopecia of the extremities/snout is seen commonly in the testing laboratory for this strain of rat and was the most common finding seen among control and treated animals in this study. The slight increase in occurrence of this finding among the treated animals was not considered indicative of a treatment-related response. No adverse effect of treatment with test material at the 15 and 50 mg/kg/day dose levels was evident from the detailed physical examinations. Generally, observations seen among animals in these groups occurred with similar incidence as control and no effect of treatment was indicated.
At the 120 mg/kg/day dose level, excessive salivation was seen with increased incidence. The incidence of high-dose females noted at least once during the treatment period with excessive salivation was 35.7 % (10/28); this finding was not seen among the control and mid-dose females throughout the study and was seen only in one low-dose female at a single interval. It was considered to represent a local response to residual amounts of test material in the buccal cavity following the dosing procedure. Other observations noted among the high-dose animals occurred at low incidence and no effect of treatment was indicated from these data.

CORPORA LUTEA AND IMPLANT DATA
A total of 12 females (three control, two low-dose, two mid-dose and five high-dose) did not have uterine implantation sites at Day 20 gestation sacrifice. The uteri of these females were then processed in an ammonium sulphide solution to reveal stained foci suggestive of implantation and early foetal death with resorption. No foci were seen in any of the uteri of these females and they were considered not pregnant.
The mean numbers of corpora lutea and uterine implantation sites per female and the mean pre-implantation loss indices for the treated groups were comparable to control. Likewise, the mean number of viable foetuses per female for the treated groups was comparable to control data. Only one dead foetus was recovered at Day 20 gestation sacrifice and that occurred in the high-dose group. This low incidence of dead foetuses in utero for the high-dose group was not considered indicative of a treatment-related response.
The mean number of resorptions, the mean resorption/implant ratios and the incidence of females with resorptions for the treated groups were higher than control; however, these differences were not statistically significant. While higher than concurrent control data, these resorption data for the treated groups were similar to recent historical control data for the laboratory, and in the concurrent control group, these data were at or outside the low range value of the recent historical control data. Since these differences in resorption data for the treated groups in comparison to concurrent control were not statistically significant, the apparent increase in these data for the treated groups was attributed to unusually low concurrent control data and not considered indicative of a treatment-related response.
Thus, no adverse effect of treatment with test material at a dose level up to and including 120 mg/kg/day was indicated from uterine implantation data.

ORGAN WEIGHTS
Mean kidney and liver weights, absolute and relative to corrected Day 20 gestation weights, for the low- and mid-dose groups were comparable to control data. In the high-dose groups, mean absolute kidney and liver weights were comparable to control; however, mean relative weights for these organs were higher than control and these differences of 7.5 and 8.5 %, respectively were statistically significant. The increases in relative weights for the kidneys and livers in the high-dose group were considered secondary to a statistically significant decrease in mean corrected Day 20 gestation body weight. Thus, no adverse effect of treatment with test material at a dose level up to and including 120 mg/kg was indicated from maternal kidney and liver weight data.

GROSS PATHOLOGY
No adverse effect of treatment with test material at a dose level up to and including 120 mg/kg/day was indicated from maternal gross postmortem evaluations. Findings noted among the treated animals occurred sporadically and were not considered related to treatment.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
FOETAL BODY WEIGHT
Mean foetal weights in the treated groups for both sexes combined and when distinguished by sex, were slightly lower than control and in most instances these differences, which were <6.0 %, were not statistically significant. The only statistical significance seen in these data was for the 5.5 % decrease in weight for female foetuses in the high-dose group. Mean foetal weight data for the treated groups were similar to recent historical control data for the laboratory, while mean foetal weight data for the concurrent control group were outside the high-range of these data. Due to the similarity in foetal weight data between the treated groups and the recent historical control data and the general lack of statistical significance in comparison of these data with concurrent control data, the slight decreases in mean foetal weights seen in the treated groups were not considered indicative of an adverse effect of treatment. Thus, no adverse effect of treatment with test material at a dose level up to and including 120 mg/kg/day was indicated from foetal weight data.

FOETAL SEX DISTRIBUTION DATA
No adverse effect of treatment with the test material at a dose level up to and including 120 mg/kg/day was indicated from foetal sex distribution data. The mean number of male and female foetuses per litter and the ratio of total male/female foetuses per group for the treated groups were comparable to control data.

EXTERNAL MALFORMATIONS
The incidences of foetuses with external malformations for the control, low-, mid- and high-dose groups were 0 % (393 foetuses), 0.3 % (1/398 foetuses), 0.3 % (1/379 foetuses) and 0 % (357 foetuses), respectively. The incidences of litters containing foetuses with external malformations for these same groups were 0 % (25 litters), 3.8 % (1/26 litters), 3.8 % (1/26 litters) and 0 % (23 litters), respectively. These incidences on both a per foetus and per litter basis, for the treated groups were considered comparable to control. In the low-dose group, one foetus from the litter of female No. 2577 had multiple defects which included the following: omphalocele; absence of eyebulges [skin over this region was removed and no eye tissue was present]; shortened hindlimb; and digital defects of the hindpaws (oligodactyly and syndactyly). In the absence of these types of malformations among foetuses at the higher dose levels, their occurrence in one low-dose foetus was not considered indicative of a treatment-related response. No other external malformations were seen among the remaining low-dose foetuses.
In the mid-dose group, one foetus from the litter of female No. 3583 had a filamentous tail. This malformation has been seen at low incidence in recent historical control data for the laboratory. In the absence of this malformation among the high-dose foetuses, its low incidence of occurrence in the mid-dose group was not considered indicative of a treatment-related response.
Thus, no adverse effect of treatment with test material at a dose level up to and including 120 mg/kg/day was indicated from the foetal external evaluations.

VISCERAL MALFORMATIONS
The incidences of foetuses with visceral malformations for the control, low-, mid- and high-dose groups were 0 % (200 foetuses evaluated), 1.5 % (3/205 foetuses), 0.5 % (1/197 foetuses) and 1.1 % (2/183 foetuses), respectively. The incidences of litters containing foetuses with visceral malformations for these same groups were 0 % (25 litters), 7.7 % (2/26 litters), 3.8 % (1/26 litters) and 8.7 % (2/23 litters), respectively. These low incidences of visceral malformations, on both a per foetus and per litter basis, for the treated groups did not differ statistically from control data.
In the low-dose group, one foetus was noted with a folded retina and another foetus from a different litter had distended lateral ventricles of the brain. Both of these malformations are seen at low incidence historically in the laboratory and in the absence of similar malformations among foetuses at the higher dose levels, their low incidence of occurrence in the low-dose group was not considered indicative of a treatment-related response. The only other visceral malformation seen in the low-dose group was the protrusion of liver tissue through the abdominal wall in the foetus noted externally with an omphalocele.
In the mid-dose group, the only malformation noted at visceral examination was cleft palate which had not been noted during the external foetal examinations. This malformation is noted at low incidence historically in the laboratory and in the absence of its occurrence in the high-dose foetuses, no treatment relationship was indicated. No other visceral malformations were seen in the mid-dose group.
In the high-dose group, unilateral microphthalmia was seen in two foetuses, one from each of two litters. This malformation though not seen in the concurrent control group, has been seen at low incidence in recent historical control data for the laboratory. In the absence of other visceral malformations among the high-dose foetuses, the low incidence of this one visceral malformation (i.e., microphthalmia) in this group was not considered indicative of a treatment-related response.

VISCERAL VARIATIONS
Visceral variations are findings that involve subtle changes in size, shape or appearance of the visceral organs/tissues. In most instances these types of observations are considered transient and not indicative of malformation. An increase in the foetal or litter incidences of one or more variations which occur in a dose-related pattern may be indicative of a foetotoxic response, i.e., delayed maturation.
The incidences of foetuses with visceral variations for the control, low-, mid- and high-dose groups were 2.0 % (4/200 foetuses), 2.0 % (4/205 foetuses), 1.0 % (2/197 foetuses) and 1.1 % (2/183 foetuses), respectively. The incidences of litters containing foetuses with visceral variations for these same groups were 16.0 % (4/25 litters), 7.7 % (2/26 litters), 7.7 % (2/26 litters) and 8.7 % (2/23 litters), respectively. These incidences on both a per foetus and per litter basis, for the treated groups were comparable to control.
The types of visceral variations seen most frequently among the control and treated group foetuses were distended renal pelvis with a papilla present and/or tortuous ureter. These findings are seen commonly in the laboratory in the Day 20 gestation rat foetus using this evaluation procedure. The incidences of these variations on both a per foetus and per litter basis for the treated groups, were comparable to or slightly lower than control data and no adverse effect of treatment was indicated.
Thus, no adverse effect of treatment with test material at a dose level up to and including 120 mg/kg/day was indicated from the foetal visceral evaluations.

SKELETAL MALFORMATIONS
The incidences of foetuses with skeletal malformations for the control, low-, mid- and high-dose groups were 0.5 % (1/193 foetuses), 0 % (194 foetuses), 4.4 % (8/183 foetuses) and 0 % (174 foetuses), respectively. The incidences of litters containing foetuses with skeletal malformations for these same groups were 4.0 % (1/25 litters), 0 % (26 litters), 23.1 % (6/26 litters) and 0 % (23 litters), respectively. These incidences for the mid-dose group were higher than control; however, only for the foetal incidence was this difference from control data statistically significant.
The skeletal malformations seen most frequently in the mid-dose group were the presence of five lumbar vertebrae or wavy rib(s). Both findings are considered minor malformations and while not seen in the concurrent control group, are seen at low incidence historically in the laboratory. Five lumbar vertebrae were seen in three of seven foetuses from the litter of mid-dose Female No. 3582. This malformation was not seen in the remaining 176 mid-dose foetuses evaluated from 25 litters. Wavy rib(s) was seen in three mid-dose foetuses, one foetus from each of three litters. This is the most commonly seen skeletal malformation in the laboratory for the Day 20 gestation rat foetus as indicated from recent historical control data. The foetal incidence of wavy rib in the mid-dose group was 1.6 % (3/183) and within the range of recent historical control data; the litter incidence of 11.5 % (3/26) was just outside this range. In one of these mid-dose foetuses with wavy rib (foetus no. 12 - female no. 3579) the rib distortion involved a subtle curve, in the other affected foetuses (foetus no. 6 - female no. 3573 and foetus no. 10 - female no. 3595) several ribs had prominent "U-shaped" bends. In the absence of these malformations (five lumbar vertebrae, wavy rib) among the high-dose foetuses, their occurrence in the mid-dose group was considered fortuitous and not indicative of a treatment-related response. The only other skeletal malformations seen in the mid-dose group were vertebral defects in the thoracic/lumbar areas in one foetus and mis-shapen lumbar vertebral transverse processes in the one foetus noted externally with a filamentous tail.
No skeletal malformations were seen in low- or high-dose foetuses. In the control group, fusions of the cervical vertebral transverse processes were seen in one foetus.

OSSIFICATION VARIATIONS
Ossification variations may represent delays in the ossification process (retarded ossification) or slight ossification irregularities which may or may not be present in the adult specimen. Ossification variations are not considered representative of malformation but an increase in the overall incidence of foetuses (or litters containing foetuses) with ossification variations or an increase in the incidence of foetuses (or litters containing foetuses) with a particular ossification variation, which occurs in a dose-responsive pattern, may be suggestive of a developmentally toxic response i.e., delayed ossification, to the test material.
The overall incidence of foetuses with ossification variations for the low-dose group was 59.8 % (116/194 foetuses) and comparable to the control incidence of 56.0 % (108/193 foetuses). In the mid- and high-dose groups, these incidences of 77.6 % (142/183 foetuses) and 79.3 % (138/174 foetuses), respectively, were higher than control and these differences were statistically significant. In comparison to recent historical control data, these foetal incidences for the mid- and high-dose groups were similar to the mean value. These overall foetal incidences for the concurrent control and low-dose groups, however, were just outside the low range of these data. The incidence of litters containing foetuses with ossification variations was 100 % in the control, mid- and high-dose groups and 96.2 % in the low-dose group; these incidences for the treated groups were comparable to control data.
No adverse effect of treatment at the low-dose level was indicated from ossification variation data. The types of ossification variations seen among the low-dose foetuses are seen commonly in the laboratory and their occurrence on both a per foetus and per litter basis, was considered similar to control data.
The types of ossification variations seen in the mid- and high-dose groups were similar to those seen in the control group. For most of these observations, the foetal and litter incidences seen in the high-dose group were similar to control data while in the mid-dose group, these incidences tended to be higher. This was most pronounced for several cranial ossifications, in particular, incompletely ossified interparietals, supraoccipitals, squamosals and parietals and unossified hyoids. There were several ossification variations in the mid- and high-dose groups that occurred with increased incidence on both a per foetus and per litter basis, in comparison to control data. These were unossified fifth sternebra and unossified sixth sternebra. These incidences, while higher than concurrent control data, were within the range of recent historical control data for the laboratory. The foetal and litter incidences of unossified fifth sternebra in the control group were at the low end of these recent historical control data and for the 21 studies that comprise these data only one study had lower foetal and litter incidences. The foetal and litter incidences of unossified fifth sternebra for the mid-dose group were similar to the mean historical values while in the high-dose group, these incidences were higher than the mean values, but within the range of individual study values. Similarly, the incidences of unossified sixth sternebra on both a per foetus and per litter basis for the mid- and high-dose groups were notably higher than concurrent control data but similar to the mean historical control value and well within the range of these data. The incidence of unossified sixth sternebra for the concurrent control was at the low range of these data. The slight increase in foetal and litter incidences for unossified fifth or sixth sternebra at the mid-dose level in comparison to concurrent control data was not considered indicative of a treatment-related response due to the similarity of these data to mean historical control data for the laboratory. In the high-dose group, the incidence data (foetal and litter) for unossified fifth or sixth sternebra were notably higher than concurrent control data, and while still within the range of historical control data, these increases were considered indicative of a treatment-related response.
The only other unusual finding during the evaluation of ossification variation data for the high-dose group was an increased incidence on both a per foetus and per litter basis of first lumbar rudimentary rib. The latter are small, discrete ossifications that are located adjacent to the transverse processes of the first lumbar vertebrae. The foetal and litter incidences of this observation in the low- and mid-dose groups were considered similar to concurrent control data and within the range of recent historical control data for the laboratory. In the high-dose group, these incidences were increased in comparison to concurrent control data and outside the range of recent historical data. As indicated from recent historical control data, there is considerable inter-study variability in the occurrence of this ossification variation.
The increased occurrence of rudimentary first lumbar rib(s) in the high-dose group was considered related to the maternal toxicity demonstrated at this treatment-level and not indicative of a developmentally toxic response.
Thus no adverse effect of treatment with test material at a dose level up to and including 50 mg/kg/day was indicated from skeletal malformation or ossification variation data. At the 120 mg/kg/day dose level, an increase in incidence of unossified sternebra (fifth or sixth) was considered indicative of delayed ossification. The increased incidence of rudimentary rib structures in this group was considered related to the maternal toxicity demonstrated in this group.

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Remarks on result:

other: Increased incidences of ossification variations were the only treatment-related developmental effects noted and were considered related to maternal toxicity.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analytical Results

- Homogeneity:

Analyses to confirm that the mixing procedure developed for this study would produce homogeneous solutions for dosing were performed at the low- and high-concentration levels (3 and 24 mg/mL, respectively). For the low-concentration level, these analyses were performed on a mock batch of solution that was eventually discarded and not used for dosing. At the high-concentration level, these analyses were performed pre-test on the mix prepared for use on study. The results from these analyses confirm that solutions, as prepared, were homogeneous.

 

- Stability:

22-day stability at ambient storage was determined during the range-finding study for dosing formulations prepared at concentration levels of 10 and 40mg/mL. Since the high-concentration for this study was within this range, it was only necessary to establish additional stability information at the low-concentration level. The mock batch of dosing solution prepared to establish homogeneity at the 3 mg/mL concentration level was stored at ambient temperature and analysed on 7, 14 and 21days post-preparation. The results of these analyses confirm stability for at least 21 days.

 

- Confirmation of Concentration Levels - Dosing Formulations

Used on Study:

Two mixes at each concentration level were used for dosing the entire study. Each of these mixes was analysed prior to use on study and the results of these analyses are summarised below:

 

Group	Nominal conc. (mg/mL)	Analytical conc. (% of nominal)
		Mix 1	Mix 2
IV	3	99.4	94.5
III	10	98.4	96.1
IV	24	102.6	99.5

 

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the No Observable Effect Level (NOEL) for maternal toxicity was 15 mg/kg/day while the NOEL for developmental toxicity was 50 mg/kg/day.

Executive summary:

The maternal toxicity, embryonal/foetal toxicity and teratogenicity potential of the test material were investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 83-3 and OECD 414.

During the study, groups of 28 bred female adult Sprague-Dawley rats were administered test material via gavage at targeted dose levels of 0 (corn oil vehicle), 15, 50, or 120 mg/kg/day on days 6 through 15 of gestation. In-life parameters evaluated included clinical signs of toxicity, feed consumption, body weights and body weight gains. On gestation day 20, all females were euthanised and examined for gross pathologic changes and alterations in liver, kidney and gravid uterus weights. The ovaries were examined to determine the number of corpora lutea and the uterine contents were evaluated for number of implantations and resorptions. The foetuses were removed, weighed, sexed and examined for external, visceral and skeletal alterations.

All animals survived to the scheduled necropsy. There was no evidence of maternal toxicity in dams given 15 mg/kg/day. Statistically significant decreases in body weight gain were noted on days 12 - 16 and 6 - 16 in rats given 50 mg/kg/day. At 120 mg/kg/day, statistically significant decreases in body weight gain occurred for the day 6 - 9 and 6 - 16 intervals. Decreased feed consumption also was observed on days 6 - 9, 12 - 16 and 16 - 20 in this group. Increased incidences of ossification variations, such as unossified fifth and sixth sternebrae and rudimentary first lumbar rib were the only treatment-related developmental effects noted in foetuses from the high dose group and the increased incidence of rudimentary rib structures in this group was considered related to maternal toxicity. No treatment-related effects were observed on reproductive or embryonal/foetal parameters at 50 or 15 mg/kg/day.

Thus, the no-observed-effect-level (NOEL) for maternal toxicity in this study was 15 mg/kg/day. The NOEL for developmental toxicity was 50 mg/kg/day.