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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 2001
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide
EC Number:
700-457-2
Molecular formula:
Unspecified
IUPAC Name:
Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide
Details on test material:
- Name of test material (as cited in study report): LIMUS-Sambaydestillation
- Test substance No.: 07/0684-1
- Batch identification: 8712 / 062
- Analytical purity: 87 %
- Storage condition of test material: At ambient temperature.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han) (outbred, SPF-Quality)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: Females were approximately 12 weeks.
- Weight at study initiation: animals of comparable seize and weight
- Housing: individually after mating
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0ºC
- Humidity (%): 40-70 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% (w/w) carboxymethyl cellulose in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
No adjustment was made for specific gravity of the test substance, vehicle, and/or formulation

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on information provided by Sponsor and trial formulations performed
at NOTOX B.V.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted once during the treatment phase (30 August 2010), according to a validated
method (NOTOX Project 494646, BASF Project 05Y0684/07X011). Samples of formulations were
analyzed for homogeneity (Groups 2 and 4) and accuracy (Groups 1-4) of preparation. Stability in
vehicle over 6 hours at room temperature was also determined (Groups 2 and 4).
The accuracy of preparation was considered acceptable if the mean measured concentrations were
85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was
≤ 10%. Formulations were considered stable if the relative difference before and after storage was
maximally 10%.
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
From Days 6 to 19 post-coitum, inclusive.
Frequency of treatment:
daily
Duration of test:
until day 20 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the doses were selected based on the available 28-day study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards. The time of onset, degree and duration was recorded. All symptoms were graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present.
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3 and 6-20 (daily) post-coitum.

FOOD CONSUMPTION: Yes
Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: as outlined in OECD 414 guideline
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
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]
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref 6) (manyto-
one t-test) based on a pooled variance estimate was applied for the comparison of the treated
groups and the control groups for each sex.
- The Steel-test (Ref 7) (many-to-one rank test) was applied if the data could not be assumed to
follow a normal distribution.
- The Fisher Exact-test (Ref 8) was applied to frequency data.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental external
variations, and each particular external malformation or variation were subjected to the Kruskal-
Wallis nonparametric ANOVA test (Ref 9) to determine intergroup differences. If the ANOVA
revealed statistically significant (p<0.05) intergroup variance. Dunn’s test (Ref 10) was used to
compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data
(scores) in the summary tables. Test statistics were calculated on the basis of exact values for means
and pooled variances. Individual values, means and standard deviations may be rounded off before
printing. Therefore, two groups may display the same printed means for a given parameter, yet display
different test statistics values.
Indices:
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as
a mean litter proportion on a total group basis, where Viable fetuses affected/litter (%) = (number of viable fetuses affected/litter)/(number of viable fetuses/litter)x 100
Historical control data:
is available in the study report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings at 300 mg/kg bw/day included lethargy, piloerection, hunched posture, (slightly) uncoordinated movements, abnormal gait, (slightly) pale feces and / or pale appearance. These findings were noted in 7 out of 21 females on one or more days during the thirs week of treatment. The other 13 out of 21 females showed no clinical signs at all.
Treatment at the lower dose levels of 30 and 100 mg/kg bw/day did not result in toxicological relevant clinical signs.
At 100 mg/kg bw/day, one female showed hunched posture, piloerection and quick breathing on one or more days from days 11 - 20 post-coitum. No clear treatment-related effects on body weight gain and food consumption were noted. Moreover, there were no gross findings at necropsy that could explain the clinical signs of this female. Hunched posture and piloerection were also seen in the high dose group. However, as at 100 mg/kg bw/day only one female was affected, this incidental finding was considered of no toxicological relevance.
Incidentally, (slight) alopecia of different parts of the body or scabs on the right flank were noted for one control female, one female at 30 mg/kg bw/day, four females at 100 mg/kg bw/day and one female at 300 mg/kg bw/day from day 10 post-coitum onwards. These findings occured within the range of background findings to be expected for rats of this age and strain which are housed an treated under the conditions in this study. At the low incidences observed and / or their slight nature, they were considered a sign of toxicological relevance.
Mortality:
mortality observed, treatment-related
Description (incidence):
Due to severe toxicity, one female treated at 300 mg/kg bw/day was euthanized on day 19 post-coitum. This female had a lean appearance and showed hunched posture, (moderately) uncoordinated movements and abnormal gait, a body weight loss of 19 % and reduced food consumption on days 6 - 9 and 15 - 17 post-coitum. At necropsy, no abnormalities were noted. This female was found to be non-pregnant.
There were no unscheduled deaths at the lower dose levels of 30 and 100 mg/kg bw/day and in the control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females treated at 300 mg/kg bw/day showed statistically significantly lower body weight and body weight gain as compared to controls from days 17 and 16 post-coitum, respectively, onwards. This effect was initially slight, but increased progressively until the end of the observation period (day 20 post-coitum). In line with this, significant body weight loss was noted for females of group 4 compared to controls (group 1) after correction for uterus weight as determined at necropsy.
No treatment-related effect on body weight was noted at the lower dose levels of 30 and 100 mg/kg bw/day.
At the individual level, there were one control female, two low dose females and one mid dose female that were pregnant but showed relatively low body weight gains until day 20 post-coitum. The reason for this was the presence of early resorptions alone or in combination with a very low number of fetuses (one or three).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment at 300 mg/kg bw/day resulted in statistically significantly lower food consumption (absolute and relative to body weight) as compared to controls in the periods from days 6 - 9 and days 15 - 20.
No treatment-related effect on food intake was noted at the lower dose levels of 30 and 100 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings included scab formation on the right flank or alopecia of several body parts, and accessory liver at the right median lobe. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.
For one control female fluid was observed in the uterus. Since this female was non-pregnant, this finding represents a normal physiological stage of the oestrus cycle.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Details on maternal toxic effects:
No significant differences were observed between control and treated groups regarding the number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, or pre- and post-implantation loss.
At the individual level, one control female had only two corpora lutea, one implantation site and one early resorption in the left uterus horn. As it was a female of the vehicle control group, this finding was not related to treatment to the test substance.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a trend towards lower fetal body weights at 300 mg/kg bw/day compared to controls. This change reached only statistical significance for male fetuses.
No treatment-related effect on fetal body weight was noted at 30 and 100 mg/kg bw/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the sex ratio of the fetuses.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no treatment-related effect on litter size.
External malformations:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on fetal external morphology.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no test substance-related fetal skeletal malformations in this study.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on fetal visceral morphology.
Details on embryotoxic / teratogenic effects:
Morphological examination of the fetuses revealed neither effects on fetal external or visceral
morphology nor skeletal malformations that could be related to treatment up to 300 mg/kg body
weight/day.
Several skeletal variations which were indicative for a developmental delay were noted at higher
incidences in the 300 mg/kg body weight/day group. These included reduced ossification of the skull,
unossified sternebra nos. 5 and/or 6, unossified hyoid, bipartite ossification of vertebral centra and
entire sternum unossified. In addition, the incidence of ossified cervical centrum no. 1 was decreased
at 300 mg/kg body weight/day compared to the concurrent control value. The reductions in ossification
noted were not statistically significant, but while considering the decreased fetal body weights at
300 mg/kg body weight/day, it is assumed that the delayed ossification occurred in this context.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study both the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance were established as being 100 mg/kg bw/day. There were no adverse fetal findings evident at a dose not producing maternal toxicity.
Executive summary:

Prenatal developmental toxicity study of the test substance in rats by oral gavage.

Mated female Wistar (Han) rats were assigned to four dose groups each containing 22 animals. The test item was administered once daily by gavage from days 6 to 19 post-coitum at doses of 30, 100 and 300 mg/kg bw/day (groups 2, 3 and 4, respectively). Rats of the control group received the vehicle, 1 % (w/w) carboxymethyl cellulose suspension in water, alone. Females were checked daily for the presence of clinical signs. Food consumption was determined at periodic intervals; body weight was determined daily during treatment and at periodic intervals in the other periods. Formulations (groups 1 - 4) prepared on one day during treatment were analyzed on accuracy, homogeneity and stability.

All animals surviving to day 20 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohystoectomy was performed on each surviving female of groups 1 - 4. Gravid uterine weights were recorded for all pregnant females, and net body weights and net body weight changes were calculated. The uteri, placentae and ovaries were examined, and the numbers of live and dead fetuses, early and late resorptions, total implantations and corpora lutea were recorded. All fetuses and placentas were weighed. The fetuses were sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses was examined for visceral anomalies and the remaining half of the fetuses was subjected to a skeletal examination.

Chemical analysis of dose preparations: Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Maternal findings: Treatment with the test substance at 300 mg/kg bw/day resulted in one unscheduled death (killed in extremis), treatment-related clinical signs (lethargy, piloerection, hunched posture, uncoordinated movements, abnormal gait, pale feces, pale and / or lean appearance), reduced body weight / body weight gain and food consumption. At necropsy, no gross findings were noted that were considered related to treatment.

No maternal toxicity was observed at the lower dose levels of 30 and 100 mg/kg bw/day.

Developmental findings: There were no treatment-related effects on viability, litter size and sex ratio up to 300 mg/kg bw/day.

Reduced fetal body weights were noted for both male (statistical significant) and female (not statistically significant) fetuses at 300 mg/kg bw/day compared to controls. This adverse effect on fetal body weight was considered related to the considerable maternal toxicity observed in the high dose group. In line with this, both mean male and female placenta weights were slightly lower at 300 mg/kg bw/day compared to controls (not statistically significant).

Morphological examination of the fetuses revealed neither effects on fetal external or visceral morphology nor skeletal malformations that could be related to treatment up to 300 mg/kg bw/day.

Several skeletal variations which were indicative for a developmental delay were noted at higher incidences in the 300 mg/kg bw/day group. These included reduced ossification of the skull, unossified sternebra nos. 5 and / or 6, unossified hyoid, bipartite ossification of vertebral centra and entire sternum unossified. In addition, the incidence of ossified cervical centrum no. 1 was decreased at 300 mg/kg bw/day compared to the concurrent control value. The reductions in ossification noted were not statistically significant, but while considering the decreased fetal body weights at 300 mg/kg bw/day, it is assumed that the delayed ossification occured in this context.

Based on the results in this prenatal developmental toxicity study both the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance were established as being 100 mg/kg bw/day. There were no adverse findings evident at a dose not producing maternal toxicity.