Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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)

GLP compliance:

yes

Test material

Test material information

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

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 / concentrations

Remarks:

Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/day
Basis:
nominal conc.

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

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
Treatment with LIMUS-Sambaydestillation at 300 mg/kg body weight/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 body weight/day.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
There were no treatment-related effects on viability, litter size and sex ratio up to 300 mg/kg body
weight/day.
Reduced fetal body weights were noted for both male (statistical significant) and female (not
statistically significant) fetuses at 300 mg/kg body weight/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 body weight/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 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