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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:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000)
Principles of method if other than guideline:
The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section).
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: HanRcc: WIST(SPF)
Details on test animals and environmental conditions:
Animals: Rat, HanRcc: WIST(SPF)
Breeder: Harlan Laboratories Ltd., Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
Number of Animals: 88 mated females (In order to complete mating within a reasonable time period, 98 female rats were obtained from the breeder. The surplus females were sacrificed after commencement of treatment for the last mated females); 22 mated females per group
Age (Day 0 Post Coitum): 11 weeks
Body Weight Range (Day 0 Post Coitum): 181 to 223 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry:
Room Number, Füllinsdorf: 08A
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz/Switzerland).
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst/Switzerland) was available ad libitum (batch no. 61/08).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Four groups of females were treated by oral gavage with SILRES® BS 1701once daily at dose levels of 0 (control), 100, 300 or 1000 mg/kg body weight/day on days 6-20 post coitum. The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar rats, Harlan Laboratories Study C16981, using dose levels of 0, 100, 300 and 1000 mg/kg/day, resulting in no clinical findings or adverse effects on dams or embryo-fetal development up to and including 1000 mg/kg body weight/day. A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose Formulations:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor. SILRES® BS 1701 was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous solution was prepared. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Storage of Dose Formulations:
Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers. Based upon the results of stability analyses performed within the (non GLP) Harlan Laboratories study C16981 (Dose Range-Finding Prenatal Developmental Toxicity Study in the Han Wistar Rat), dose formulations were stable for at least 7 days.

Analysis of Dose Formulations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analyzed by GC coupled to a flame ionisation detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analysed samples were not discarded without written consent from the study director.

SILRES® BS 1701 application formulations investigated during the study were found to comprise the test item in the range of 97.8% to 102.2%. All samples met the required content limit of ±20% with reference to the nominal concentration. The proper preparation of dose formulations was confirmed by the analysis of samples collected during the last week of the treatment (recovery estimated in these samples confirm concentration of the test item in the range between 96.7% and 102.0% of the nominal concentration). The homogeneous distribution of SILRES® BS 1701 in the application formulations was approved because single results did not deviate more than 1.9% (<15%) from the corresponding mean.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum. Male rats of the same source and strain were used only for mating. These male rats are in the possession of Harlan Laboratories and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.
Duration of treatment / exposure:
On days 6-20 post coitum
Frequency of treatment:
daily
Duration of test:
Up to day 21 post coitum
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
22 mated females/dose group
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
The following observations were recorded as follows:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
Food Consumption: Recorded at 3-day intervals: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.
Body Weights: Recorded daily from day 0 until day 21 post coitum.

At the scheduled necropsy on day 21 post coitum, females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section. Post mortem examination included gross macroscopic examination of all internal organs.
Ovaries and uterine content:
Post mortem examination included emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
Fetal examinations:
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. Microdissection technique (sectioning/dissection technique). At least one half of the fetuses from each litter was fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.

2. The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in plastic vials.

If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.

Terminology Used in the Assessment of the Data:
Empty Implantation Site: Very early resorption or aborted implantation
Embryonic Resorption: Amorphous mass being resorbed
Fetal Resorption: Clearly defined fetal body being resorbed
Dead Fetus: Appearance of live fetus but without induced respiration or movement
Live Fetus: Breathing and/or moving fetus
Abnormality: A structural change in a fetus that would probably impair its health or development.
Variation: A fetal change that is unlikely to adversely affect survival or health. This includes a delay in growth or morphogenesis that has otherwise followed a normal pattern of development.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Indices:
The following parameters were calculated: pre- and post-implantation losses, embryonic and fetal deaths, live and dead fetuses, abnormal fetuses, fetal sex ratios and fetal body weights.

For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean fetal weights were calculated from the individual weights both on a per group and on a per litter basis.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
General Tolerability: All dams survived until the scheduled necropsy. No clinical symptoms related to treatment with the test item were noted during the study at any dose level.

Food Consumption: No effects on mean food consumption were noted at any dose level. The overall differences in food consumption during the treatment period were by +0.5%, +4.2% and -0.9% in order of ascending dose levels (percentages refer to the value of the control group).

Body Weights: Mean body weight and mean body weight gain were not affected by treatment with the test item at any dose level. The overall differences in mean body weight gain were by +49.5%, +45.7%, +47.1 and +46.4% in order of ascending dose levels (percentages refer to the alterations within the treatment period). Mean corrected body weight gain (corrected for the weight of the gravid uterus) was similar in all dose groups: 12.4%, 10.3%, 12.8% and 10.1% in order of ascending dose levels.

Reproduction Data: No test item-related effects on the relevant reproduction data (post implantation loss and number of live fetuses at termination) were observed at any dose level. Incidental statistically significantly lower number of embryonic resorptions was observed at the dose level of 100 mg/kg. This effect was considered to be a result of biological variability. Mean number of live fetuses was similar in all groups and was 13.1, 12.2, 12.1 and 12.4 in order of ascending dose levels.

Macroscopic Findings: No findings were noted during macroscopic examination at any dose level.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
External Examination: No test item-related abnormal findings were noted during external examination of the fetuses at any dose level. A malrotaded hind limb was found in 1 fetus at the dose of 100 mg/kg and 2 fetuses at the dose of 300 mg/kg. Because of lack of the dose-correlation, this finding was considered to be incidental.

Sex Ratios: No effects on sex ratio of the fetuses were observed at any dose level. Proportions of male fetuses were 50.0%, 46.1%, 45.9% and 46.5% in order of ascending dose levels.

Body Weights: No test item-related effects on mean weights of live fetuses were observed at any dose level. Slightly but statistically significantly higher mean body weights of live fetuses were observed at the dose levels of 100 and 1000 mg/kg. Mean fetal body weights calculated on an individual basis were 4.8 g in both groups compared to 4.7 g in the control group. Both values were in the range of historical control data (mean fetal body weight in control groups comprised values from 4.7 to 4.9 g) therefore this effect was considered not to be test item-related but a result of biological variability.

Visceral Examination of Fetuses (Microdissection Technique): During visceral examination of fetuses, findings were noted in:
34% examined fetuses (in 100% litters) in the control group
36% examined fetuses (in 91% litters) at the dose level of 100 mg/kg
31% examined fetuses (in 86% litters) at the dose level of 300 mg/kg
35% examined fetuses (in 100% litters) at the dose level of 1000 mg/kg
The type and frequencies of the noted variations were similar in the groups receiving the test item and the control group and did not indicate any dose-dependency, therefore these findings were considered not to be test item-related. All found abnormalities (situs invertus noted in 2 fetuses/2 litters, small pituitary noted in 1 fetus and interventricular septal defect of the heart noted in 1 fetus) were noted in the control group.

Skeletal Examinations of Fetuses - Bone and Cartilage Abnormalities and
Variations: During skeletal examination of fetuses, findings were noted in:
17% examined fetuses (in 55% litters) in the control group
27% examined fetuses (in 73% litters) at the dose level of 100 mg/kg
18% examined fetuses (in 68% litters) at the dose level of 300 mg/kg
20% examined fetuses (in 62% litters) at the dose level of 1000 mg/kg
The type and frequencies of the noted skeletal variations were similar in the groups receiving the test item and the control group and did not indicate any dose-dependency, therefore they were considered not to be test item-related. No test item-related abnormalities were observed. A supernumerary greater horn of hyoid arch was noted in 1 fetus in the control group. A malpositioned and/or shortened and fused costal cartilage was found in 1 fetus each at the dose levels of 100 mg/kg and 1000 mg/kg. This finding was considered to be incidental.

Bone Examination of Fetuses (Ossification Stage and Supernumerary Ribs): During bone examination of fetuses, findings were noted in:
16% examined fetuses (in 50% litters) in the control group
22% examined fetuses (in 68% litters) at the dose level of 100 mg/kg
17% examined fetuses (in 64% litters) at the dose level of 300 mg/kg
19% examined fetuses (in 57% litters) at the dose level of 1000 mg/kg
No test item-related effects on the ossification stage and supernumerary ribs were noted at the dose level of 100 mg/kg.

When compared to the control values statistically significantly lower numbers of non-ossified cervical and caudal vertebrae and incompletely ossified sternal bodies were noted at the dose level of 1000 mg/kg. Statistically significantly lower number of non-ossified digits and toes was noted in all groups treated with the test item. Most of these values were in the range of the historical control data and therefore these findings were considered not to be test item-related but a result of biological variability. Compared to the control values, a statistically significantly increased incidence of supernumerary rudimentary ribs was noted at the dose levels of 300 and 1000 mg/kg when calculated on an individual basis. This effect did not correlate with the dose levels; it was most pronounced at the dose level of 300 mg/kg. When calculated on a litter basis, statistically significant increase was noted only at the dose level of 300 mg/kg. The increased numbers of supernumerary ribs exceeded the historical control data and were considered to be test item-related. An increase of rudimentary supernumerary ribs indicate only minor and not adverse developmental disturbance as the rudimentary thoracolumbar supernumerary ribs are known to be transient, therefore this effect was considered not to be adverse.

Cartilage Examination of Fetuses (Additional Variations): During cartilage examination of fetuses, findings were noted in:
2% examined fetuses (in 14% litters) in the control group
10% examined fetuses (in 41% litters) at the dose level of 100 mg/kg
2% examined fetuses (in 14% litters) at the dose level of 300 mg/kg
4% examined fetuses (in 19% litters) at the dose level of 1000 mg/kg
No test item-related effects on the cartilage development were noted at the dose level of 100 mg/kg. At the dose level of 1000 mg/kg, statistically significantly lower number of skull cartilaginous structures with small hole was observed when compared to the control value. This value remained in the range of the historical control data and was therefore considered not to be test item-related but a result of biological variability. Compared to the control value, an increased incidence of long or interrupted costal cartilages was noted at the dose levels of 300 and 1000 mg/kg when calculated on an individual basis. This effect did not correlate with the dose levels; it was most pronounced at the dose level of 300 mg/kg. When calculated on a litter basis, statistically significant increase was noted only at the dose level of 300 mg/kg. The increased numbers of long or interrupted costal cartilages exceeded the historical control data and were considered to be test item-related. Although long or interrupted costal cartilages are considered as permanent structural changes, they are minor and most unlikely to adversely affect further development and postnatal live of the animal therefore this effect was considered not to be adverse.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the variations on development of axial skeleton

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

No clinical signs or observations were noted up to and included dose level of 1000 mg/kg. Food consumption, body weight and corrected body weight gain (corrected for the gravid uterus weight) were not affected by treatment with the test item up to and included dose level of 1000 mg/kg. No test item-related effects on the relevant reproduction parameters were observed up to and included dose level of 1000 mg/kg. No test item-related effects on development of embryos or fetuses were observed at the dose level of 100 mg/kg. An increased incidence of supernumerary rudimentary ribs and long or interrupted costal cartilages observed at the dose levels of 300 and 1000 mg/kg indicate a treatment-related minor disturbance in the development of the axial skeleton at these dose levels. Both types of variations belong to the most common seen in the control groups. They are of minor nature and not likely to affect further development and postnatal live of the animal. Therefore the increase of their incidence was considered not to be adverse.

Applicant's summary and conclusion

Conclusions:
Under the conditions described for this study, NOEL (No Observed Effect Level) for pregnant rat was considered to be 1000 mg/kg body weight/day. Based on the variations on development of axial skeleton, NOEL for embryo and fetal development was considered to be 100 mg/kg body weight/day whereas NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day. Consequently, SILRES® BS 1701 was considered not to reveal teratogenic potential up to and including a dose of 1000 mg/kg.