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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013-2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study perforemed according to OECD 414 and GLP guidelines and considered to be reliable, adequate and relevant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Sodium trichloracetate, Sodium trichloracetate (STCA) Gran
- Physical state: white powder
- Analytical purity: 99.9*%
- Impurities (identity and concentrations): see confidential details
- Composition of test material, percentage of components: see confidential details
- Purity test date: 2013-08-23
- Lot/batch No.: DEFM119007
- Expiration date of the lot/batch: date of production: 2013-03-19; durability: stable for 1095 days after production
- Stability under test conditions: the formulations: stable for at least 24h
- Storage condition of test material: At +10°C to +25°C. In original, tightly sealed container in a dry, cool and well-ventilated place, protected from light.
- Manufacturer: CABB GmbH Ludwig-Hermann-Str. 100 86368 Gersthofen Germany

Test animals

Species:
rat
Strain:
other: CD® / Crl: CD (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sandhofer Weg 7, 97633 Sulzfeld Germany GmbH
- Age on day 0 of pregnancy: 61 - 64 days
- Weight on day 0 of pregnancy: 194.4 – 252.0 g
- Housing: Except during the mating period, the dams were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were cleaned and changed once a week.
- Diet (e.g. ad libitum): Commercial ssniff R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany was offered daily ad libitum.
- Water (e.g. ad libitum): Tap water (in drinking bottles) was offered daily ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 28, 2013 To: November 21, 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: tap water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in the vehicle (tap water) to the appropriate concentrations of 20, 60 and 200 mg/mL and was administered at a constant volume (5mL/kg bw/day) once daily. The pH values of the first prepared test item formulations were as follows:
- Group 2 (20 mg/mL): pH 7.63
- Group 3 (60 mg/mL): pH 7.54
- Group 4 (200 mg/mL): pH 7.23
The amount of the test item was adjusted to the animals current body weight daily.
The control animals received the vehicle at the same administration volume daily in the same way.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item formulations, samples of approximately 10 mL were taken at the following times and stored at -20°C or colder until dispatch to Allessa GmbH for analysis:
At start of the administration period:
-Analysis of stability and concentration
Immediately after preparation of the formulation as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 sample/dose level group; groups 2-4). Number of samples : 3 x 3 = 9
- Homogeneity
At start of administration, during (middle) administration and before administration to the last animal of the group (3 samples/dose level group; groups 2-4)
Number of samples: 3 x 3 = 9
At study termination (at a time when the majority of animals was dosed):
- Analysis of concentration
During the treatment with the test item always before administration to the last animal/group (1 sample/dose level group; groups 2-4).
Number of samples: 1 x 3 = 3
Sum of all Samples: 21
The samples were labelled with the study number, species, type of sample, concentration, sampling time and date.

The analysis of the stability, homogeneity and concentrations of the test item Sodium trichloracetate was performed by Allessa GmbH.
The results confirmed that the test item formulations were correctly prepared and the concentrations were in good agreement to those expected. The homogeneity of the samples was warranted during the procedure of treatment and the formulations were stable for at least 24h. In detail, the concentrations of Sodium trichloracetate in the low dose samples were between 97.6% and 99.1%, those of the intermediate samples between 93.6% and 99,5% and those of the high
dose samples between 98.0% and 100.1% of the nominal concentrations.
The investigation of the validation parameters indicated that the method employed was suitable for the determination of Sodium trichloroacetate in test item formulations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner.
- This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant (3 control animals out of 25 control animals) were excluded from the analysis of the results and replaced by spare animals. The number of non-pregnant animals of this study was within the normal range of variation. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
- Verification of same strain and source of both sexes: yes. Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.


Duration of treatment / exposure:
Day 6 to 19 of gestation
Frequency of treatment:
Once daily
Duration of test:
From the 6th to the 19th day of pregnancy
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20 ( pregnant females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a key repeated dose oral toxicity study, male and female rats were dosed in the diet at 250, 630, 1600, 4000 and 10000 ppm for 4 months. Under the experimental conditions, no toxic damages were observed in any treated group. In the highest dose group (10000 ppm), only a slight loss of body weight was observed. The NOEL was determined as 4000 ppm (approximately 365 mg/kg b.w./day). (Study report No. HOE 69.0154, 1969).
There is information available from a developmental study on the substance Trichloroacetic acid, member of the chloroacetates category (Smith et al., 1989, Teratogenic Activity of Trichloroacetic Acid in the Rat, Teratology (1989) 40, 445-451). Doses were 0, 330, 800, 1200 and 1800 mg/kg b.w./day. Based on these results trichloroacetic acid was considered to be developmentally toxic in the pregnant rat. Results after treatment of pregnant rats from 6th to the 15th day of gestation showed maternal and embryonic toxicity from 330 mg/kg body weight and from 800 mg/kg b.w. also embryo-lethality. In all dose groups there was a dose-dependent increase in visceral anomalies, particularly in the cardiovascular system. Maternal spleen and kidney weights increased in a dose-related manner. The mean frequency of soft tissue malformations especially in the cardiovascular system, ranged from 9% at the low dose (330 mg/kg b.w./day) to 97% at the high dose (1800 mg/kg b.w. /day). Taking into account difference in molecular weight between trichloroacetic acid (163.39) and sodium trichloroacetic acid (185.36), these doses correspond to approx. ca. 370 and 900 to 2040 mg/kg b.w./day.
In the latter study, two of the four doses were above limit dose and therefore considered less relevant to developmental toxicity study. On the other hand, no severe maternal toxicity nor mortality was observed in the above study, therefore 1000 mg/kg b.w. can be considered as highest dose for the dose-range-finding study.
The dose levels were further selected based on the results of a dose-range-finding study in rats (LPT Study No. 30400). Dose levels of 0 (vehicle), 100, 300 and 1000 mg/kg b.w. were given to 3 females/group at the age of 6 weeks. At the dose of 1000 mg/kg b.w., an increased discharge of faeces was noted from test day 5 and increased intake of drinking water was noted from test day 8 or 12 onwards. Both symptoms were observed daily until necropsy on test day 15. Also slight reductions of relative food consumption were noted in the first test week (10% below the vehicle control). Finally, increases were noted for the mean absolute/relative kidney weights (>20% versus vehicle control) and absolute/relative liver weight (>15% versus vehicle control). No such increases were seen in the 100 and 300 mg/kg b.w. dos groups.
Based on the available existing information and information from the above dose-range-finding study dose levels selected were 100, 300 and1000 mg/kg b.w./day.
- Rationale for animal assignment (if not random):
Day 0 of pregnancy, the animals were assigned to the test groups by mating day using a Provantis (Version 8.2.08) generated randomisation.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: : Individual animals were observed daily for behaviour, external appearance and nature of the faeces. Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighings - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20). Furthermore the net weight change from day 6 is given.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The ovaries and the uteri of the dams were removed; the uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations.

OTHER:
Viability:
Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way would have been examined for abnormal development, whenever possible.



Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
individual data per fetus; mean per litter; mean per group; litter mean per group; litter mean per sex and group
- Number of corpora lutea: Yes
number per dam; absolute number per group; mean per group
- Number of implantations: Yes
number per dam; distribution in the uterine horns; absolute number per group; mean per group
- Number of early resorptions: Yes
number per dam; distribution in the uterine horns; absolute number per group; mean per group; mean % per group; early resorptions <2 mm; late resorptions >2 mm
- Number of late resorptions: Yes
- Other:
Resorption rate % = (resorptions / implantations) x 100
Weight of placentae
Fetal examinations:
- Weight of fetuses
individual data per fetus; mean per litter; mean per sex and litter; litter mean per group; litter mean per sex and group
- Number Fetuses
number per dam (alive and dead); number of fetuses per sex and dam; distribution in the uterine horns; absolute number of fetuses alive per group;
mean number of fetuses alive per group; mean % of fetuses alive per group; mean % per sex and group
- Dead fetuses
number per dam; mean per group
- Runts
number per dam; mean per group
- External examinations: Yes: all
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: included in other examinations
Statistics:
The data were captured, whenever possible, using the departmental computerized systems (Provantis® Integrated preclinical software, version 8.2.0.8, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups (2 to 4) were compared with the control group (1).
The following statistical methods were used:
STUDENT's t-test: All numerical values (p≤0.01)
Multiple t-test based on DUNNETT, C. W. (New tables for multiple Comparisons with a control Biometrics, 482-491 (Sept 1964)): Body weight / Food consumption (p ≤ 0.05 and p ≤0.01)

For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance was p ≤ 0.01.
For the comparison of classification measurements (for example malformation-, resorption-, retardation- and variation rate) the FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥ 100 (p ≤ 0.05 and p ≤0.01) were employed.
These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding

Indices:
Resorption rate % = (resoptions/ implantations) x 100
Malformation rate % = (malformed fetuses/ fetuses) x 100
Variation rate % = (fetuses with variations/ fetuses) x 100
Retardation rate % = (fetuses with retardations/fetuses) x 100
Pre-implantation loss % = ((corpora lutea – implantations)/ implantations) x 100
Post-implantation loss % = ((implantations- living fetuses)/ implantations) x 100
Historical control data:
Results of the 55 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH) performed at LPT in the years 2000 to October 2013

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
None of the dams died prematurely.
The administration of the test item revealed pale and/or pultaceous faeces in 8 of 20 dams of the intermediate dose group (300 mg Sodium trichloracetate/kg bw/day) and in 20 of 20 dams of the high dose group (1000 mg Sodium trichloracetate/kg bw/day).
An increased drinking water consumption (visual observation) was noted for several dams of the intermediate (3/20) and the high dose group (14/20).
Accordingly, a reduction in body weight gain between gestation days 6 and 9 by 29.5% was noted in the high dose group (1000 mg Sodium trichloracetate/kg bw/day).
A test item-related reduction in food consumption by maximal 27.7% on gestation day 7 was noted after the start of treatment for the dams of the high dose group (1000 mg Sodium trichloracetate/kg bw/day). Food consumption increased again from gestation day 9 onwards and reached the level of the control group on gestation day 13.
An enlarged spleen was noted in 6 of 20 dams of the high dose group (1000 mg Sodium trichloracetate/kg bw/day) during macroscopic inspection at laparotomy.
A reduction in the gravid uterus weight by 12.2% was noted in the high dose group (1000 mg Sodium trichloracetate/kg bw/day) due to a reduction in fetal body weight.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: embryotoxicity at 1000 mg/kg bw

Details on embryotoxic / teratogenic effects:
The number of resorptions and live fetuses was not influenced by the test item in any of the test item treated groups.
No dead fetus was noted in the control group and in any of the test item treated groups.
A reduction in fetal weight by 22.2% was noted in the high dose group (1000 mg Sodium trichloracetate/kg bw/day).
No malformation was noted in any of the test groups during external / internal macroscopic examination, skeletal examination (according to DAWSON) and soft tissue examination (according to WILSON).
No test item-related variations were noted in any of the test groups during external / internal macroscopic examination.
Variations noted during the skeletal examination according to DAWSON were in the range of LPT background data.
Test item-related soft tissue variations were noted in the high dose group (1000 mg Sodium trichloracetate/kg bw/day) in form of an increased fetal incidence of dilated cerebral ventricle during the examination of the sectioned fetuses according to WILSON. The fetal incidence of total soft tissue variations was not statistically significantly increased
Examination of the fetuses from the high dose group (1000 mg Sodium trichloracetate/kg bw/day) according to DAWSON revealed test item-related increased fetal and/or litter incidences of skeletal retardations of the skull, the sternebae (not or incompletely ossified), the thoracic vertebral bodies (dumbbell shaped), the lumbar vertebral arches (not ossified, the pelvic vertebral bodies (less than 5 ossified), the caudal vertebral bodies (no body ossified), the os ischii and os pubis (not ossified) and the metatarsalia. The relevance of these findings is limited for following reasons. First, human skeletal development takes place postnatally, so these findings are not applicable to human situation. Secondly, studies in rats have demonstrated that delayed ossifications do not persist postnatally, so they are reversible (Carney and Kimmel, 2007).

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
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:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg Sodium trichloracetate/kg bw/day for the dams.
The NOAEL for the fetal organism was also 300 mg Sodium trichloracetate/kg bw/day.
The NOAEL for teratogenicity was 1000 mg Sodium trichloracetate/kg bw/day.
Executive summary:

In this prenatal developmental toxicity study, the test item Sodium trichloracetate was administered to female rats at dose levels of 100, 300 or 1000 mg/kg bw/day (administration volume: 5 mL/kg bw/day), orally by gavage from the 6th to 19th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg Sodium trichloracetate/kg bw/day for the dams.

At 1000 mg Sodium trichloracetate/kg b.w./day, a marked reduction in food consumption and a slight reduction in body weight and body weight gain were noted for a few days after the start of treatment. Six of 20 dams revealed an enlarged spleen during macroscopic examination at laparotomy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was also 300 mg Sodium trichloracetate/kg b.w./day.

Test item-related embryotoxic effects were only noted at the materno-toxic dose level of 1000 mg Sodium trichloracetate/kg bw/day in the form of reduced fetal body weights, increased fetal and/or litter incidences of skeletal retardations (retarded ossification) and an increased fetal incidence of fetuses with a dilated cerebral ventricle, classified as a soft tissue variation.

No malformation was noted at any of the test item treated groups.

The test item did not possess any teratogenic effect. The no-observed adverse-effect level (NOAEL) for teratogenicity was 1000 mg Sodium trichloracetate/kg bw/day.