Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: Spermhead morphology assay (reproductive function)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Test method was not according to any guideline. It provides information on the observed effects on the spermhead morphology. No data on GLP.
Principles of method if other than guideline:
A spermhead morphology assay was performed in mice with sodium trichloroacetate. B6C3F1 mice (24/group) were given 0, 625, 1250, or 2500 mg/kg bw/day of test substance for five consecutive days by oral gavage. Twelve mice per treatment group were sacrificed at 21 and 35 days after the first treatment. These sacrifice times correspond to treatment of cells at the late spermatogonial and primary spermatocyte periods, respectively, assuming no treatment-related delays in sperm development. Body weights were recorded for each animal during the dosing phase and at sacrifice, and testes and cauda epididymidal weights were recorded at sacrifice. Ten animals from each treatment/sacrifice group were randomly selected to be evaluated for sperm count and morphology. The types and frequencies of spermhead abnormalities were based on the scoring of 500 sperm per animal.
GLP compliance:
not specified
Limit test:
yes
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, lnc., Gilroy, CA, USA.
- Age at study initiation: Eleven-week-old
- Weight at study initiation: 20-30 g
- Housing: The animals were housed six per cage with wood shavings used for bedding, and beddings were changed weekly.
- Diet (e.g. ad libitum): Certified Purina Lab Chow, #5002C, ad libitum.
- Water (e.g. ad libitum): Recirculated, deionized, UV -treated water was available to the animals throughout the study.
- Acclimation period: min. 5 days

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 hrs dark / 12 hrs light.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
A forty percent (w/v) solution of the acid as its sodium salt was prepared by dissolving sodium trichloroacetate in distilled water and titrating the pH to 7.0 with NaOH.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
5 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
625 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1250 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2500 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
24 male mice per group were used.
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Twelve mice per treatment group were sacrificed at 21 and 35 days after the first treatment. These sacrifice times correspond to treatment of cells at the late spermatogonial and primary spermatocyte periods, respectively, assuming no treatment-related delays in sperm development.
Body weights were recorded for each animal during the dosing phase and at sacrifice, and testes and cauda epididymidal weights were recorded at sacrifice.
Sperm parameters (parental animals):
Ten animals from each treatment/sacrifice group were randomly selected to be evaluated for sperm count and morphology.
The types and frequencies of spermhead abnormalities were based on the scoring of 500 sperm per animal.
Statistics:
Initial and sacrifice body weights, testes and cauda epididymal weights, testes to final body weight ratios, and sperm concentration/mg of cauda epididymis were analyzed by one-way analysis of variance (ANOVA) at a significance level of p < 0.05. When significant differences were found, Dunnett's test was applied to compare the different treatment group means with the negative control means. The numbers of morphologically abnormal sperm were analyzed by the Kruskall-Wallis and Mann-Whitney U tests, with differences considered significant at the p < 0.05 level. These latter tests are nonparametric alternatives to ANOVA and Dunnett's test, respectively.
Clinical signs:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
The testes-to-final-body weight ratio, the number of sperm per milligram of cauda epididymis, and the percentage of abnormal spermheads were unaffected in the treated animals either at 21 or 35 days following oral gavage administration.
Dose descriptor:
NOEL
Effect level:
>= 2 500 mg/kg bw/day
Sex:
male
Basis for effect level:
other: overall effects
Remarks on result:
other: Generation not specified (migrated information)
Reproductive effects observed:
not specified

The NOEL value at 21 and at 35 days wqas equal or greater than 2500 mg/kg bw/day.

Conclusions:
The NOEC value at 21 and at 35 days was equal or greater than 2500 mg/kg bw/day.
Executive summary:

A spermhead morphology assay was performed in mice with sodium trichloroacetate. B6C3F1 mice (24/group) were given 0, 625, 1250, or 2500 mg/kg bw/day of test substance for five consecutive days by oral gavage. Twelve mice per treatment group were sacrificed at 21 and 35 days after the first treatment. These sacrifice times correspond to treatment of cells at the late spermatogonial and primary spermatocyte periods, respectively, assuming no treatment-related delays in sperm development. Body weights were recorded for each animal during the dosing phase and at sacrifice, and testes and cauda epididymidal weights were recorded at sacrifice. Ten animals from each treatment/sacrifice group were randomly selected to be evaluated for sperm count and morphology. The types and frequencies of spermhead abnormalities were based on the scoring of 500 sperm per animal. Initial and sacrifice body weights, testes and cauda epididymal weights, testes to final body weight ratios, and sperm concentration/mg of cauda epididymis were analyzed by one-way analysis of variance (ANOVA) at a significance level of p < 0.05. When significant differences were found, Dunnett's test was applied to compare the different treatment group means with the negative control means. The numbers of morphologically abnormal sperm were analyzed by the Kruskall-Wallis and Mann-Whitney U tests, with differences considered significant at the p < 0.05 level. These latter tests are nonparametric alternatives to ANOVA and Dunnett's test, respectively.

The testes-to-final-body weight ratio, the number of sperm per milligram of cauda epididymis, and the percentage of abnormal spermheads were unaffected in the treated animals either at 21 or 35 days following oral gavage administration.

The NOEL value at 21 and at 35 days was equal or greater than 2500 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 000 mg/kg bw/day
Study duration:
subacute
Species:
mouse
Quality of whole database:
Reliable
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A reproductive screening study was not available, however a spermhead morphology assay was conducted with read across substances sodium trichloroacetate in B6C3F1 mice orally dosed by gavage up to 2500 mg/kg bw/day for five consecutive days (Meier et al., 1997). The types and frequencies of spermhead abnormalities were based on the scoring of 500 sperm cells per animal. In addition, body weights, testes and cauda epididymal weights, testes to final body weight ratios, and sperm concentration/mg of cauda epididymis were analyzed. The testes-to-final-body weight ratio, the number of sperm per milligram of cauda epididymis, and the percentage of abnormal spermheads were unaffected in the treated animals either at 21 or 35 days following oral gavage administration. The NOEL value at 21 and at 35 days was equal or greater than 2500 mg/kg bw/day.

 

Further, key oral repeated dose toxicity studies in rats and dogs with sodium trichloroacete (see Section 7.5) did not indicate reproductive toxicity potential. When administered in rats for 4 months (Scholz and Kozlik, 1969)and for 2 years (Scholz & Shultes, 1972) there were no effects on male and female reproductive organs up to 10000 ppm in the feed. Severe body weight loss was observed at the highest dose levels, leading to NOEL of 4000 ppm (approximately 365 mg/kg bw and 1600 ppm (approximately 160 mg/kg bw) for the 4-month and 2-year study, respectively. When Sodium trichloroacetate was studied in dogs in the feed for 90 days at 500, 2000, 4000 and 8000 ppm, male animals were more affected than the females (Scholz and Brunk, 1970). Next to mortality (in all high dosed males and one high dosed female), body weight loss and changes were observed in liver, heart muscles, skeletal muscles and testes (spermatogenesis) in animals from the 2000 ppm group upwards. Based on these results, the no-effect-level in this study was 500 ppm (approximately 30 mg/kg bw/day). As similar effects were not seen in the rat 90-day and lifetime studies conducted at doses of around 3x the LOAEL for testicular toxicity derived from the dog study, the testicular findings in dogs are considered to be secondary to body weight loss and severe paternal toxicity, therefore no indications for reproductive toxicity were derived from repeated dose toxicity studies in rats and dogs. No NOAELs were determined for reproductive toxicity.

Supporting information from a 90-day oral repeated dose toxicity study in rats dosed with trichloroacetic acid via the drinking water, only showed increased liver and kidney organ to body weight ratios and histopathologic changes after 5000 ppm, corresponding with 355 mg/kg bw (Mather et al., 1990). An additional study for 90 days in rats dosed with trichloroacetic acid via the drinking water at ca. 785 mg/kg bw , showed variable degrees of alterations in the lung and liver of treated animals: in the liver, morphological changes were predominantly localized to the portal triads, which were mildly to moderately enlarged with random bile duct proliferation, extension of portal veins, fibrosis, edema, and occasional foci of inflammation. In the lungs, minimal alterations were observed as foci of perivascular inflammation on small pulmonary veins (Bath et al., 1991).

Based on this comparative information between sodium trichloroacete and trichloroacetic acid, no relevant reproductive changes were seen after subchronic dosing for both substances. As most information was available for sodium trichloroacetate, this substance is selected as the source substance for read across.

 

According to REACH 1907/2006 legislation Annex IX (amended Feb. 20, 2015 by Commission Regulation (EU) 2015/282) an Extended One-Generation Reproductive Toxicity Study (B.56 of the Commission Regulation on test methods as specified in Article 13(3) or OECD 443), basic test design (cohorts 1A and 1B without extension to include a F2 generation), one species, most appropriate route of administration, having regard to the likely route of human exposure, is requested if the available repeated dose toxicity studies (e.g. 28-day or 90-day studies, OECD 421 or 422 screening studies) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity. Based on the negative outcome of STCA in a spermhead morphology assay in mice dosed up to 2500 mg/kg bw (2212 mg/kg bw TCA equivalents) and the absence of reproductive findings in rats up to highest tested doses of 365 mg/kg and 80 mg/kg bw (322 and 70.5 mg/kg bw TCA equivalents) after subchronic and chronic (lifetime) exposure, and the confirmatory absence for TCA of reproductive findings up to highest tested dose of 355 mg/kg bw in a subchronic rat study, there are no indications for adverse effects on reproductive organs or tissues. Therefore further testing for this endpoint is waived.


Short description of key information:
Based on the negative outcome of STCA in a spermhead morphology assay in mice dosed up to 2500 mg/kg bw (2212 mg/kg bw TCA equivalents) and the absence of reproductive findings in rats up to highest tested doses of 365 mg/kg and 80 mg/kg bw (322 and 70.5 mg/kg bw TCA equivalents) after subchronic and chronic (lifetime) exposure, and the confirmatory absence for TCA of reproductive findings up to highest tested dose of 355 mg/kg bw in a subchronic rat study, there are no indications for adverse effects on reproductive organs or tissues. Therefore further testing for this endpoint is waived.

Justification for selection of Effect on fertility via oral route:
Key study

Effects on developmental toxicity

Description of key information
Available data indicate that TCA is a developmental toxicant in the pregnant rat at doses of> 300 mg/kg bw. Maternal and fetal toxicity were observed from 330 mg/kg bodyweight, and from 800 mg/kg also embryo-lethality. In all dose groups there was a dose-dependent increase in visceral anomalies, particularly in the cardiovascular system. In contrast, female Sprague Dawley rats dosed at 300 mg/kg showed significantly reduced fetal body weights on GD 21, but did not demonstrate cardiac malformations. Other studies have been published on the developmental toxicity of TCA in rats and in vitro. Many of  these studies were conducted with excessively high TCA concentrations. The malformations seen were possibly due to too high toxic dose levels, therefore resulting in secondary findings. In fact there was no NOAEL for maternal and developmental toxicity defined for TCA. 
A key prenatal developmental toxicity study was conducted with read across substance Sodium trichloracetate in Sprague Dawley rats , resulting in NOAEL of 300 mg/kg bw/day for the dams and the fetuses. Test item-related embryotoxic effects were only noted at the materno-toxic dose level of 1000 mg/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.
A key prenatal development study was also conducted in pregnant New Zealand white rabbits with Sodium trichloroacetate, administered orally to female rabbits at dose levels of 5, 15 and 45 mg/kg bw/day during the critical phase of organogenesis and embryo/fetal development from the 6th to 28th day of pregnancy. The NO(A)EL was 15 mg/kg bw/day for the dams as well as for the fetal organism. This NOAEL was impacted by the gastro-intestinal senstiivity of the dams.
Link to relevant study records
Reference
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.
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
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
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
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.
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
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.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
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).
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
Abnormalities:
not specified
Developmental effects observed:
not specified
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.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A prenatal developmental toxicity study conducted in female Long-Evans rats from 6th to the 15th day of gestation dosed at oral doses of 330, 800, 1200 and 1800 mg/kg bw (Smith et al., 1989). Maternal and fetal toxicity (decreased fetal weight and length ) were observed from 330 mg/kg bodyweight, and from 800 mg/kg 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/day) to 97% at the high dose (1800 mg/kg/day).

In contrast, Fisher et al. (2001) dosed female Sprague Dawley rats from 6thto the 15thday of gestation, and observed significantly reduced fetal body weights on GD 21, but did not observe treatment-related effects on the incidence of cardiac malformations. The reason for the inconsistent findings is unknown. Smith et al. (1989) considered the cardiac malformation (levocardia) to be an ill-defined malformation and possibly of trivial appearance as found in Bouin’s fixed slices, therefore they are of uncertain biological relevance. In addition, the available data also did not permit identification of NOAEL values for the developmental or maternal toxicity of TCA, since in each study, adverse effects were observed at the lowest or only dose tested.

Other studies have been published on the developmental toxicity of TCA in rats exposed via the oral route. Some of these studies were conducted with excessively high TCA concentrations (e.g. Singh, 2006) or with a single dose of TCA (e.g.Johnson et al., 1998), and therefore provide limited information useful for informing the dose-response relationship for TCA in the low-dose region (EPA, 2011).

In vitro studies with mouse and rat whole embryo cultures were used to assess the potential for developmental toxicity of TCA (Hunter et al., 1996: Saillenfait et al., 1995). TCA induced a variety of morphologic changes in mouse and rat whole embryo cultures, supporting the appearance of soft-tissue malformations observed in vivo at maternally toxic doses. However, because of the high concentrations used in these assays, in vitro test systems are limited in their utility to predict adverse developmental effects and associated toxic potencies in intact organisms.

A key prenatal developmental toxicity study was conducted with read across substance Sodium trichloracetate in Sprague Dawley rats dosed at 100, 300 or 1000 mg/kg bw/day by oral gavage from the 6th to 19th day of pregnancy (Hansen, 2014). The NOAEL was 300 mg/kg bw/day for the dams. At 1000 mg /kg bw/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 NOAEL for the fetal organism was also 300 mg/kg bw/day. Test item-related embryotoxic effects were only noted at the materno-toxic dose level of 1000 mg/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 malformations were noted at any of the test item treated groups; the test item did not possess any teratogenic effect. The NOAEL for teratogenicity was 1000 mg/kg bw/day.

According to REACH 1907/2006 legislation Annex IX and X, pre-natal developmental toxicity testing in a second species is based on the outcome of the first test and all other relevant available data.According to OECD TG 414, the preferred non-rodent species is the rabbit. Data were also available from read-across substance Sodium trichloroacetate.

A supporting/dose-range-finding study was conducted in the rabbit by oral administration to select the dose levels for the main prenatal developmental toxicity study. Sodium trichloroacetate was administered orally to female rabbits at dose levels of 60, 100, 300 and 1000 mg/kg bw/day, during the ‘critical’ phase of organogenesis and embryo/fetal development from the 6th to 28th day of pregnancy (Hansen, 2015a). Treatment with 300 mg and 1000 mg/kg bw/day caused pronounced clinical signs of toxicity in the dams. Effects on the reproductive performance were noted in the dams and dosing in groups 3 and 4 was discontinued on gestation day 10 and a fifth group was added to the study. Group 5 was treated with 60 mg/kg bw/day. At 60 mg Sodium trichloroacetate/kg bw/day, one dam died on gestation day 29 immediately before laparotomy. One further dam aborted on gestation day 22 and was sacrificed on the following morning. At 100 mg Sodium trichloroacetate/kg bw/ day, one dam aborted on gestation day 26 and was sacrificed on the following morning. At 300 mg Sodium trichloroacetate/kg bw/ day, one dam was found dead on gestation day 10. Clinical and macroscopic observations in the dams were observed from 60/kg bw/day onwards, including lesions of the stomach, which are considered to be test item-related. Also in the foetuses, effects were observed from 60 mg/kg bw/day, such as increases late and total resorptions and slightly decreased fetal and placental weights. Based on the data obtained in this study, dose levels are suggested for the prenatal developmental toxicity study in rabbits with Sodium trichloroacetate by oral administration were 5, 15 and 45 mg/kg bw/day.

A key prenatal development study was conducted in pregnant New Zealand white rabbits. Sodium trichloroacetate was administered orally to female rabbits at dose levels of 5, 15 and 45 mg/kg bw/day during the critical phase of organogenesis and embryo/fetal development from the 6th to 28th day of pregnancy (Hansen, 2015b). At 45 mg/kg bw/day, an increase in the incidence of abortions was noted. Slight reductions were noted for body weight and body weight gain and distinct reductions were noted for the food consumption of the dams. Necropy of the dams treated with the high dose revealed gastric lesions, hepatic changes and an enlarged spleen. At the materno-toxic dose level of 45 mg Sodium trichloroacetate/kg bw/day, an increased post-implantation loss was noted due to an increased incidence of resorptions assicated with a reduced incidences of fetuses. Developmental delays were observed in the form of reduced fetal body weights and an increased incidence of retarded ossification of the sternum. There was no test item-related increase in the incidence of fetal malformations or variations during the macroscopic external or internal inspections at laparotomy, during the skeletal examination (according to DAWSON) or soft tissue examination of the fetal head (according to WILSON) at any tested dose level. Under the present test conditions, the no-observed-effect level (NOEL) as well as the no-observed-adverse-effect level (NOAEL) was 15 mg Sodium trichloroacetate/kg bw/day for the dams. The no-observed-adverse-effect level (NOAEL) for the fetal organism was also 15 mg/kg bw/day.

The dosing in rabbits was technically limited by the gastro-intestinal tolerance to the substance, therefore a low NOAEL was derived compared to the rat study. The rabbit is known as a difficult species as maternal gastro-intestinal disturbance may prevent testing higher dose levels, or gastro-intestinal disturbance may have impact on physiology and gestation. Therefore, although the rabbit study is also entered as a key study, the NOAEL is not considered to be fully equally representative as the rat NOAEL.


Justification for selection of Effect on developmental toxicity: via oral route:
Key study

Justification for classification or non-classification

Based on the read across between trichloroacetate and sodium trichloroacetate, and the absence of malformations in the prenatal developmental toxicity study in rats with sodium trichloroacetate there is no need to classify the substance for reproductive toxicity in accordance to criteria listed in the CLP Regulation (EC 1272/2008).