Registration Dossier

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:
8 January 2019 -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 25 June 2018
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
Commission Regulation (EU) No. 260/2014 adopted 24 January 2014, published in the Official Journal of the European Union L81, dated 19 March 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test material contains 41.2% solid content, therefore a correction factor 2.43 was applied.
See confidential information.

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, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age on day 0 of pregnancy: 62 - 70 days
- Weight on day 0 of pregnancy: 197.5 g - 275.6 g
- Fasting period before study: not reported
- Housing: The animals 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. The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages. Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
- Diet (e.g. ad libitum): Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, served as food. This food was offered daily ad libitum.
- Water (e.g. ad libitum): Drinking water (in drinking bottles) was offered ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%):55% ± 10% (maximum range)
- Air changes (per hr): between fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From start of mating: 14 January 2019
To termination of in-life part: 13 February 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: tap water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentrations and was administered orally at a constant volume/kg bw once daily from the 6th to the 20th day of gestation.
The amount of the test item was adjusted to the animal's current body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.
In addition, the homogeneity and concentration of the test item mixture were monitored.
The male rats for mating remained untreated.
Administration volume: 5 mL/kg bw/day

VEHICLE
- Concentration in vehicle: The test item was diluted in the vehicle to the appropriate concentrations and was administered orally at a constant volume/kg bw once daily from the 6th to the 20th day of gestation.
The amount of the test item was adjusted to the animal's current body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.
- Amount of vehicle (if gavage): Administration volume: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approximately 10 mL were taken at the following times and stored at -20 °C ± 10 % until analysis at LPT:
-At start of dosing:
Analysis of concentration: During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples:1 x 3 = 3 (Sampling date: 22 January 2019)
Homogeneity: At the start of treatment, during (middle) administration and before administration to the last animal of the test item group. (3 samples/test item group)
Number of samples: 3 x 3 = 9 (Sampling date: 22 January 2019)
- At the end of the dosing period, at a time when the majority of the animals was dosed:
Analysis of concentration: During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples:1 x 3 = 3 (Sampling date: 04 February 2019)
Sum of all samples:=15
The samples were labelled with the study number, species, type of sample, concentration, sampling time, date and test day.
The analytical method was validated in LPT Study No. 36867. The samples were analysed at LPT.
-Results:
Concentration before administration of the last animal of each dose group at start of dosing and at a time when the majority of animals was dosed: 99.2% - 101.6% nominal concentration.
Homogeneity at the start of administration, during administration and before administration to the last animal of each dose group: 98.5% - 100.2% nominal concentration.
The measured actual concentrations of the test item in the test item vehicle-mixtures were between 98.5% and 101.6% of the nominal concentrations, indicating correctly prepared and homogenous formulations.
Details on mating procedure:
- Impregnation procedure: cohoused
- If 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 25 mated dams were available for each groups.
- Any non-pregnant rats would have been excluded from the analysis of the results and replaced with the designated spare animals of each group treated and mated together with the other animals. 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: 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:
15 administration days from gestation days 6 to 20
Frequency of treatment:
once daily
Duration of test:
5 adaptation days, 1 mating day, 15 administration days from gestation days 6 to 20, laparotomy on gestation day 21= 22 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 other: mg act. ingr./kg bw/day actual ingested
Dose / conc.:
300 other: mg act. ingr./kg bw/day actual ingested
Dose / conc.:
1 000 other: mg act. ingr./kg bw/day actual ingested
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for this study had been selected to be 100, 300 and 1000 mg act. ingr./kg bw/day by the Sponsor based on available toxicological data of an OECD 422 study (LPT study report no. 28640). The dose of 1000 mg/kg b. was considered to be a maximum tolerated dose.
In this OECD 422 study, the test item was administered orally to rats at dose levels of 100, 300 and 1000 mg act. ingr./kg bw/day. The administration started two weeks before mating on test day one and ended the day before sacrifice. Males were dosed up to test day 36 and females were dosed up to test day 54 (day 3 post-partum).
- Rationale for animal assignment (if not random): Four (4) groups of pregnant rats were established, each obtained from matings which were carried out on a daily basis. Vaginal smears were taken each morning. Day 0 of pregnancy was the day on which sperm was found in the vaginal smear. When positive, the animals were assigned to the test groups by mating day using a Provantis® -generated randomisation based on the body weight of the animals.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: In addition to detailed clinical observations, 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.
Further checks were made early in the morning and again in the afternoon of each working day 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 were examined for abnormal development, whenever possible. No abortion or premature delivery occurred in the study.

DETAILED CLINICAL OBSERVATIONS: Yes. Individual animals were observed daily for behavioural changes, reaction to treatment, or illness.
- Time schedule: 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.
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 weighing - always at the same time of the day.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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.
The relative food consumption (g/kg bw/day) was calculated using the following formula:
Daily food consumption [g/kg bw/day]= Total food intake in g / Body weight in kg

WATER CONSUMPTION: Visual
- Time schedule for examinations: Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. Dehydration of the dams was avoided.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21. The rats were laparotomised under CO2 narcosis
- Organs examined: . The thyroids (including parathyroids) and the gravid uterus (in toto) of the dams were removed and weighed.
In order to check for possible test item effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. The thyroids and any organs with macroscopic findings of all dams (including deceased or prematurely sacrificed animals) were fixed in 7% neutral buffered formalin.
- The thyroids of all evaluated dams were examined histopathologically after preparation of haematoxylin-eosin stained paraffin sections.

OTHER:
BODY WEIGHT GAIN: The body weight gain was calculated in intervals (i.e. gestation day 0-3, 3 6, 6-9, 9-12, 12 15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 is given. These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.

THYROID HORMONE (T3, T4, TSH) DETERMINATION: In order to obtain approximately 2 x 150 µL serum for each endocrine endpoint (T3, T4, TSH), a sufficient volume of blood was taken from the retrobulbar ve-nous plexus under isoflurane anaesthesia from animals fasted overnight following a randomisation scheme. Blood samples were taken always at the same time of day (approximately from 7:00 a.m. to 10:00 a.m.). The samples were divided into aliquots and stored at -20°C ± 10% at LPT until analysis using ELISA.
The T3, T4 and TSH ELISA (commercial kits: IBL International GmbH , instrument: Tecan Sunrise ) was conducted at LPT.

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
- Other: Weight of placentae
Fetal examinations:
- External examinations: Yes: all per litter ((dead and alive)
- Soft tissue examinations: Yes: The remaining 50% of the number of fetuses (cfr skeletal examinations) in each litter were examined for soft tissue anomalies.
- Skeletal examinations: Yes: 50% of the number of fetuses in each litter
- Head examinations: No

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per sex and litter
- mean per group
- mean per sex and group

Fetuses
- number of evaluated dams with viable fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive + dead) per sex and dam
- number of fetuses (alive + dead) per sex and group
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group per dam
- mean % of fetuses alive per group per dam
- male/female ratio (alive + dead) per litter
- male/female ratio (alive + dead) per group

Runts
- number per dam
- number per group
Statistics:
-Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
-Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test: FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01) or Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01).
The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external or internal macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
The statistical evaluation of the pre- and post-implantation index per group using the number of corpora lutea, implantation sites and/ or fetuses per was done using StatXact 4.0.1 software.
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:
Total malformation rate (%) = (malformed fetuses per group / fetuses per group) x 100
Total variation rate (%) = (fetuses per group with variations / fetuses per group) x 100
Total retardation rate (%) = (fetuses per group with retardations / fetuses per group) x 100
Pre-implantation loss (%) = ((Corpora lutea (per group) – implantations (per group)) / Corpora lutea (per group)) x 100
Post-implantation loss (%) = ((Implantations (per group) – living fetuses (per group)) / Implantations (per group)) x 100
Litter indices:
Pre-implantation loss [%] =Sum of pre-implantation losses per litter in a group [%] / Number of litters in a group
Post-implantation loss [%] = Sum of post-implantation losses per litter in a group [%] / Number of litters in a group



Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
(No toxicologically relevant effects). No adverse influences on the behaviour, external appearance or faeces were noted for the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day). However, at 1000 mg act. ingr./kg bw/day, salivation was noted for 16 of 20 females. This was not considered to be an adverse effect.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related premature deaths were noted in the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group (300 mg act. ingr./kg bw/day), female no. 67 was prematurely sacrificed on its gestation day 12 due to animal welfare reasons. Female no. 67 was noted with clinical observations in form of piloerection and slightly reduced motility on GD 11. Also, a reduced food consumption from GD 7 onwards and a reduced body weight from GD 8 onwards were noted. Necropsy revealed macroscopic changes in form of a haemorrhagic nose and canthus of the left eye, dark red discoloured lungs and a stomach ulcus. The death of the animal was regarded to be due to a misgavage.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg act. ingr./kg bw/day, a reduced body weight and a reduced body weight gain were noted from GD 16 until study termination on GD 21 (at maximum 6.1% below the value of the control group for the body weight and 22% below the value of the control group from GD 6 to GD 21 for the body weight gain).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg act. Ingr./kg bw/day, a test item-related transient decrease in food consumption was noted between GD 6 and GD 10.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
No test item-related differences were noted.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Thyroid hormone concentration: No toxicologically relevant changes for serum T3, T4 and TSH levels were noted.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Uterus and carcass weights: No test item-related differences were noted for the gravid uterus weight. In the high dose group (1000 mg act. ingr./kg bw/day), a reduction was noted for the carcass weight (7.0% below the value of the control group, p ≤ 0.01).
Thyroid weights: No test item-related differences were noted.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic inspection of the dams at necropsy revealed no adverse effects at 100 and 300 mg act. ingr./kg bw/day, but test item-related changes in the cardiac region of the stomach in form of a (partly) whitish thickening or white deposits for 18 of 20 high dose animals at 1000 mg act. ingr./kg bw/day. However, due to the absence of a forestomach in humans this finding is of no noteworthy relevance for humans.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathologic examination of the thyroids revealed no test item-related changes.
Histopathological findings: neoplastic:
no effects observed
Details on results:
- Behaviour, external appearance, faeces:
No changes in behaviour, the external appearance or the faeces were noted in the control group and the low dose group (100 mg act. ingr./kg bw/day) and no toxicologically relevant changes were noted for the intermediate and high dose groups (300 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group female no. 67 was prematurely sacrificed on its gestation day 12 due to animal welfare reasons; two further animals (nos. 59 and 60) were noted with clinical observations. However, as only 3 of 22 animals including prematurely sacrificed animal no. 67 were affected and the clinical signs were noted only for a few days , the clinical signs were considered to be not toxicologically relevant. In addition, animal no. 59 was identified with 19 dead fetuses at laparotomy. However, it is unknown whether the fetuses died first and were the cause for the observations noted for animal no. 59 or the death of all fetuses was caused by the poor health of the dam. Nevertheless, this animal can be considered as an outlier.
In the high dose group (1000 mg act. ingr./kg b.w./day), clinical observations were noted in form of piloerection, salivation and increased water consumption. As piloerection and increased water consumption were noted only for a few group 4 females, these two clinical observations were not considered to be an adverse effect. Salivation was noted for 16 of 20 animals. However, as animals were affected not more than two days each and salivation lasted only shortly, the observations of salivation are regarded to be test item-related but not considered to be an adverse effect.
- Mortality:
No test item-related premature deaths were noted in the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day). In the intermediate dose group (300 mg act. ingr./kg bw/day), female no. 67 was prematurely sacrificed on its gestation day 12 due to animal welfare reasons. Female no. 67 was noted with clinical observations in form of piloerection and slightly reduced motility on GD 11. Also, a reduced food consumption from GD 7 onwards and a reduced body weight from GD 8 onwards were noted. Necropsy revealed macroscopic changes in form of a haemorrhagic nose and canthus of the left eye, dark red discoloured lungs and a stomach ulcus. The death of the animal was regarded to be due to a misgavage.
- Body weight and body weight gain:
No test item-related differences in body weight were noted between the dams of the control group and low and intermediate dose groups (100 or 300 mg act. ingr./kg bw/day).
In the intermediate dose group (300 mg act. ingr./kg bw/day), a reduced but not statistically significant body weight was noted from GD 18 until GD 21 (at maximum 5.9% below the value of the control group). However, as this was mainly due to the two females nos. 59 (animal with 19 dead fetuses) and female no. 60 that were in a poor health conditions; the reduced body weight was considered to be not test item-related. In the high dose group (1000 mg act. ingr. /kg bw/day), a decreased body weight was noted from GD 16 until study termination on GD 21 (at maximum 6.1% below the value of the control group on GD 21, statistically significant for GD 20 and GD 21 at p ≤ 0.05 or 0.01) for nearly all animals. This constantly decreased body weight was considered to be test item-related.
No test item-related difference between the control group and the animals treated with 100 or 300 act. ingr./kg bw/day was noted for the body weight gain from GD 0 to 21. At 300 mg act. ingr./kg bw/day, a slight reduction was noted for the body weight gain (statistically significant for the body weight gain of the treatment period from GD 6 to GD 21, p ≤ 0.01). However, this was mainly due to only two females (nos. 59 and 60). Animal no. 59 delivered 19 dead fetuses with distinctly reduced body weights and also the fetuses of animal no. 60 had a lower body weight due to the poor health of this dam. The decreased body weights of the fetuses led to decreased maternal body weights and therefore, the reduced body weight gain was considered to be not test item-related. At 1000 mg act. ingr./kg bw/day, a statistically significant reduction was noted for the body weight gain for the whole study period (GD 0 to GD 21) and for the treatment period (GD 6 to GD 21) at p ≤ 0.01 for nearly all animals. As this was in accordance with the test item-related reduction in the body weight, also the reduced body weight gain was considered to be test item-related.
There were no test item-related influences on the gravid uterus weight. Therefore, the gravid uterus weight had the same influence on the body weight gain for all groups
- Food and drinking water consumption:
No test item-related difference was noted between the control group and the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day). In the high dose group, a statistically significantly (at p ≤ 0.05 or 0.01) decreased food consumption was noted between GD 6 to GD 10 (at maximum 25.8% below the value of the control group on GD 7 to GD 8). It reflects the decreased body weight and body weight gain observed in this group. As the food consumption recovered thereafter, this transient reduction in food consumption was considered to be test item-related. Furthermore, a statistically significant increase in food consumption was noted for the high dose group (GD 2 to GD 3) and a statistically significant decrease was noted for the low dose group (GD 12 to GD 13). However, as these differences were either before start of treatment (high dose group) or lasted for only one day (low dose group) these differences were considered to be spontaneous.
Although female no. 86 of the high dose group was noted with increased water consumption on gestation day 20 and 21, no test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.
- Thyroid hormone concentration:
No test item-related differences were noted for the serum concentration of T4 in all dose groups (100, 300 or 1000 mg act. ingr./kg bw/day) and for T3 and TSH in the low dose group.
In the intermediate and high dose group (300 or 1000 mg act. ingr./kg bw/day), a dose-related increase was noted for the serum concentration of T3 (64.4% and 92.2% above the value of the control group, both statistically significant at p < 0.01) and TSH (8.8% and 19.8% above the value of the control group, not statistically significant). However, no difference was noted for the thyroid weights and the histopathological examination of the thyroids revealed no pathologic changes. Therefore, as it is known that histopathological examination of the thyroids is usually more sensitive than hormone levels (Beekhuijzen et al., 2016) the increases in serum levels of T3 and TSH were considered to be of no toxicological relevance.
- Necropsy findings:
No test item-related observations were noted for the dams of the low and intermediate dose groups (100 or 300 mg act. ingr./kg bw/day) during the macroscopic inspection of the organs and tissues.
In the intermediate dose group, macroscopic changes were noted for three females: No. 59 that delivered only dead fetuses revealed changes in form of a haemorrhagic nose and snout, rough fur , an anus that was soiled with faeces and inflated intestines filled with yellow liquid. Female no. 60 was noted with rough fur11 and few haemorrhagic foci in the stomach and the prematurely sacrificed female no. 67 was noted with a nose and the canthus of the left eye that was haemorrhagic, dark-red discoloured lungs and a stomach ulcus.
All findings were regarded to be spontaneous (animals no. 59 and 60) or related to the misgavage (animal no. 67). It is unknown whether the dead fetuses of animal no. 59 were the cause or the result of the bad health condition of the dam. Although the occurrence of dead fetuses is a very rare incident, this incident with all fetuses being dead is considered to be not test item-related as it was observed in only 1 animal of the intermediate dose group and therefore regarded to be an outlier.
In the high dose group (1000 mg act. ingr./kg bw/day), necropsy revealed for single animals changes in the stomach in form of ulcers (no. 80) and detachment of the mucosa (no. 93). Furthermore, a (partly) whitish thickening of the cardiac region of the stomach was noted for 18 of 20 examined animals.
Due to the high incidence of stomach observations, the changes related to the cardiac region of the stomach observed in the high dose animals were considered to be test item-related. However, due to the absence of a forestomach in humans this finding is of no noteworthy relevance for humans.
- Histopathology:
No test item-related morphological lesions were noted during histopathologic examination of the thyroids of the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
In all groups, histopathologic examination revealed microfollicular structures with cells in the follicular lumen of the thyroids and squamous cell cysts of the thyroid follicle. However, these findings were considered to be coincidental and not test item-related.
- Thyroid weights
No test item-related differences to the control group were noted for the thyroid weights of the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Gravid uterus weight, carcass weight and body weight gain from day 6:
No test item-related differences were noted between the gravid uterus weight of the control dams and the dams of the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
For the carcass weight, a statistically significant decrease was noted for the high dose animals (7.0% below the value of the control group, p ≤ 0.01). As this was in accordance with the lower body weight, the reduced carcass weight was considered to be test item-related.
No test item-related differences were noted for the net body weight gain from GD 6 to 21 between the dams of the control group and the dams of the low and intermediate dose groups (100 or 300 mg act. ingr./kg bw/day).
In the intermediate dose group, a statistically significant decrease (p ≤ 0.01) was noted for the net body weight gain. However, as also the low net body weight gain was mainly due to the animals nos. 59 and 60, the reduction was considered to be not test item-related.
At 1000 mg act. ingr./kg bw/day, the distinctly decreased net body weight gain (9.2 g compared to 37.0 g in the control group, p ≤ 0.01) was considered to be test item-related as it was in accordance with the reduced carcass weight and the reduced weight of the whole body (gravid uterus weight + carcass weight).

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
In the intermediate dose group, a statistically significantly increased value was noted for the post-implantation loss (p ≤ 0.01). As this was mainly due to one animal that delivered only dead fetuses (animal no. 59), the increased post-implantation loss was considered to be spontaneous and not test item-related.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
In the intermediate dose group, a statistically significantly increased value was noted for the post-implantation loss (p ≤ 0.01). As this was mainly due to one animal that delivered only dead fetuses (animal no. 59), the increased post-implantation loss was considered to be spontaneous and not test item-related.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
In the intermediate dose group, one animal that delivered only dead fetuses (animal no. 59), which was considered to be spontaneous and not test item-related.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) was noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group, a statistically significantly increased value was noted for the post-implantation loss (p ≤ 0.01). As this was mainly due to one animal that delivered only dead fetuses (animal no. 59), the increased post-implantation loss was considered to be spontaneous and not test item-related.

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:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
not specified

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weight of the fetuses and the placentae: No test item-related differences were noted between the control group and the dose groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the low and high dose group (100 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group (300 mg act. ingr./kg bw/day), dam no. 59 delivered only dead fetuses (11 male and 8 female fetuses). External inspection revealed one female fetus (no. 59-08) with a malformation in form of an anencephaly. However, as no other influences on the reproduction were noted and no dead fetuses were observed in the high dose group, the occurrence of one litter with only dead fetuses was considered to be not test item-related.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related differences between the ratio of male and female fetuses were noted between the control group and the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test item-related malformations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions).
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test item-related malformations were noted during the skeletal examination according to DAWSON.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test item-related malformations were noted during the soft tissue examination according to WILSON.
Other effects:
no effects observed
Description (incidence and severity):
Mortality: No test item-related death of fetuses was noted.
Variations: The macroscopic examinations at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON revealed no test item-related variations
Retardations: No test item-related retardations (delays in ossification) were noted during the skeletal examination according to DAWSON.
Details on embryotoxic / teratogenic effects:
- Mortality:
No dead fetus was noted in the control group and in the low and high dose group (100 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group (300 mg act. ingr./kg bw/day), dam no. 59 delivered only dead fetuses (11 male and 8 female fetuses). External inspection revealed one female fetus (no. 59-08) with a malformation in form of an anencephaly. However, as no other influences on the reproduction were noted and no dead fetuses were observed in the high dose group, the occurrence of one litter with only dead fetuses was considered to be not test item-related.
- Sex distribution of the fetuses:
No test item-related differences between the ratio of male and female fetuses were noted between the control group and the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Weight of placentae and fetuses:
The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Number of runts:
No runts were noted for the control group and the intermediate and high dose groups (300 or 1000 mg act. ingr./kg bw/day).
In the low dose group (100 mg act. ingr./kg bw/day), two runts (nos. 26-15 and 36-11) were noted. The occurrence of two runts in the low dose group is within the normal range of biological variability and therefore, was considered to be not test item-related.
- Ano-genital distance:
No test item-related differences to the control group were noted for the fetal ano-genital distance of the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Macroscopic inspection of the fetuses at laparotomy:
1) External inspection at laparotomy
No macroscopically visible external malformations were noted for the fetuses of the low and high dose group (100 or 1000 mg act. ingr./kg bw/day) during the external inspection at laparotomy.
In the intermediate dose group (300 mg act. ingr./kg bw/day), one fetus (no. 59 08, dead at birth) was noted with a malformation in form of an anencephaly and one fetus (no. 54-19) was noted with multiple malformations in form of a bifurcated tail, supernumerary extremities at the back (presumably a conjoined twin), a cleft palate and an anencephaly. The occurrence of two fetuses with malformations was considered to be spontaneous and not test item-related.
2) Gross inspection of the organs and tissues at laparotomy
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
3) Testicular development
No cryptorchidism and no testicular malposition was noted during assessment of the testicular development of the male fetuses of the control group and the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Skeletal examination according to DAWSON:
1) Skeletal malformations
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (100, 300 or 1000 mg act. ingr./kg bw/day) during the skeletal examination according to DAWSON.
2) Skeletal variations
Skeletal variations were noted for the ribs (ribs isolated, ribs short or ribs wavy) and the sternum (bipartite or misaligned to a slight degree).
No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
3) Skeletal retardations
Retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite, reduced in size or dumbbell-shaped), the caudal vertebral bodies (only one body ossified or all bodies unossified), the lumbar vertebral bodies (unossified), the sacral vertebral bodies (unossified), the lumbar vertebral arches (incompletely ossified), the os ischii (unossified or incompletely ossified), the os pubis (unossidied or incompletely ossified) and the metacarpalia/metatarsalia (absence of ossification in metacarpalia/metatarsalia 2 to 5).
No test item-related increase in the incidence of skeletal retardations at 100, 300 or 1000 mg act. ingr./kg bw/day was noted during skeletal examination according to DAWSON.
In the intermediate dose group, statistically significantly increased incidences were noted for skeletal retardations that were outside the range of the LPT background data (all caudal vertebral bodies unossified, os ischii unossified, os pubis unossified and sacral vertebral bodies unossified). However, the majority of the mentioned retardations were noted for dam no. 59 that littered only dead fetuses. Therefore, the retardations were considered to be not test item-related.
- Soft tissue examination according to WILSON:
1) Malformations
No malformations were noted for the fetuses of the control group and the fetuses of the low and high dose groups (100 or 1000 mg act. ingr./kg bw/day) during the soft tissue examination according to WILSON.
In the intermediate dose group (300 mg act. ingr./kg bw/day), one fetus (no. 59 8) was noted an anencephaly and one fetus (no. 54 19) was a conjoined twin with two spinal cords, extremities in the back region, a bifurcated tail, anencephaly, an open eye, a cleft palate and a missing anus.
However, as no malformations were noted in the high dose group, the occurrence of two fetuses with malformations in the intermediate dose group was considered to be spontaneous and not test item-related.
2) Variations
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis or malpositioned) and the liver (haemorrhagic focus/foci).
No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
3) Unclassified observations
No unclassified observations were noted for the control group and for the low and high dose group (100 or 1000 mg act. ingr./kg bw/day).
An unclassified observation in form of a thoracic cavity filled with blood was noted for two fetuses (nos. 60-1 and 60-2) of the intermediate dose group (300 mg act. ingr./kg bw/day). This observation was considered to be a preparation-induced artefact and not test item-related.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: highest tested dose

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg act. ingr./kg bw/day for the dams.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mg act. ingr./kg bw/day.
Under the conditions of the study, Butanedioic acid, 2(or 3)-sulfo-,4-[2-[(1-oxo(C12-C8 (even numbered) and C18 (unsaturated)alkyl) amino]ethyl]esters, disodium salts did not show any teratogenic potential in rats
Executive summary:

The aim of the study was to obtain information of the influence of the test item on the pregnant rat and the fetus when administered orally during the critical period of organogenesis and the fetal development (6th to 20th day of gestation) according to OECD Guideline 414. The test item was administered orally by gavage to rats at dose levels of 100, 300 and 1000 mg active ingredient/kg bw/day starting from the 6th and lasting until the 20th day of pregnancy. Their development was observed during the gestation period (day of mating is day 0 of pregnancy). On gestation day 21 (one day before the calculated date of parturition), the dams were laparotomised and examined for corpora lutea, implantation sites and resorptions in the uterus and for the condition of the fetuses.

 

Examination of the dams:

No test item-related premature deaths were noted in any of the test groups.

No adverse influences on the behaviour, external appearance or faeces were noted for the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day). However, at 1000 mg act. ingr./kg bw/day, salivation was noted for 16 of 20 females.

At 1000 mg act. ingr./kg bw/day, a reduced body weight and a reduced body weight gain were noted from GD 16 until study termination on GD 21 (at maximum 6.1% below the value of the control group for the body weight and 22% below the value of the control group from GD 6 to GD 21 for the body weight gain).

At 1000 mg act. ingr./kg bw/day, a test item-related transient decrease in food consumption was noted between GD 6 and GD 10.

No test item-related differences were noted in drinking water consumption.

Macroscopic inspection of the dams at necropsy revealed no adverse effects at 100 and 300 mg act. ingr./kg bw/day, but test item-related changes in the cardiac region of the stomach in form of a (partly) whitish thickening or white deposits for 18 of 20 high dose animals at 1000 mg act. ingr./kg bw/day. However, due to the absence of a forestomach in humans this finding is of no noteworthy relevance for humans.

No test item-related differences were noted in thyroid weights.

No toxicologically relevant changes for serum T3, T4 and TSH levels were noted.

Histopathologic examination of the thyroids revealed no test item-related changes.

No test item-related differences were noted for the gravid uterus weight.

In the high dose group (1000 mg act. ingr./kg bw/day), a reduction was noted for the carcass weight (7.0% below the value of the control group, p ≤ 0.01).

No test item-related influence was noted on the reproductive parameters (number of implantation sites, resorptions and fetuses).

 

Examination of the fetus:

No test item-related death of fetuses was noted.

No test item-related differences in body weight of the fetuses and the placentae were noted between the control group and the dose groups.

 

Fetal alterations:

No test item-related malformations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions), and no malformations were noted during the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.

The macroscopic examinations at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON revealed no test item-related variations.

No test item-related retardations (delays in ossification) were noted during the skeletal examination according to DAWSON.

 

Analysis of test item formulations:

The measured actual concentrations of the test item in the test item vehicle mixtures were between 98.5% and 101.6% of the nominal concentrations, indicating correctly prepared and homogeneous formulations.

 

Conclusion

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

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mg act. ingr./kg bw/day.

Under the conditions of the study, Butanedioic acid, 2(or 3)-sulfo-,4-[2-[(1-oxo(C12-C8 (even numbered) and C18 (unsaturated)alkyl) amino]ethyl]esters, disodium salts did not show any teratogenic potential in rats.