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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2022-09-07 to 2022-10-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2022-05-18 to 2022-06-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
yes
Remarks:
please refer to 'principles of method if other than guideline'
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
please refer to 'principles of method if other than guideline'
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
yes
Remarks:
please refer to 'principles of method if other than guideline'
Principles of method if other than guideline:
- Principle of test:
14 days dose range finder for full OECD 414 study:
10 sperm positive females per dose group (4 dose groups) were treated from gestation day 5 to 19
- Parameters analysed / observed:
Mortality, clinical signs, necropsy and histopathology findings of dams, food consumption, body weight (gain), corrected body weight for uterine weight, intrauterine mortality, fetal- and placental weight, external examination of fetuses and placentas
GLP compliance:
no
Remarks:
This study was not performed according to GLP compliances, however, the principles of GLP were followed and all data were recorded and retained.
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han: WIST
Details on test animals or test system and environmental conditions:
EST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: females: ca. 9 weeks of age at start of the mating period; males: ca. 13 – 14 weeks of age at start of the mating period
- Weight at study initiation: females: ca. 170 –190 g
- Fasting period before study: no
- Housing:
before mating: 1 – 2 females/cage; during mating: 1 male and 1 – 2 females/cage; during gestation: 2 sperm positive females/ cage, if not possible 1 sperm positive female/ cage;
cages: Type II polypropylene/polycarbonate;
bedding: Certified laboratory wood bedding (Safe 3/ 4-S-FASERN produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany)
- Diet: ad libitum, ssniff SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 – 22.8
- Humidity (%): 51 – 69
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2022-05-18 To: 2022-06-08
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water). Formulations were prepared beforehand not longer than for three days and stored at room temperature until use.

VEHICLE
- Justification for use and choice of vehicle: Water (Aqua purificata) has been shown to be a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 60, 140 and 200 mg/mL (calculated by the concentration of active ingredient)
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 2202-8205
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male/ one to two females
- Length of cohabitation: 2 - 4 h
- After unsuccessful pairing replacement of first male by another male with proven fertility: not performed
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
14 days
The sperm positive females were treated from gestational day 5 to 19.
Frequency of treatment:
daily
Duration of test:
The animals were sacrificed by anaesthesia on gestation day 20.
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
700 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
A total number of 100 females was used for mating to achieve the sufficient number of females per group of at least 8 spermium positive females per group.

The number of mated and of pregnant females was as follows:
Control: mated: 11, pregnant: 10
300 mg/kg bw/d: mated: 11, pregnant: 11
700 mg/kg bw/d: mated: 12, pregnant: 9
1000 mg/kg bw/d: mated: 12, pregnant: 8
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels of 1000, 700 and 300 mg/kg bw/day were selected based on the results of the results of a preliminary repeated dose toxicity study and a reproduction toxicity screening study respectively. Additionally, the available short-term repeated dose toxicity study was taken into consideration. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

- Rationale for animal assignment (random):
Females were randomly assigned to dose groups on the basis of their body weight on the day of mating in such a way that the group averages of the body weight were as similar as possible on the first day (day 0) of gestation.

- Fasting period before blood sampling for dam thyroid hormones: No fasting period described.
- Time of day for dam blood sampling: Not performed
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day from GD 0 to 20, on treatment days after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. When signs of toxicity were observed, animals were observed more frequently.
Individual observation included the check of behavior and general condition.
Duration and severity of the clinical signs were recorded.
Observations for signs of morbidity and mortality were made twice daily from GD 1 to 19, at the beginning and end of the working period, and once on GD 0 and 20.

DETAILED CLINICAL OBSERVATIONS: Yes, see above.

BODY WEIGHT: Yes for females; No for males
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).

FOOD CONSUMPTION: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: 20
- Organs examined: The uterus with cervix was removed and weighed. The ovaries were placed into a Petri dish after removal. After removing the uterus, gross pathology of dams' viscera was performed.
Organs and tissues with undiagnosed macroscopic findings were fixed in 4 % (v/v) buffered formalin solution at necropsy for possible histological examination. Control organs were fixed in 4 % (v/v) buffered formalin solution and preserved for comparison.
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: Live fetuses and fetal deaths were counted.
Blood sampling:
Not performed
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: Yes: all per litter (external)
- Anogenital distance of all live rodent pups
Statistics:
Due to the low number of litters in the DRF study, statistical analysis was not performed.
Indices:
- Pre-implantation loss: ((Number of corpora lutea - Number of implantations)/Number of corpora lutea)*100
- Post-implantation loss: ((Number of implantations - Number of live fetuses)/Number of implantations)*100
- Sex distribution: (Number of Male (Female) fetuses/Number of fetuses)*100
- External abnormalities/litter: (Number of fetuses with abnormality/Number of fetuses)*100
Historical control data:
please refer to 'Overall remarks, attachments'
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The deceased animals had clinical signs related to observed or supposed reflux of the test item formulation. Clinical signs such as dyspnoea, reduced activity, hunched back and/or piloerection were recorded for two females in the 300 mg/kg bw/day group (reflux was observed in one female, both died), for five in the 700 mg/kg bw/day group (for 3 reflux was observed for 1 reflux was supposed, 1 died and 1 was moribund) as well as for three in the 1000 mg/kg bw/day dose group and in addition squealing for two of these animals (reflux was recorded for two animals, all survived). In the cases where no reflux of the test item was seen, also aspiration was assumed based on the similar clinical signs.
Since no dose-relation was observed and reflux and aspiration of the test item was attributed solely to the application via gavage, the observed findings were considered to be non-related to oral exposure to the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two animals died at 300 mg/kg bw/day and one at 700 mg/kg bw/day, as well as one was moribund in latter group. The cause of death was supposed as secondary exposure by aspiration. In case of one of the 300 mg/kg bw/day dams (No.: 149) the cause of death was likely mis-gavage (bloody content in the thoracic cavity). There was no mortality at 1000 mg/kg bw/day.
Since no dose-relation was observed and reflux and aspiration of the test item was attributed solely to the application via gavage, the observed findings were considered to be non-related to oral exposure to the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower body weight was seen at 700 mg/kg bw/day at G.D. 8, 17 and 20, a test item effect was not proved. From G.D. 8, at 1000 mg/kg bw/day, the mean body weight of the dams was moderately lower than in the other groups.
The body weight gain was significantly lower at 700 mg/kg bw/day and weight loss was observed at 1000 mg/kg bw/day from G.D. 5 to 8. At 1000 mg/kg bw/day the body weight gain was lower which was significant between G.D. 11 and 17, as well as if the whole treatment period observed (G.D. 5 to 20 or 0 to 20).
The corrected body weight/gain was slightly lower in the test item treated groups at a similar level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food consumption was observed at 700 and 1000 mg/kg bw/day between G.D. 5 and 11 with a dose response.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
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):
Gravid uterine weights were comparable in all groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item related findings as thicker cardia (2 at 700 and 5 at 1000 mg/kg bw/day) were observed. These findings were attributed to the irritative properties of the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histological examination revealed squamous cell hyperplasia in the forestomach of all surviving animals in the middle and high dose. In addition, erosion (700 mg/kg bw/day, No.: 187, 194; 1000 mg/kg bw/day, No.: 113, 151, 158, 169), and -inflammatory cell infiltrates occurred as well.
The development of squamous cell hyperplasia was presumed to be in connection with the local effect of 700 mg/kg bw/day and 1000 mg/kg bw/day of the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Other findings like bloody dried fluid at nasal region (1 at 700 mg/kg bw/day), compact formation next to salivary gland (1 at 300 mg/kg bw/day), point-like bleedings in the thymus (1 at 700 mg/kg bw/day), foamy discharge in the esophagus (1 at 700 mg/kg bw/day), reddish mottled or dark red lungs (1 at 300 and 2 at 700 mg/kg bw/day), bloody content in the thoracic cavity (1 at 300 mg/kg bw/day), yellowish content in the stomach and duodenum (1 at 300 mg/kg bw/day) and dark red liver (1 at 300 mg/kg bw/day) were not judged to be a consequence of a direct test item effect.
Number of abortions:
no effects observed
Description (incidence and severity):
None of the dams had total post-implantation loss.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The mean of pre-implantation loss was slightly higher at 1000 mg/kg bw/day, as well as the mean number of implantations was slightly lower in this group. Considering the slight difference, a test item effect was not proved.
Post-implantation loss was higher in the test item treated groups without a dose response.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item related increase in resorptions was noted.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no dose response indicated in early or late embryonic death.
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item related increase in dead fetuses was observed.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
All animals were sacrificied on GD 20.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Control: mated: 11, pregnant: 10
300 mg/kg bw/d: mated: 11, pregnant: 11
700 mg/kg bw/d: mated: 12, pregnant: 9
1000 mg/kg bw/d: mated: 12, pregnant: 8

Slightly fewer pregnant animals were observed in the high dose group. Since test item treatment of the pregnant animals starts at GD 5, no test item relation was proved.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
DRF study
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The weight of the male and female fetuses was similar in the experimental groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of viable fetuses was comparable in all groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no dose response indicated in sex ratio of fetuses.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The were no differences in litter size and weights.
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no variations and malformations observed. There was no dose response in the incidence of body weight retarded (small) fetuses.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
DRF study
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the preliminary observations obtained in this dose range finding study, the doses for the main Prenatal Developmental Toxicity Study were selected as follows:
Group 1 Vehicle control
Group 2 100 mg/kg bw/day
Group 3 300 mg/kg bw/day
Group 4 1000 mg/kg bw/day
Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of eleven sperm-positive female Han: WIST rats were treated with Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated) -N,N-dibutylamide, monosulphated, sodium salts by oral administration daily at three dose levels of 300, 700 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of eleven sperm positive females was included and the animals were given the vehicle water (Aqua purificata). The treatment volume was 5 mL/kg bw.


During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day (G.D.) 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were examined externally.


Proceeding from 10, 11, 9 and 8 pregnant females, after death, euthanasia or exclusion due to a technical reason (reflux of the test item formulation), on gestation day 20, a total of 10, 9, 6 and 8 litters in the control, 300, 700 and 1000 mg/kg bw/day groups, respectively, were evaluated.


Two animals died at 300 mg/kg bw/day and one at 700 mg/kg bw/day, as well as one was moribund in latter group. The cause of death was supposed as secondary exposure by aspiration, and in case of one of the 300 mg/kg bw/day dams (No.: 149) mis-gavage was assumed. None of the dams died at 1000 mg/kg bw/day.


The animals which died or were moribund had reflux of the test item formulation related clinical signs such as dyspnoea, reduced activity, hunched back, piloerection and squeling which were observed in most cases after detecting reflux of the test item. Also, three survived females in the 700 mg/kg bw/day group had similar clinical signs.


Reduced food consumption was observed at 700 and 1000 mg/kg bw/day between G.D. 5 11 with a dose response.


At 1000 mg/kg bw/day, the mean body weight of the dams was moderately lower. Slightly lower body weight at 700 mg/kg bw/day at G.D. 8, 17 and 20 was observed.


Weight loss was observed at 1000 mg/kg bw/day from G.D. 5 to 8, as well as the body weight gain was lower which was significant between G.D. 11 and 17, as well as between G.D. 5 to 20 or 0 to 20. The body weight gain was significantly lower at 700 mg/kg bw/day from G.D. 5 to 8.


The corrected body weight/gain was slightly lower than in the control in the test item treated groups at a similar level.


Test item related findings as thicker cardia (2 at 700 and 5 at 1000 mg/kg bw/day) as well as white coated stomach mucosa (1 at 700 mg/kg bw/day) were observed.


Histological examination revealed squamous cell hyperplasia and inflammatory cell infiltrates in the forestomach in the middle and high dose groups.


In this study the development of squamous cell hyperplasia could be in connection with the local effect of 700 mg/kg bw/day and 1000 mg/kg bw/day of the test item.


Other findings like bloody dried fluid at nasal region, compact formation next to salivary gland, point-like bleedings in the thymus, foamy discharge in the esophagus, reddish mottled or dark red lungs, bloody content in the thoracic cavity, yellowish content in the stomach and duodenum and dark red liver with low incidences in the 300 and 700 mg/kg bw/day dose groups were not judged to be a consequence of a direct test item effect.


The mean of preimplantation loss was slightly higher at 1000 mg/kg bw/day, as well as the mean number of implantations was slightly lower in this group. Considering the slight difference, a test item effect was not proved. Post-implantation loss was higher in the test item treated groups without a dose response. None of the dams had total post-implantation loss. The sex ratio of fetuses was similar in all groups. There was no test item effect indicated. There were no fetal variations and malformations observed. There was no dose response in the incidence of body weight retarded (small) fetuses.


In one litter at 300 mg/kg bw/day 6 edemateous placentas were seen (in a litter where the dam had clinical signs and slighter weight loss).


Based on these preliminary observations, the doses for the main Prenatal Developmental Toxicity Study were selected as follows:
Group 1 Vehicle control
Group 2 100 mg/kg bw/day
Group 3 300 mg/kg bw/day
Group 4 1000 mg/kg bw/day

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2023
Report date:
2023

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dibutyloleamide and oleic acid, sulphated, sodium salts
EC Number:
915-926-9
Molecular formula:
C18H34Na2O6S + C26H52NNaO5S
IUPAC Name:
N,N-dibutyloleamide and oleic acid, sulphated, sodium salts
Test material form:
liquid
Details on test material:
The water content of the REACH registration substance is 3.8% (w/w).

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han: WIST
Details on test animals or test system and environmental conditions:
EST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: females: ca. 10 weeks of age at start of the mating period; males: ca. 31 – 32 weeks of age at start of the mating period
- Weight at study initiation: females: The group averages of the body weight of the females were as similar as possible on the first day of gestation
- Fasting period before study: no
- Housing:
before mating: 2 females/cage, 2 maless/ cage; during mating: 1 male and 1 – 2 females/cage; during gestation: 2 sperm positive females/ cage, if not possible 1 sperm positive female/ cage;
cages: Type II polypropylene/polycarbonate;
bedding: Certified laboratory wood bedding (Safe 3/ 4-S-FASERN produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany)
- Diet: ad libitum, ssniff SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 24.3
- Humidity (%): 53 – 69
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2022-09-07 To: 2022-10-05

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water). Formulations were prepared beforehand not longer than for three days and stored at room temperature until use.

VEHICLE
- Justification for use and choice of vehicle: Water (Aqua purificata) has been shown to be a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 20, 60 and 200 mg/mL (calculated by the concentration of active ingredient)
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 2206-8278
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (verification of concentrations and homogeneity) was performed twice during the study. Five aliquots of 5 mL (first occasion) of each formulation and five aliquots of 5 mL of the control substance (vehicle) were taken and analyzed. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (92 and 96 % relative to nominal concentrations) at 20 mg/mL and at 500 mg/mL, respectively.
Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated) -N,N-dibutylamide, monosulphated, sodium salts proved to be stable in distilled water at the intended concentrations at room temperature for three days.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male/ one to three females
- Length of cohabitation: 2 - 4 h
- After unsuccessful pairing replacement of first male by another male with proven fertility: not performed
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
14 days, from gestational day (G.D.) 5 to 19
Frequency of treatment:
daily
Duration of test:
The animals were sacrificed by anaesthesia on gestation day 20.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
A total of 104 females were used for mating to achieve the sufficient number of females per group of at least 26 spermium positive females per group.

The number of mated and of pregnant females was as follows:
Control: mated: 26, pregnant: 22
300 mg/kg bw/d: mated: 26, pregnant: 22
700 mg/kg bw/d: mated: 26, pregnant: 22
1000 mg/kg bw/d: mated: 26, pregnant: 20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen on the basis of the results of “Dose Range Finding Prenatal Developmental Toxicity Study with Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated)-N,N-dibutylamide, monosulphated, sodium salts in the Rat by Oral (Gavage) Administration (please refer to 'Cross-reference'). In this dose range finding study, death of two animals in the 300 and one in the 700 mg/kg bw/day group, as well as moribund state of one animal in the 700 mg/kg bw/day group was attributed to a technical reason (secondary exposure by aspiration). In case of one of the 300 mg/kg bw/day dam, mis-gavage was assumed. None of the dams died at 1000 mg/kg bw/day.

- Rationale for animal assignment:
The sperm positive females were allocated to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. Females paired with the same male were allocated to different groups on the same mating day.

- Fasting period before blood sampling for dam thyroid hormones: No fasting period described.
- Time of day for dam blood sampling: in the morning on the day of necropsy

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day from G.D. 0 to 20, on treatment days after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. When signs of toxicity were observed, animals were observed more frequently.
Individual observation included the check of behavior and general condition.
Duration and severity of the clinical signs were recorded.
Observations for signs of morbidity and mortality were made twice daily from G.D. 1 to 19, at the beginning and end of the working period, and once on G.D. 0 and 20.

DETAILED CLINICAL OBSERVATIONS: Yes, see above.

BODY WEIGHT: Yes for females; No for males
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).

FOOD CONSUMPTION: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: 20
- Organs examined: The uterus with cervix was removed and weighed. The ovaries were placed into a Petri dish after removal. After removing the uterus, gross pathology of dams’ viscera was performed. Thyroid glands of all sperm positive females were weighed and histological examination was performed on the thyroid glands of all females.
Organs and tissues with undiagnosed macroscopic findings were fixed in 4 % (v/v) buffered formalin solution at necropsy for possible histological examination. Control organs were fixed in 4 % (v/v) buffered formalin solution and preserved for comparison.
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: Live fetuses and fetal death were counted.
Blood sampling:
Blood samples collected for TSH and Thyroid Hormones (FT3 and FT4) measurements were drawn from all sperm positive females from the sublingual vein in the morning on the day of necropsy.
- Plasma: No
- Serum: Yes
- Volume collected: at least 2.5 mL blood
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, half per litter
- Anogenital distance of each fetus was determined.
Statistics:
Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
Current historical control data were used to enhance interpretation of study results (please refer to 'Overall remarks, attachments'). Means and standard deviations and/or percentages were calculated.
Statistical analysis were performed with IBM SPSS 25 software.
The heterogeneity of variance between groups was checked by Levene homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test. Chi2 test was performed if feasible.
Indices:
- Pre-implantation loss: ((Number of corpora lutea - Number of implantations)/Number of corpora lutea)*100
- Post-implantation loss: ((Number of implantations - Number of live fetuses)/Number of implantations)*100
- Sex distribution: (Number of Male (Female) fetuses/Number of fetuses)*100
- External- (including placentas), visceral and skeletal abnormalities abnormalities/litter: (Number of fetuses with abnormality/Number of fetuses)*100
Historical control data:
Historical control data on prenatal developmental toxicity from studies in the rat strain conducted in the same laboratory from 2017-2021 were included in the study report and used as reference (please refer to 'Overall remarks, attachments').

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The most common clinical sign on different days of gestation for the survived females was reduced activity of 2, 4 and 7 animals in the 100, 300 and 1000 mg/kg bw/day dose groups (together with the excluded animals). In most cases this clinical sign was accompanied with piloerection (1, 3 and 7 animals in the 100, 300 and 1000 mg/kg bw/day dose groups respectively). In addition, noisy respiration was recorded for one animal in the 300 and one in the 1000 mg/kg bw/day group, as well as hunched back for one non-pregnant in the 1000 mg/kg bw/day dose group. The clinical signs lasted one to six days long.
Reflux of the test item directly after treatment was observed in three females from four with clinical signs in the 300 and for five from seven with clinical signs in the 1000 mg/kg bw/day group.
In the cases where no reflux of the test item was seen directly after treatment, but clinical signs occurred (2 from 2, 2 from 4 and 1 from 7 in the 100, 300 and 1000 mg/kg bw/day dose groups respectively), also aspiration was assumed based on the similar clinical signs.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1000 mg/kg bw/day: 1/20 (G.D. 20);
No clinical sings were recorded priorly and blood around the snout was observed. It was assumed to be due to a technical reason and test item relation could not be proven.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower body weight was observed in the 1000 mg/kg bw/day dose group from the first treatment which was statistically significant (p<0.05) on G.D. 8 and 11.
Weight loss was indicated in the 1000 mg/kg bw/day dose group between G.D. 5 and 8 (p<0.01) and statistically significantly (p<0.05) lower body weight gain was seen if calculated from the first treatment to up to the end of the in-life phase (G.D. 5 to 20). Also, the for gravid uterine weight corrected body weight gain was statistically significantly lower (p<0.01) in the 1000 mg/kg bw/day group and lower corrected body weight was observed without a statistical significance.
Statistically significant increases were observed in the body weight gain (p<0.05 and p<0.01) in the 100 and 300 mg/kg bw/day groups respectively, as well as if calculated for between G.D. 5 and 20 (p<0.05) in the 300 mg/kg bw/day group. They were presumed to be of no toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food consumption (-13 to -38% versus control, p<0.01) was observed in the 1000 mg/kg bw/day group between G.D. 5 and 14 (for all three days periods (5 to 8, 8 to 11 and 11 to 14). Also, if the mean values were evaluated, the reduced food consumption in the 1000 mg/kg bw/day dose group (-16%) was statistically significant (p<0.05) between G.D. 5 and 20.
Statistical significances were revealed for the slightly lower food consumption in the 300 and 100 mg/kg bw/day group (p<0.01, -10 and -6 % respectively).
Based on the slight manner this was not considered as adverse, which was supported by the body weight and corrected body weight/gain parameters which were unaffected.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
please refer to 'Endocrine findings'
Clinical biochemistry findings:
not examined
Endocrine findings:
no effects observed
Description (incidence and severity):
The mean FT4 level was statistically significantly lower (p<0.05) in the 300 mg/kg bw/day group without a dose response, hence was not considered as adverse. There were no significant differences in the FT3 and TSH levels.

There were no significant differences in the mean values of thyroid weight in the experimental groups.

No lesion of the thyroid tissue in any dam was detected at histological examination.
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):
The gravid uterine and thyroid weights (please refer to 'Endocrine findings') were comparable in all groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Thicker cardia was observed in 10 of 19 evaluated as well as in 4 of 7 excluded females in the 1000 mg/kg bw/day dose group. Dark discoloration in the stomach mucosa was seen in one, and thicker cardia mucosa in another female in the 1000 mg/kg bw/day group.
These finding were absent in the lower dose groups and were attributed to the irritative effect of the test item.

Dilated renal pelvis was observed in one pregnant female in the 100 and in one non-pregnant in the 1000 mg/kg bw/day dose group. Hydrometra was recorded for one female in the control group. These findings were not attributed to the treatment based on the low incidence and lack of dose response or occurrence in the control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
For thyroid results, please refer to 'Endocrine findings'.

Histopathological examination of deceased animal wil be included in an update.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There was no statistically significant or dose related increase indicated.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no statistically significant increase indicated in the pre- implantation loss.
There were two females with total post-implantation loss, one in the 100 (No.: 188) and one in the 1000 (No.: 227) mg/kg bw/day dose group. Considering the lack of dose response, this was not proved to be test item related.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no statistically significant increase indicated.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no statistically significant increase indicated in the early- and late embryonic death.
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no statistically significant increase indicated in the number of dead fetuses.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number and percentage of pregnant females compared to mated females showed similar values in all groups.
Control: mated: 26, pregnant: 22 (84.6%)
100 mg/kg bw/day: mated: 26, pregnant: 22 (84.6%)
300 mg/kg bw/day: mated: 26, pregnant: 22 (84.6%)
1000 mg/kg bw/day: mated: 26, pregnant: 20 (76.9 %)
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the fetal weights.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the number of viable fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The distribution of the two sexes was similar in the groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in litter size and weights.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in ano-genital distance.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of evaluated fetuses was 213, 214, 247 and 181 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.
At external examinations there were 2/2, 0/0, 1/1 and 1/1 fetuses/litters found in the control, 100, 300 and 1000 mg/kg bw/day group respectively.
One fetus in the 1000 mg/kg bw/day dose group (No.: 0617179/12) had microcephalia, microphthalmia and absence of mandible.
One hypoplastic mandible was found in the 300 mg/kg bw/day dose group (No.: 1250123/8). Also, absence of mandible was recorded for one control fetus (No.: 2073101/2).
Considering the low incidence, these malformations were not judged to be treatment related. Moreover, hypoplastic or absent mandible was present in the control group, and absent or hypoplastic mandible (agnathia or retrognathia) and also microphthalmia may occur in control fetuses according to the Background Pregnancy and Fetal Data (please refer to 'Overall remarks, attachments').
Umbilical hernia was found in one control fetus (No.: 01402127/10) as an incidental finding.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 108, 105, 123 and 190 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.
The number of malformed fetuses/litters was 2/2, 0/0, 3/2 and 3/3 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

In the 1000 mg/kg bw/day dose group fetus No. 0104177/1 and 1046104/12 had hemicentric ossification of the thoracic vertebra IX as a malformation. Fetus No. 0410141 had an interrupted rib and the 2nd rib was not connected to the sternum.

In the 300 mg/kg bw/day dose group fetus No. 1250123/1 had three misshapen thoracic vertebrae and two of these were fused and inclined. Fetus No. 1250123/8 in the same litter, which was found with a markedly hypoplastic mandible, was observed with absence of mandible and in addition cleft palate. Fetus No. 1457170/7 in another litter had a misshapen- and cleft palate.

There were no skeletal malformations found in the 100 mg/kg bw/day group.

In the control group fetus No. 0411154/5 had a split xiphoid of the sternum. Absence mandible of fetus No. 2073101/2 was confirmed by skeletal examination and in addition misshapen, cleft palate and mal-positioned tympani were observed in the same fetus.

The skeletal malformations in the 1000 and 300 mg/kg bw/day dose group were not proved to be test item related, based on the low incidence or presence of the malformation also in the control group (absent or hypoplastic mandible (agnathia, retrognathia) and cleft palate. Moreover, malformation of vertebrae (including hemicentric ossification), as well as if the second rib is not connected to sternum or if a rib is interrupted may occur in control fetuses according to the Background Pregnancy and Fetal Data (please refer to 'Overall remarks, attachments').
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 105, 109, 124 and 91 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.
The number of malformed fetuses/litters was 3/3, 0/0, 0/0 and 1/1 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

Fetus No. 0617179/12 in the 1000 mg/kg bw/day group, which was found as malformed also by external examination, was allocated to visceral examination. Examination of the head and brain revealed dilated lateral brain ventricles, displacement of inner and mid ear, anophthalmia of one eye (eye was not visible, histopathology was not performed to confirm of complete absence), microphthalmia and disposition of the other eye, as well as absence of the nasal region.
In the control group, umbilical hernia (the same as at external examination No.: 01402127/10) and markedly dilated lateral brain ventricles (No.: 1340105/9) as well as malformation of the great arteries (from left carotid artery originated left subclavian artery, and high positioned aortic arch accompanied with the variation short brachiocephalic trunk (No.: 2283112/9) were observed in one fetus each.

There were no visceral malformations found in the 300 and 100 mg/kg bw/day groups.

Considering the single occurrence of the head/brain malformations at soft tissue evaluation of the 1000 mg/kg bw/day dose group fetuses, which were partially present in a control fetus (dilated brain ventricles (No.: 1340105/9) and mal-positioned ear bones (No.: 2073101/2, skeletal examination), as well as that microphthalmia may occur in control fetuses according to the Background Pregnancy and Fetal Data (please refer to 'Overall remarks, attachments'), the malformations in Fetus No. 0617179/12 in the 1000 mg/kg bw/day group were not judged to be test item related.
Other effects:
no effects observed
Description (incidence and severity):
There was no dose related increase in incidence of variations. The incidence of fetuses with overall variations (if external, visceral and skeletal taken into consideration) was statistically significantly higher (p<0.05) in the 300 mg/kg bw/day group which was not attributed to the test item based on lack of dose response.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
other: no effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

please refer to 'Overall remarks, attachments'

Applicant's summary and conclusion

Conclusions:
Oral treatment of pregnant Han: WIST rats from gestation day 5 up to day 19 (the day before Caesarean section) with Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated) -N,N-dibutylamide, monosulphated, sodium salts at the dose level of 1000 mg/kg bw/day has not been proven to cause mortality. Death of one female in the 1000 mg/kg bw/day dose group was attributed to a technical reason (mis-gavage or aspiration of the test item) or to a local effect in the gastrointestinal tract according to the necropsy findings. The test item at the dose level of 1000 mg/kg bw/day caused reduced food consumption and body weight development as well as macroscopic changes in maternal stomachs (thicker cardia) as a local effect. The slight reductions in the food consumption at the dose levels of 300 and 100 mg/kg bw/day were not considered as adverse, based on the unaffected body weight parameters. Clinical signs such as reduced activity, piloerection and or noisy breath or hunched back which occurred in the 1000, 300 and 100 mg/kg bw/day dose groups were in majority with association of reflux of the test item or similar to the observations made in such animals and was assumably attributed to aspiration. The dose levels of 1000, 300 and 100 mg/kg bw/day with the test item had no impact on the maternal FT3, FT4 and TSH hormone levels, the weight of thyroids and it did not cause changes in thyroids which could have been observed at histopathological evaluation. The treatment with the test item had no impact on the intrauterine parameters. There were no test item-related effects proven on external, visceral and skeletal examination of fetuses.

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 300 mg/kg bw/day
NOAEL (developmental toxicity
including teratogenicity): 1000 mg/kg bw/day
Executive summary:

Groups of 26 sperm-positive female Han:WIST rats were treated with the test item in water (Aqua purificata) by oral gavage administration at three dose levels of 100, 300 and 1000 mg/kg bw/day, respectively, from gestation day 5 to 19. A concurrent control group of 26 sperm positive females was treated with the vehicle alone. The dose volume was 5 mL/kg/bw. Dosing formulations were analysed for homogeneity and achieved concentrations of the test item two times during the treatment period using a validated by high performance liquid chromatography coupled with RID detection method. The determined test item concentrations were within 10% of the nominal concentration (90.4 – 102 %) and, therefore, the dosing formulations were considered acceptable. A homogenous distribution of the test item in the vehicle was demonstrated for all analyzed dosing formulations.


For mating, the females were cohoused in the mornings with males of the same strain and source (1 male: 1-2 females) and the day of detection of sperm in the vaginal smear of females was regarded as gestation day 0. During the study, the animals were checked daily twice for mortality and at least once for clinical signs. Body weight and food consumption of the dams were recorded repeatedly. Blood collection for determination of thyroid hormone FT3 (free T3) and FT4 (free T4), as well as TSH level was performed on gestation day 20. A Caesarean section and gross pathology were performed on gestation day 20. Organs of the dams were examined macroscopically. Thyroid glands of all sperm positive females were weighed and histological examination was performed on the thyroid glands of all females.


The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and external abnormalities. The ano-genital distance was measured. The placentas were weighed and examined externally. The body of about half of each litter was subjected to visceral examination by means of a dissecting microscope after fixation in Sanomiya mixture. The heads were examined by Wilson's free-hand razor blade method. After double staining, the skeletons of the remaining fetuses (not assigned to visceral examination) were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.


There were 22 pregnant females in the control, 100 and 300 mg/kg bw/day group as well as 20 in the 1000 mg/kg bw/day group respectively. After deduction of two females with total post-implantation loss, (100 and 1000 mg/kg bw/day) and one female which died assumably due to a technical reason in the 1000 mg/kg bw/day group, the number of evaluated litters was 22 in the control and 300 mg/kg bw/day, 21 in the 100 and 18 in the 1000 mg/kg bw/day respectively.


Death of one female in the 1000 mg/kg bw/day dose group which was found dead on G.D. 20 after lack of any clinical signs priorly, was assumably attributed to a technical reason (administration of the test item) based on the presence of dried blood around the snout. Considering the stomach irritative properties of the test item, a local effect in the stomach and intestines was also not excluded. The recorded clinical signs (one to six days long in different periods of the gestation) such as reduced activity, piloerection, hunched back and noisy breath occurred in most of the cases in animals where reflux of the test item directly after treatment was observed (or in some cases assumed) in the 100, 300 and 1000 mg/kg. Macroscopic observations in the stomach such as thicker cardia in 14 of 26 females (as well as dark discoloration in the stomach mucosa and thicker cardia mucosa in two different females) observed in the 1000 mg/kg bw/day dose group could be in connection with the local effect of the test item based on the similar findings and histopathological results in the DRF study (non GLP). Treatment related slightly lower body weight on G.D. 8 and 11, weight loss between G.D. 5 and 8 as well as lower body weight gain between G.D. 5 and 20, as well as reduced corrected body weight gain in the 1000 mg/kg bw/day dose group was attributed to the treatment. Reduced food consumption (-13 to -38%) in the 1000 mg/kg bw/day group between G.D. 5 and 14 as well as 5 and 20 was judged as treatment related. Statistical significances in the slightly lower food consumption in the 300 and 100 mg/kg bw/day group for between G.D. 5 and 8 (-10 and -6% respectively) were not considered as adverse based on the small difference and the unaffected body weight parameters. There was no test item effect indicated in the above hormone levels. There were no significant differences in the mean values of thyroid weight in the experimental groups. No lesion of the thyroid tissue in any dam was detected at histological examination. There were no statistically significant differences observed in intrauterine mortality, number of viable fetuses and their sex distribution. Based on lack of dose response, occurrence of one female in the 1000 and one in the 100 mg/kg bw/day dose group with total post-implantation loss was not proved to be test item related.


There were no statistically significant differences observed in the body weights of fetuses, placental weights and ano-genital distances. There was no dose related increase in incidence of malformations or variations in the external, visceral and skeletal examination. The malformations and variations were found with low incidences or/and also present in the current control group or and in the historical control data.


 


Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:


 


NOAEL (maternal toxicity): 300 mg/kg bw/day


NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day