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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A screening for reproductive/developmental toxicity study and Extended one-generation reproductive toxicity study in rats were waived based on REACH Annex VIII (column 2) and Annex IX (column 1).

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A screening for reproductive/developmental toxicity study in rats (OECD 421 or 422) was waived based on column 2 of REACH Annex VIII as an OECD 414 pre-natal developmental toxicity study (Annex IX, 8.7.2), is available. 

An Extended one-generation reproductive toxicity study was waived based column 1 of REACH Annex IX because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity.

Effects on developmental toxicity

Description of key information

A key prenatal developmental toxicity study using the registered substance in rats rats dosed via the diet at 250, 500 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy did not show any teratogenic or embryotoxic potential in rats. The NOAEL was 500 mg/kg bw/day for the dams, whereas the NOAEL for the fetal organism was above 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 February 2020 to 25 June 2021 (Final draft report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A final draft report is attached. During validation of the analytical method, the measured concentrations of freshly prepared samples of the test item-diet mixtures were well within the range of the expected concentrations. However, the recovery of the stability samples was outside the acceptance criteria. An interaction between the test item and the diet during storage was assumed and therefore, the extraction method has to be adapted and additional validation work is needed.
Qualifier:
according to guideline
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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 25 June 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Manufacturer/Supplier Evonik Nutrition & Care GmbH, Goldschmidtstraße 100, 45127 Essen, Germany
Batch no. S019844264
- Content of solid: 34.4%
- Correction factor: 2.91

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, in tightly closed container and stored in a well ventilated place. Protected from direct sunlight and heat.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: For the determination of the test item in the exposure diets , samples of exposure diets (approximately 2 x 5 g aliquots) were taken at the following times and stored at -20°C ± 10% until shipment to Analytisches Zentrum Biopharm Berlin GmbH:
Determination of Concentration and homogeneity in the test item-diet mixture immediately after preparation: 1st and 2nd week of treatment: 1 sample was taken from each of 3 areas (top, middle and bottom) of the bucket for in total 3 dosages and 1 control (control: only one sample as the diet was used as supplied): 10 samples (20 aliquots) were taken at the 2 time points.
Determination of Concentration and stability (left-over diet): End of the 1st week and End of 2nd week of treatment: taken from left-over diet – food which had been available to the animals for 7 days: 4 samples (8 aliquots) were taken at the 2 time points.
Total number of samples (aliquots): 28 (56)
No additional sampling was carried out in the 3rd and 4th week of treatment since treatment of the majority of the animals ended in the middle of the 3rd week of dosing.
The samples were labelled with the study number, species, type of sample, aliquot number, concentration, test day, location (top/middle/bottom), if applicable, and date.
Only one aliquot was analysed, the second aliquot was retained as a back-up.
Following advance notice via e-mail, the exposure diet samples were shipped on dry ice on 12 March 2020 to Test Site 1.
The results of the test item-formulation analyses for the investigated parameters are still pending and will be given in the next version of the report.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The appropriate amount of test item was weighed into a glass bottle. Some of the test item and diet was mixed with an impact mill to produce a premix. This process was repeated until the whole quantity of test compound was distributed in the diet. Then the premix was added to the diet, mixed with a mixer (Röhnradmischer; Typ ELTE 650; J. Engelsmann AG, Ludwigshafen, Germany) for 15 minutes and then transferred to a closable bucket. Each bucket was labelled according to group and dose.
Species:
rat
Strain:
other: CD® / Crl:CD (SD)
Remarks:
The male animals remained untreated.
Details on test animals or test system 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: 70 – 78 days
- Weight on day 0 of pregnancy: 242.7 g - 315.2 g
- Fasting period before study: no
- Housing: Except during the mating period, 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. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL. 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): A certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered daily ad libitum. Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL at least twice a year. No contaminants above the limitations were noted.
- Water (e.g. ad libitum): Drinking water (in drinking bottles) was offered ad libitum.
Samples of drinking water were taken periodically by the Wasserwerk Wankendorf (24601 Wankendorf, Germany), and periodic analyses were performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung 2018' [German Regulations on drinking water 2018 ]. In addition, drinking water samples taken at Provivo were analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2018, Anlage 1' [German Regulations on Drinking Water 2018, Addendum 1].
No contaminants above the limitations were noted.
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 19°C and 23°C
- Humidity (%): between 50% and 65%.
- Air changes (per hr): between fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

IN-LIFE DATES:
From: Start of mating on 17 February 2020
To: Termination of the in-life part on 18 March 2020


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
The test item was mixed in the diet.
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): The test item formulations were freshly prepared once a week.
- Mixing appropriate amounts with (Type of food): A certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food.
The appropriate amount of test item was weighed into a glass bottle. Some of the test item and diet was mixed with an impact mill to produce a premix. This process was repeated until the whole quantity of test compound was distributed in the diet. Then the premix was added to the diet, mixed with a mixer (Röhnradmischer; Typ ELTE 650; J. Engelsmann AG, Ludwigshafen, Germany) for 15 minutes and then transferred to a closable bucket. Each bucket was labelled according to group and dose.
The control animals received the standard diet only.
The test item concentrations in the diet of the first dosing week were calculated based on a daily food intake of 75 g/kg bw resulting in concentrations of 3.33 g/kg, 6.67 g/kg and 13.33 g/kg in the diet mixture. The test item concentrations in the diet of the following dosing weeks were calculated based on the amount of food consumed in the previous week for the respective groups.
Test item concentration in the diet [g/kg food]:
Group 2:
-Week 1: 3.33 g/kg food
-Week 2: 3.48 g/kg food
-Week 3: 3.67 g/kg food
-Week 4: not applicable (no further animals)
Group 3:
-Week 1: 6.67 g/kg food
-Week 2: 7.22 g/kg food
-Week 3: 7.67 g/kg food
-Week 4: 7.81 g/kg food
Group 4:
-Week 1: 13.3 g/kg food
-Week 2: 15.02 g/kg food
-Week 3: 14.95 g/kg food
-Week 4: 14.45 g/kg food
- Storage temperature of food: The untreated food was stored at room temperature. The test item-diet mixtures prepared for the dosing week were stored in a closable bucket, in the same room the animals were housed.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the determination of the test item in the exposure diets, samples of exposure diets (approximately 2 x 5 g aliquots) were taken at the following times and stored at -20°C ± 10% until shipment to Analytisches Zentrum Biopharm Berlin GmbH:
Determination of Concentration and homogeneity in the test item-diet mixture immediately after preparation: 1st and 2nd week of treatment: 1 sample was taken from each of 3 areas (top, middle and bottom) of the bucket for in total 3 dosages and 1 control (control: only one sample as the diet was used as supplied): 10 samples (20 aliquots) were taken at the 2 time points.
Determination of Concentration and stability (left-over diet): End of the 1st week and End of 2nd week of treatment: taken from left-over diet – food which had been available to the animals for 7 days: 4 samples (8 aliquots) were taken at the 2 time points.
Total number of samples (aliquots): 28 (56)
No additional sampling was carried out in the 3rd and 4th week of treatment since treatment of the majority of the animals ended in the middle of the 3rd week of dosing.
The samples were labelled with the study number, species, type of sample, aliquot number, concentration, test day, location (top/middle/bottom), if applicable, and date.
Only one aliquot was analysed, the second aliquot was retained as a back-up.
Following advance notice via e-mail, the exposure diet samples were shipped on dry ice on 12 March 2020 to Test Site 1.
The results of the test item-formulation analyses for the investigated parameters are still pending and will be given in the next version of the report.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 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.
- The non-pregnant rats were excluded from the analysis of the results and replaced by 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:
Day 6 to 20 of gestation
Frequency of treatment:
Daily
Duration of test:
= 43 days (20 adaptation days, 1 mating day, 6 days pre-implantation, 15 administration days from gestation days 6 to 20, laparotomy on gestation day 21)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
solid content (A correction factor of 2.91 was used.) Only females were dosed.
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
solid content (A correction factor of 2.91 was used.) Only females were dosed.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
solid content (A correction factor of 2.91 was used.) Only females were dosed.
Dose / conc.:
235 mg/kg bw/day (actual dose received)
Remarks:
mean test item intake for the whole dosing period
Dose / conc.:
471.9 mg/kg bw/day (actual dose received)
Remarks:
mean test item intake for the whole dosing period
Dose / conc.:
1 002.6 mg/kg bw/day (actual dose received)
Remarks:
mean test item intake for the whole dosing period
No. of animals per sex per dose:
25 mated females per group.
20 evaluated litters per group.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels for this study were selected in agreement with the Sponsor based on the results of a dose-range-finding study for a prenatal developmental toxicity study in rats (Provivo study report no. 37820).
In this dose-range-finding for a prenatal developmental toxicity study, the test item was administered orally via the diet to female rats at target dose levels of 300, 600 or 1000 mg act. ingr./kg bw/day from the 6th to 20th day of pregnancy.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 600 mg act. ingr./kg bw/day for the dams.
None of the dams died prematurely.
No changes in behaviour or external appearance were noted.
Slightly reduced body weight, body weight gain and carcass weights were noted at the high dose level. No test item-related changes were noted for the food consumption.
No pathological changes were noted in the macroscopic examination during laparotomy.
The mean actual intake of the test item via the diet over the dosing period was 276.3, 539.1 and 963.7 mg act. ingr./kg bw/day. Hence, the actual values were in the range of the nominal values with 300, 600 or 1000 mg act. ingr./kg b.w./day.
The no-adverse-observed-effect level (NOAEL) for the fetal organism was 1000 mg act. ingr./kg bw/day.
The reproductive parameters (number of implantation sites, number of resorptions, number of fetuses and sex distribution) were not influenced by the test item.
No test item-related influence was noted on the weights of the placentae or the fetuses. No runts were noted in any of the dose groups.
No dead fetuses, no malformations and no variations were noted.
Hence, the dose levels of 250, 500 or 1000 mg/kg were selected for the main OECD 414 study by the Sponsor.
- 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 lavages were taken each morning. Day 0 of pregnancy was the day on which sperm was found in the vaginal lavage. When positive, the animals were assigned to the test groups by mating day using a Provantis® -generated randomization based on the body weight of the animals.
- Fasting period before blood sampling for (rat) dam thyroid hormones: Yes, overnight.
- Time of day for (rat) dam blood sampling: Blood samples were taken always at the same time of day (approximately from 6:00 a.m. to 9:00 a.m.)
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Individual animals were observed daily for behavioural changes, reaction to treatment, or illness. Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, would have been sacrificed for humane reasons based on guidance document on humane endpoints (ENV/JM/MONO 2000/7). However, none of the animals of this study had to be prematurely sacrificed.
Special attention was paid to ascertain if there were any signs of irritation after oral dosing, such as increased salivation, redness of the oral cavity etc.
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 would have 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 were sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible.

DETAILED CLINICAL OBSERVATIONS: No

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 approximately at the same time of the day.
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 18-20) and for the whole study (gestation day 0 - 20). Furthermore the carcass weight and the net weight gain from day 6 are given.
These values are stated in the report.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The test item concentrations in the diet of the first dosing week were calculated based on a daily food intake of 75 g/kg bw resulting in concentrations of 3.33 g/kg, 6.67 g/kg and 13.33 g/kg in the diet mixture. The test item concentrations in the diet of the following dosing weeks were calculated based on the amount of food consumed in the previous week for the respective groups

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No drinking water study.
- 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. On the 21st day of gestation the rats were laparotomized by a CO2 overdose following randomization. The ovaries, thyroid and the uteri were removed.
- Organs examined:
The following organs were to be weighed: Thyroid (1) (including parathyroids); Gravid uterus including cervix.
In order to check for possible test item effects, a dissection with macroscopic examination of the internal organs of the dams was carried out on the day of sacrifice. The thyroid and any organs with macroscopic findings of all dams (including prematurely deceased or prematurely sacrificed animals) were fixed in 7% neutral buffered formalin.
The following organs were to be preserved: Thyroid (2) including parathyroids; Gross lesions

OTHER:
-Thyroid hormone (T3, T4, TSH) determination
In order to obtain 2 aliquots of 150 µL serum for each endocrine endpoint (T3, T4, TSH), a sufficient volume of blood was taken from the retrobulbar venous 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 6:00 a.m. to 9:00 a.m.)
Sampling schedule for thyroid hormones: Before sacrifice: all evaluated dams: 80 samples/ 160 aliquots.
The samples were divided into aliquots, if possible, and stored at -20°C ± 10% at Provivo until analysis.
The T3, T4 and TSH ELISA (commercial kits: IBL International GmbH , instrument: Tecan Sunrise , software: Magellan V7.27) was conducted at Provivo.

-Histopathology
The thyroids of all evaluated dams were examined histopatholgically after preparation of hematoxylin-eosin stained paraffin sections.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, Gravid uterus including cervix was weighed.
- Number of corpora lutea: Yes, number per dam, absolute number per group, mean per group
- Number of implantations: Yes, number per dam, distributions in the uterine horns, absolute number per group, mean per group
- Number of early resorptions: Yes (< 2 mm, number and % per litter)
- Number of late resorptions: Yes (> 2 mm, number and % per litter)
- Other: weight of placenta

Fetal examinations:
- External examinations: Yes: all per litter. All fetuses (dead and alive) were inspected externally for damages, especially for malformations.
- Skeletal examinations: Yes: half per litter. 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Fetuses were eviscerated and skinned before staining procedure. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
- Soft tissue examinations: Yes: half per litter. The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON. The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.
- Head examinations: No (not applicable)
- Anogenital distance of all live rodent pups: The anogenital distance (AGD) of all live fetuses was determined using a scale.
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).
A comparative statistical evaluation of the pre- and post-implantation loss with the group values (number of corpora lutea, implantation sites and fetuses per group) using the Chi² test and the StatXact software as stated in the Study Plan was not performed. This was due to the fact that the OECD Guideline 414 recommends only the use of the litter as the unit for data analysis.
Hence a comparative statistical evaluation of the pre- and post-implantation loss was performed on the level of the dams using an ANOVA/DUNNETT test.
The percentages of pre- and post-implantation loss for the group level are presented without any statistical comparisons.

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.
Group Pre-implantation loss [%] = [(Corpora lutea (per group) - implantations (per group)) / Corpora lutea (per group)] x 100.
Group Post-implantation loss [%] = [(Implantations (per group) - living fetuses (per group)) / Implantations (per group)] x 100
Litter Pre-implantation loss [%] = Sum of pre-implantation losses per litter in a group [%] / Number of litters in a group.
Litter Post-implantation loss [%] =Sum of post-implantation losses per litter in a group [%] / Number of litters in a group.

Historical control data:
Provivo Background Data##: Summarized results of the 76 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH) performed at Provivo in the years 2000 to July 2017.
## These background data have not been audited by the Quality Assurance Unit.
- General Reproductive Indices (laparotomy on gestation day 21, since 2016)
- External Malformations (laparotomy on gestation day 20 or 21)
- External Variations (laparotomy on gestation day 20 or 21)
- Skeletal Malformations (laparotomy on gestation day 20 or 21)
- Skeletal variations (laparotomy on gestation day 21)
- Skeletal Retardations (laparotomy on gestation day 21, since 2016)
- Visceral Malformations (laprotomy on gestation day 20 or 21)
- Visceral Variations (laparotomy on gestation day 20 or 21)

Clinical signs:
no effects observed
Description (incidence and severity):
No changes in behaviour, external appearance or the faeces were noted in any of the test groups.
Mortality:
no mortality observed
Description (incidence):
None of the animals died prematurely.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Noteworthy reductions in the body weight, the body weight gain and the carcass weight were noted for the dams of the high dose group (1000 mg act. ingr./kg bw/day).
In the high dose group (1000 mg act. ingr./kg bw/day), a decrease was noted for the body weight (at maximum 12.3% below the value of the control group on GD 19 and 20, statistically significant at p ≤ 0.01).
Accordingly, also the body weight gain of the high dose group was decreased (40.8% below the value of the control group for the dosing period between GD 6 and GD 20, statistically significant at p ≤ 0.01).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related difference was noted between control group and the dose groups.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry 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):
No test item-related differences were noted for the thyroid weights. No test item-related differences were noted between the gravid uterus weight of the control dams and the dams of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
For the carcass weight and the net body weight gain, a decrease was noted (7.0% below the value of the control group for the carcass weight and -17.3 g net body weight gain of the high dose group compared to -2.4 g in the control group, both statistically significant at p ≤ 0.01) for the high dose group (1000 mg act. ingr./kg bw/day).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic inspection of the dams at necropsy revealed no pathological changes in the dose groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathologic examination revealed no test item-related changes in the thyroid.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Histopathologic examination restricted to the thyroids revealed no test item-related changes.
Details on results:
- Clinical signs: No changes in behaviour or the external appearance were noted in the control group and the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
- Mortality: No premature deaths were noted in the control group and the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
- Body weight and weight changes:
*Body weight:
No test item-related differences in body weight were noted between the dams of the control group and the low dose group (250 mg act. ingr./kg bw/day).
In the intermediate dose group (500 mg act. ingr./kg bw/day), a slight reduction was noted for the body weight (at maximum 6.9% below the value of the control group, statistically significant at p ≤ 0.01). This slight reduction has to be considered as a beginning test item-related effect on the body weight that was pronounced at the high dose level. However, as the effect was only slight and no other effects were noted for the intermediate dose group, this reduction is only of minor toxicological relevance and not considered for the determination of the NOAEL.
In the high dose group (1000 mg act. ingr./mg bw/day), a more pronounced decrease for the body weight was noted from GD 8 to GD 20 (at maximum 12.3% below the value of the control group on GD 19 and GD 20, statistically significant at p ≤ 0.01). This distinct and constant decrease for the body weight at the high dose level was considered to be test item-related.
*Body weight gain from GD 0 to 20:
In accordance with the body weight, also no test item-related difference between the control group and the animals treated with 250 act. ingr./kg bw/day was noted for the body weight gain from GD 0 to 20 and in the dosing period from GD 6 to GD 20.
In the intermediate dose group (500 mg act. ingr./kg bw/day), a beginning test item-related effect was noted also for the body weight gain from GD 0 to 20 and from GD 6 to 20 (17.0% or 19.4%, respectively below the value of the control group, statistically significant at p ≤ 0.01). As the difference to the control group was only slight, the reductions were considered to be of minor toxicological relevance and were not considered for the NOAEL.
In the high dose group (1000 mg act. ingr./kg bw/day), a statistically significantly lower body weight gain was noted between GD 0 and GD 20 and also between GD 6 and GD 20 (34.6% or 40.8, respectively below the value of the control group, p ≤ 0.01).
The distinctly and statistically significantly reduced body weight gain that was noted for the high dose group was considered to be test item-related.
The reduction in the body weight was due to the test item-related decrease of the carcass weight.
- Food consumption and compound intake (if feeding study): No test item-related difference was noted between the control group and the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
Statistically significant differences were noted for the intermediate dose group between GD 6 and GD 7 (16.6% below the value of the control group, p ≤ 0.05) and for the high dose group between GD 6 to GD 8 (19.6% or 13.9% below the value of the control group, p ≤ 0.05) and between GD 13 to GD 14 (30.4% above the value of the control group, p ≤ 0.01). However as these differences were only transient, the differences for the food consumption on the first 3 days of administration were considered to be not directly test item-related but possibly caused by the taste and/or smell of the test item mixed with the diet.
The test item was administered orally via the diet. The concentration of the test item in the diet was adjusted weekly based on the mean daily food consumption of the week before. The mean actual test item intake via the diet for the whole dosing period was in the range of the nominal test item intake. In the fourth dosing week, the actual test item intake in the intermediate and high dose group was above the nominal concentration. However, the dosing in dosing week 4 was only for two intermediate dose dams and for one high dose dam for one day each.
- Drinking water consumption: Visual appraisal revealed no test item-related influences on drinking water consumption for any dose group.
- Thyroid hormone level analysis: No test item-related differences were noted for the serum levels of T3, T4 and TSH in any of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
- Necropsy findings: No observations were noted for the dams of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day) during the macroscopic inspection of the organs and tissues. In the control group dam no. 15 was noted with the right thyroid being reduced in size.
- Histopathology: No test item-related morphological changes were noted during histopathological examination of the thyroids of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
In all groups, histopathological examination of the thyroids revealed single or multiple keratinized cysts. Additionally, a focal lymphocytic infiltration into the left thyroid was noted for dam no. 62 of the intermediate dose group. However, these findings were considered to be coincidental and not test item-related as these were within the historical background data range (0 to 12 animals affected out of 20) and they were also found in the control group, in addition, no dose response-relationship was present.
-Thyroid weights: No test item-related differences to the control group were noted for the absolute thyroid weights of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
- Gravid uterus weight, carcass weight and body weight gain from day 6:
* Gravid uterus weight and Carcass weight:
No test item-related differences were noted between the gravid uterus weight of the control dams and the dams of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
For the carcass weight, no differences were noted between the control group and the low dose group.
In the intermediate and high dose group (500 or 1000 mg act. ingr./kg bw/day), a decreased carcass weight was noted (5.9% or 7.0% below the value of the group, statistically significant at p ≤ 0.01). However, these slightly decreased carcass weights for the intermediate and high dose group were considered to be only of minor toxicological relevance.
* Net body weight gain from GD 6 to 21
For the net body weight gain from GD 6 to 21, no test item-related differences were noted between the dams of the control group and the dams of the low dose group (250 mg act. ingr./kg bw/day).
In the intermediate dose group (500 mg act. ingr./kg bw/day), a decrease was noted for the net body weight gain between GD 6 and GD 21 (-17.3 g in the intermediate compared to -2.4 g in the control group, statistically significant at p ≤ 0.01). However, this slightly reduced net body weight gain was considered to be only of minor toxicological relevance.
In the high dose group (1000 mg act. ingr./kg bw/day), the decrease for the net body weight gain between GD 6 and 21 was more pronounced (-21.7 g compared to -2.4 g in the control group, statistically significant at p ≤ 0.01). This reduction was considered to be test item-related.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
Details on maternal toxic effects:
- Reproduction data of the dams: No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
In the high dose group, a lower number per dam was noted for the corpora lutea and as a consequence also for the number of implantations and fetuses. However, as the development of corpora lutea and the implantation is completed before start of treatment and no influence was noted on the number of resorptions, the reduced numbers of corpora lutea, implantation sites and fetuses were considered to be spontaneous.

- Noteworthy reductions in the body weight, the body weight gain and the carcass weight were noted for the dams of the high dose group (1000 mg act. ingr./kg bw/day).
In the high dose group (1000 mg act. ingr./kg bw/day), a decrease was noted for the body weight (at maximum 12.3% below the value of the control group on GD 19 and 20, statistically significant at p ≤ 0.01).
Accordingly, also the body weight gain of the high dose group was decreased (40.8% below the value of the control group for the dosing period between GD 6 and GD 20, statistically significant at p ≤ 0.01).
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
other: solid content
Remarks:
A correction factor of 2.91 was used.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The fetal body weights showed no test item-related differences between the control group and the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
In the high dose group, a statistically significantly increased body weight was noted for the female fetuses (6.8% above the value of the control group, p ≤ 0.05). However, an increased fetal weight was considered to be not adverse.
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 dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
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 (250, 500 or 1000 mg act. ingr./kg bw/day).
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
In the high dose group, a lower number per dam was noted for the corpora lutea and as a consequence also for the number of implantations and fetuses. However, as the development of corpora lutea and the implantation is completed before start of treatment and no influence was noted on the number of resorptions, the reduced numbers of corpora lutea, implantation sites and fetuses were considered to be spontaneous.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No macroscopically visible external observations were noted for the fetuses of the of the intermediate and high dose groups (500 or 1000 mg act. ingr./kg bw/day) during the external inspection at laparotomy.
One fetus each of the control group and the low dose group (250 mg act. ingr./kg bw/day) were noted with multiple malformations: Fetus no. 16-4f of the control group displayed a generalized edema and an omphalocele. The low dose fetus no. 36-3f was noted with a cranioschisis leading to an exencephaly, furthermore, the fetus had a spina bifida and the eyes were absent.
As no external observations were noted for the fetuses of the intermediate and high dose group and also one fetus of the control group displayed malformations the single occurrence of one low dose fetus with multiple malformations was considered to be spontaneous.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (250, 500 or 1000 act. ingr./kg bw/day) during the skeletal examination according to DAWSON.
Visceral malformations:
no effects observed
Description (incidence and severity):
No soft tissue malformations were noted for the fetuses of the control group and the fetuses of the intermediate and high dose groups (500 or 1000 mg act. ingr./kg bw/day) during the soft tissue examination according to WILSON.
In the low dose group (250 mg act. ingr./kg bw/day), the fetus no. 36-3 was noted with multiple malformations in form of an encephalocele (cranium absent, prolapse of brain), a cleft palate (approx. 2 mm in diameter), an anophthalmia and a spina bifida. However, as no malformations were noted in the intermediate and high dose group, the occurrence of one fetus with malformations in the low dose group was considered to be spontaneous and not test item-related.
Other effects:
no effects observed
Description (incidence and severity):
No cryptorchidism and no testicular malposition were noted during assessment of the testicular development of the male fetuses of the control group and the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
Details on embryotoxic / teratogenic effects:
-Mortality: No dead fetus was noted in the control group and in the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
-Twins: One pair of twins (nos. 67-11f and 67-12m) was noted in the intermediate dose group (500 mg act. ingr./kg bw/day).
- 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 (250, 500 or 1000 mg act. ingr./kg bw/day).
-Weight of placentae and fetuses: The placental weights and the fetal body weights showed no test item-related differences between the control group and the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
In the high dose group, a statistically significantly increased body weight was noted for the female fetuses (6.8% above the value of the control group, p ≤ 0.05). However, an increased fetal weight was considered to be not adverse.
-Number of runts: No test item-related difference in the number of runts were noted for dose groups (250, 500 or 1000 mg ingr. act./kg bw/day) in comparison to the control group.
Two runts each were noted in the control group (nos. 16-4f and 20-1m), the low dose group (nos. 36-3f and 43-19f) and the intermediate dose group (nos. 61-3f and 67-11f) and one runt was noted in the high dose group (no. 88-18m).
-Ano-genital distance: No test item-related differences to the control group were noted for the fetal ano-genital distance of the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group, a decreased relative ano-genital distance was noted for the male fetuses (4.9% below the value of the control group, p ≤ 0.05). This slight difference was considered to be spontaneous.
-Macroscopic external inspection at laparotomy: No macroscopically visible external observations were noted for the fetuses of the of the intermediate and high dose groups (500 or 1000 mg act. ingr./kg bw/day) during the external inspection at laparotomy.
One fetus each of the control group and the low dose group (250 mg act. ingr./kg bw/day) were noted with multiple malformations: Fetus no. 16-4f of the control group displayed a generalized edema and an omphalocele. The low dose fetus no. 36 3f was noted with a cranioschisis leading to an exencephaly, furthermore, the fetus had a spina bifida and the eyes were absent.
As no external observations were noted for the fetuses of the intermediate and high dose group and also one fetus of the control group displayed malformations, the single occurrence of one low dose fetus with multiple malformations was considered to be spontaneous.
-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 (250, 500 or 1000 mg act. ingr./kg bw/day).
-Testicular development: No cryptorchidism and no testicular malposition were noted during assessment of the testicular development of the male fetuses of the control group and the dose groups (250, 500 or 1000 mg act. ingr./kg bw/day).
-Skeletal malformations: No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (250, 500 or 1000 act. ingr./kg bw/day) during the skeletal examination according to DAWSON
-Skeletal variations: Skeletal variations were noted for the 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 (250, 500 or 1000 mg act. ingr./kg bw/day).
-Skeletal retardations: Retardations (delayed ossifications) were related to the skull (incomplete ossification of nasal, 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, dumbbell-shaped or unossified), the caudal vertebral bodies (only one body ossified or all bodies unossified), the sacral vertebral arches (incompletely ossified), the sacral vertebral bodies (unossified), the os ischii (incompletely ossified or unossified), the os pubis (unossified) and the metacarpalia/metatarsalia (absence of ossification in metacarpalia/metatarsalia 2 to 5).
No test item-related difference in the incidence of skeletal retardations at 250, 500 or 1000 mg act. ingr./kg bw/day was noted during skeletal examination according to DAWSON.
-Soft tissue malformations: No malformations were noted for the fetuses of the control group and the fetuses of the intermediate and high dose groups (500 or 1000 mg act. ingr./kg bw/day) during the soft tissue examination according to WILSON.
In the low dose group (250 mg act. ingr./kg bw/day), the fetus no. 36-3 was noted with multiple malformations in form of an encephalocele (cranium absent, prolapse of brain), a cleft palate (approx. 2 mm in diameter), an anophthalmia and a spina bifida. However, as no malformations were noted in the intermediate and high dose group, the occurrence of one fetus with malformations in the low dose group was considered to be spontaneous and not test item-related.
-Soft tissue 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 statistically significant differences in the incidences of observed variations were noted between the control group and the low dose group (250 mg act. ingr./kg bw/day).
In the intermediate and high dose group (500 or 1000 mg act. ingr./kg bw/day) increased incidences were noted for the dilatation of the renal pelvis, for the malpositioned kidney and for the total soft tissue variations. However, these incidences, with the exception of the incidence of the malpositioned kidney in the high dose group, were within the range of the Provivo background data. Therefore, the dilatations of the renal pelvis and of the malpositioned kidney were considered to be not test item-related but spontaneous.
-Unclassified soft tissue observations: No unclassified observations were noted for the control group and the intermediate and high dose groups (500 or 1000 mg act. ingr./kg bw/day).
Two unclassified observations were noted for the low dose group (250 mg act. ingr./kg bw/day): The fetus no. 32-5 was noted with the thoracic cavity filled with blood and fetus no. 42-1 was noted with a hemorrhagic area in the spinal cord. However, the observation of the thoracic cavity filled with blood was considered to be a preparation-induced artefact and the single occurrence of one low dose fetus with a hemorrhagic area in the spinal cord was considered to be spontaneous.
-ABSTRACT and ASSESSMENT of fetal alterations: No test item-related malformations or variations were noted during the macroscopic inspection at laparotomy (including an external inspection and a gross inspection of the organs), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON).
Furthermore, no test item-related retardations (delay in ossification) were noted in any of the dose groups (250, 500 and 1000 mg act. ingr./kg bw/day).
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
other: solid content
Remarks:
A correction factor of 2.91 was used.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
Developmental effects observed:
no

Table1:Summary of the method validation – preliminary data

Analytical Validation ReportLocations

VAL19_019

n.a.

Shortdescriptionofthemethod

LC/MS/MS

Matrixforcalibration andQCsamples

Reconstitutionsolution

Matrixforrecoveryandstability samples

Diet mixture

Analyte

Locationof productcertificate

Rewopol B 2003n.a.

Calibrationconcentrations(Units)

75.0 –3000.0ng/mL

Lower limit of quantification (Units),accuracy,precision

75.0 ng/mL105.8%,8.3%

QC concentrations (Units)RewopolB 2003

75.0ng/mL,225.0ng/mL,

900.0ng/mL,2250.0ng/mL

Between-runaccuracy

94.6%-105.8%

Between-runprecision

2.2%-8.3%

Within-runaccuracy

93.6%- 109.5%

Within-runprecision

1.6%-12.8%

Recovery of analyte in diet mixturesamples(Accuracy%)

 

60.42-90.34

Short term stability in diet mixture atsample processing temperature(Accuracy%)

Confirmed up to 6 hours at room temperature49.77-54.64

Freeze and thaw stability(Accuracy%)

-20°C±5°C,1cycle

57.05-78.76

Autosampler storage stability(Accuracy%)

Confirmed up to 24 hours93.8-95.7

Stock solution long-term stability(Accuracy%)

Confirmed up to 441 days97.9

Stock solution benchtop stability(Accuracy%)

Confirmed up to 24 hours101.5

Working solution benchtop stability(Accuracy%)

Confirmed up to 24 hours97.5 -100.0

 

 

Table 2: Summary of animals examined

Treated dams

25

25

25

25

Not pregnant dams

0

0

0

0

Dams with premature delivery

0

0

1 #

0

act. ingr.

Group 1

Control

Group 2

250 mg/kg

Group 3

500 mg/kg

Group 4

1000 mg/kg

Not evaluated dams

(spare animals)

5

5

4

5

Evaluated litters

20

20

20

20

#: Dam no. 51 delivered prematurely and was excluded from evaluation.

 

 

Table3: Body weight gain from GD 0 to GD 20 and from GD 6 to GD 20

Group

Time interval

Gestation day 0 - 20

(whole study period)

Time interval

Gestation day 6 - 20

(whole treatment period)

 

Gain

in g

Gain

in %

Difference to control %

Gain

in g

Gain

in %

Difference to control %

Control

+155.4

+56.4

n.a.

+126.0

+41.4

n.a.

Group 2

250 mg/kg

+149.0

+54.3

-4.1

+116.7

+38.0

-7.4

Group 3

500 mg/kg

+129.0**

+47.1

-17.0

+101.6**

+33.7

-19.4

Group 4

1000 mg/kg

+101.6**

+36.9

-34.6

+74.7**

+24.7

-40.8

n.a.: Not applicable

**: Statistically significant at p ≤ 0.01 (ANOVA/DUNNET test).

 

 

Table4: Actual test item intake via the diet per group per day

Dosing week

Nominal concentrations [mg/kg bw/day]

250

500

1000

Actual concentrations (group mean)

[mg/kg bw/day]

1

239.1

449.0

890.8

2

228.2

466.7

1033.7

3

244.0

504.9

1070.1

4

n.a. #2

652.3

1744.3

Whole dosing period

235.0

471.9

1002.6

% difference to nominal

-6.0

-5.6

+0.3

n.a.: Not applicable

#: Dosing of Group 2 was completed in dosing week 3.

 

 

Table5: Overview on reproduction parameters



Parameter

Group 1

Control

(n=20)

Group 2

250

mg/kg

(n=20)

Group 3

500

mg/kg

(n=20)

Group 4

1000 mg/kg

(n=20)

Corpora lutea

total

mean per dam

349

17.5

340

17.0

357

17.9

314

15.7

Implantation sites

total

mean per dam

343

17.2

329

16.5

346

17.3

298

14.9

Resorptions

total

mean per dam

23

1.2

17

0.9

23

1.2

13

0.7

Early resorptions

total

mean per dam

19

1.0

17

0.9

18

0.9

12

0.6

Late resorptions

total

mean per dam

4

0.2

0

0.0

5

0.3

1

0.1

Live fetuses

total

mean per dam

320

16.0

312

15.6

324

16.2

285

14.3

Dead fetuses

total

0

0

0

0

Pre-implantation loss [%]

per group

mean per dam

1.7

1.6

3.2

3.6

3.1

2.5

5.1

4.5

Post-implantation loss [%]

per group

mean per dam

6.7

6.7

5.2

4.8

6.6

6.6

4.4

4.7

Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.

Conclusions:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 500 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.
Executive summary:

In this prenatal developmental toxicity study, the test item was administered orally via the diet to female CD rats at dose levels of 250, 500 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 500 mg act. ingr./kg bw/day for the dams.
Noteworthy reductions in the body weight, the body weight gain, the carcass weight and the net body weight gain were noted for the dams of the high dose group (1000 mg act. ingr./kg bw/day).
None of the animals of the test groups died prematurely.
No changes in behaviour, external appearance and the faeces were noted in any of the dose groups.
No test item-related influences were noted on the food and drinking water consumption of the dose groups.
No test item-related differences were noted for the reproduction parameters (number of implantations, number of resorptions, number of fetuses).
Macroscopic post mortem examination during necropsy revealed no pathologic changes in any of the dose groups.
No test item-related differences were noted for the serum levels of the thyroid hormones T3, T4 and TSH and also no test item-related differences were noted for the thyroid weights. Furthermore, histopathologic examination revealed no test item-related changes in the thyroid.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mg act. ingr./kg bw/day.
No deaths of fetuses and no test item-related malformations, variations or retardations were noted.
No test item-related influence was noted on the developmental parameters (fetal body weight, ano-genital distance, testicular development for the male fetuses).
Under the conditions of the study, the test item did not show any embryotoxic potential in rats.

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

A key prenatal developmental toxicity study was conducted using the registered substance  administered orally via the diet to female CD® (Crl: CD(SD) rats at dose levels of 250, 500 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy (Quentin, 2021). The dose levels are expressed as solid content of the registered substance. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 500 mg/kg bw/day for the dams. Maternal toxicity was evident by noteworthy reductions in the body weight, the body weight gain, the carcass weight and the net body weight gain for the dams of the high dose group (1000 mg/kg bw/day). None of the animals of the test groups died prematurely. No changes in behaviour, external appearance and the faeces were noted in any of the dose groups.
No test item-related influences were noted on the food and drinking water consumption of the dose groups. No test item-related differences were noted for the reproduction parameters (number of implantations, number of resorptions, number of fetuses). Macroscopic post mortem examination during necropsy revealed no pathologic changes in any of the dose groups. No test item-related differences were noted for the serum levels of the thyroid hormones T3, T4 and TSH and also no test item-related differences were noted for the thyroid weights. Furthermore, histopathologic examination revealed no test item-related changes in the thyroid.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mg/kg bw/day. No deaths of fetuses and no test item-related malformations, variations or retardations were noted. No test item-related influence was noted on the developmental parameters (fetal body weight, ano-genital distance, testicular development for the male fetuses). Under the conditions of the study, the test item did not show any embryotoxic potential in rats.

A supporting dose-range-finding study in pregnant rats was conducted to select the dose levels for the main OECD 414 study (Hansen, 2021). The registered substance was administered orally via the diet to female rats at target dose levels of 300, 600 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 600 mg kg bw/day for the dams. None of the dams died prematurely. No changes in behaviour or external appearance were noted. Slightly reduced body weight, body weight gain and carcass weights were noted at the high dose level. No test item-related changes were noted for the food consumption. No pathological changes were noted in the macroscopic examination during laparotomy. The mean actual intake of the test item via the diet over the dosing period was 276.3, 539.1 and 963.7 mg/kg bw/day. Hence, the actual values were in the range of the nominal values with 300, 600 or 1000 mg/kg bw/day. The no-observed-adverse-effect level (NOAEL) for the fetal organism was 1000 mg/kg bw/day. The reproductive parameters (number of implantation sites, number of resorptions, number of fetuses and sex distribution) were not influenced by the test item. No test item-related influence was noted on the weights of the placentae or the fetuses. No runts were noted in any of the dose groups. No dead fetuses, no malformations and no variations were noted.

Mean actual test item intake values in the key OECD 414 study were calculated to be 235, 472 and 1003 mg/kg bw, respectively, therefore they approximated the nominal values of 250, 500 and 1000 mg/kg bw. During validation of the analytical method, the measured concentrations of freshly prepared samples of the test item-diet mixtures were well within the range of the expected concentrations. However, the recovery of the stability samples was outside the acceptance criteria. An interaction between the test item and the diet during storage was assumed and therefore, the extraction method needs to be further investigated. The results of the test item-formulation are still pending and will be given in the final report (a statement by the contract laboratory for prolongation is attached to the endpoint record).

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the effects observed in animals are regarded as relevant for humans.

Justification for classification or non-classification

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), the test item does not have to be classified and has no obligatory labelling requirement for reproductive and developmental toxicity.

Additional information