Acetic acid, chloro-, sodium salt, reaction products with 4,5-dihydro-2-undecyl-1H-imidazole-1-ethanol and sodium hydroxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 December 2021 to 26 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Regulation (EC) 440/2008 of May 2008

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

Reference substance name:

Acetic acid, chloro-, sodium salt, reaction products with 4,5-dihydro-2-undecyl-1H-imidazole-1-ethanol and sodium hydroxide

EC Number:

271-794-6

EC Name:

Acetic acid, chloro-, sodium salt, reaction products with 4,5-dihydro-2-undecyl-1H-imidazole-1-ethanol and sodium hydroxide

Cas Number:

68608-66-2

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11 alkyl) derivs. and sodium hydroxide and chloroacetic acid

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability in the vehicle (Elix water): Stability for at least 24 hours at room temperature under normal laboratory light conditions, 8 days in the refrigerator and of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 2 to 200 mg/mL, Test Facility Study No. 20293261.
Stability for at least 24 hours at room temperature (15 to 25°C) under normal laboratory light conditions and for at least 8 days in the refrigerator (set to maintain 4°C) is confirmed at a concentration of 231 mg/mL (solutions, 370 mL bulk volume), Test Facility Study No. 20293261.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at initiation of dosing: 11-15 weeks old
- Weight at initiation of dosing: 187-273 g
- Fasting period before study: not specified
- Housing: individually housed in polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. For psychological/environmental enrichment and nesting material, animals were provided with paper and with aspen wooden sticks.
- Diet: pellets of SM R/M-Z (from SSNIFF® Spezialdiäten GmbH, Soest, Germany) were given ad libitum.
- Water: municipal tap water was freely available via water bottles.
- Acclimation period: 5-6 days

Results of analyses confirmed the absence of contaminants in the feed, water and enrichments that could have interfered with the objectives of the study.

ENVIRONMENTAL CONDITIONS (ACTUAL)
- Temperature: 19-20°C
- Humidity: 48 to 56%
- Air changes: at least 10 air changes per hour
- Photoperiod: 12-hours light and 12-hours dark

IN-LIFE DATES: From: 20 December 2021 To: 07 January 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(Elix)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dose formulations were prepared at least weekly in daily portions and stored at 4°C. For the high dose group (1000 mg/kg bw/day) pure test item was used. Adjustment was made for specific gravity of the test item. Dose calculations were corrected for purity, therefore, a correction factor of 2.83 was used for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: the test item showed to be stable in the vehicle. Further, the vehicle is not toxic to animals and does not interfere with the objectives of the study.
- Concentration in vehicle: 38.5. 115.5 and 385.2 mg/mL for the low-, mid- and high-dose groups respectively.
- Amount of vehicle: 2.596 mL/kg bw. For the high dose group, undiluted test material was administered, in order to minimize the chance of foam formation and regurgitation, as observed in the OECD 422 study for the C8-18 analogue.. The dose volume was calculated as: 2830 mg/kg bw/day / density (1090 mg/mL) = 2.596 mL/kg. This dose volume was kept the same for all groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During week 1 of treatment, from all groups samples were taken for concentrations analysis and from the low- and mid-dose groups samples were taken for homogeneity analysis. Analyses were not conducted for the high-dose group as pure test item was used.
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20293261).
Acceptance criteria were also established for concentration (mean sample results within or equal to ± 10% of theoretical concentration) and homogeneity (coefficient of variation of ≤ 10%).

Details on mating procedure:

Animals were purchased time-mated. Untreated females were mated at the Supplier and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).

Duration of treatment / exposure:

From Day 6 to Day 20 post-coitum.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

low-dose group

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

mid-dose group

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

high-dose group

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Refer to Table 1 under "Any other information on materials and methods incl. tables" for a summary of the experimental design.

JUSTIFICATION OF TEST SYSTEM AND NUMBER OF ANIMALS
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for developmental toxicity testing by regulatory agencies. The Test Facility has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

JUSTIFICATION OF ROUTE AND DOSE LEVELS
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on results of a Dose Range Finder Study with oral exposure of Acetic acid, chloro-, sodium salt, reaction products with 4,5-dihydro-2-undecyl- 1H-imidazole-1-ethanol and sodium hydroxide in rats (Test Facility Study No. 20293263), and in an attempt to produce graded responses to the test item.
In the Dose Range Finding Study, 6 females per group were treated with 0, 300, 600 and 1000 mg/kg bw/day. At 600 mg/kg bw/day, a single female was found dead after a mis-gavage. Abnormal breathing sounds were noted for individual females at all treatment groups. Mean body weights, body weight gain and weight gain corrected for gravid uterus were unaffected by treatment up to 1000 mg/kg bw/day. Mean food consumption was slightly lower than control at 600 and 1000 mg/kg bw/day between Day 6-9 post-coitum (7% and 6%, respectively; not statistically significant), but recovered over the treatment period to levels similar as control. No macroscopic findings were noted at scheduled necropsy. Mean fetal body weights (male, female and combined) were slightly decreased at 1000 mg/kg bw/day (not statistically significant for males). Visceral examination of fetal hearts was included but showed no abnormalities.
Based on these results, and in consultation with the Sponsor, dose levels were selected at 0, 100, 300 and 1000 mg/kg bw/day for the current study. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality was checked for all animals twice daily starting at the beginning upon arrival through termination/release. Other observations were performed for all animals at least once daily 0 to 1 hours post-dose, starting on Day 6 post-coitum up to and including the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were removed from their cages on Days 2, 6, 15 and 21 post-coitum.

BODY WEIGHT: Yes
- Time schedule for examinations: for all animals on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. Body weight gains were calculated for the following intervals: Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, and 6 to 21 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured for all animals over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: for all animals on regular basis by visual inspection of water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 post-coitum by carbon dioxide inhalation.
- All animals (including those found dead or sacrificed before planned necropsy and females with delivery prior to necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.
- Gravid uterus adjusted to body weight was determined as follows: Body weight on Day 21 post-coitum - body weight on Day 6 post-coitum - gravid uterus weight.
- The thyroid gland was weighed at necropsy for all scheduled euthanasia animals, except for females that delivered their offspring prior to necropsy. Thyroid weights were also not be recorded for animals found dead, euthanized in poor condition or in extremis. Organ weight as a percent of body weight (using the body weight on Day 21 post-coitum) were calculated.
- The thyroid gland and macroscopic abnormalities were collected from all animals and preserved in 10% buffered formalin. Thyroid glands of all animals were then prepared for microscopic examinations.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not for animals found dead, sacrificed before planned necropsy or that started to deliver)
- Number of corpora lutea: Yes
- Number of implantations: Yes (in case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites).
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and distribution of live and dead fetuses

Blood sampling:

- Plasma: no
- Serum: Yes. Blood was sampled on Day 21 post-coitum between 07.00 and 09.00 from all parental animals reaching planned euthanasia. Animals were not fasted and the blood was sampled from the jugular vein. Blood was then processed to serum before thyroid hormones analysis.
- Volume collected: 1.0 mL
- Other: T3, T4 and TSH were measured from serum samples. IMMULITE® 1000 analyser was used for TSH measurements. For measurement of T3 and T4 an LC-MS system was employed. Measurement were performed according to the bioanalytical method validated in Test Facility Study No. 20213516.
The LC-MS analysis will be based on the following guidelines:
• European Medicines Agency (EMA). Guideline on Bioanalytical Method Validation. EMEA/CHMP/EWP/192217/2009, 01 February 2012.
• Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM), May 2018.

Fetal examinations:

Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity. In case this was not possible due to a malformation, the fetus was euthanized by decapitation or by interscapular injection of sodium pentobarbital. Recognizable live fetuses of females found dead or sacrificed before planned necropsy or pups from females that delivered prior to necropsy, were euthanized by decapitation.

- External examinations: Yes: all per litter. Performed on viable and non-viable fetuses of dams surviving until scheduled necropsy, late resorptions and fetuses of dams not surviving until Day 21 post-coitum. Malformed late resorptions were collected and fixed in 10% buffered formalin. Late resorptions without malformation were discarded.
- Soft tissue examinations: Yes: for viable and non-viable fetuses of dams surviving until scheduled necropsy. All fetuses were subjected to internal sex confirmation; 50% of these fetuses was subjected to visceral body examinations after fixation in 96% aqueous ethanol, macerated in potassium hydroxide, and stained (Alizarin Red S);
- Skeletal examinations: Yes: for viable and non-viable fetuses of dams surviving until scheduled necropsy. 50% percent of fetuses with head was subjected to skeletal examination after fixation with 96% aqueous ethanol, macerated in potassium hydroxide and stained (Alizarin Red S).
- Head examinations: Yes: for viable and non-viable fetuses of dams surviving until scheduled necropsy. 50% percent of fetuses was subjected to visceral head examination after fixation in Bouin's solution.
- Anogenital distance (AGD) of all live rodent pups: yes, for viable fetuses of dams surviving until scheduled necropsy. AGD was normalized to the cube root of the fetal body weight.
- Body weights: measured for viable and non-viable fetuses of dam surviving until scheduled necropsy.
- External sex determination of fetuses, viable and non-viable, was based on anogenital distance if possible (not for animals found dead, sacrificed before planned necropsy or that started to deliver). Internal sex determination was performed on viable and non-viable fetus of dam surviving
until scheduled necropsy on Day 21 post-coitum.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Analyses were performed according to Table 2 under "Any other information on materials and methods incl. tables" when possible but excluded any group with less than 3 observations.

PARAMETRIC/NON-PARAMETRIC
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

NON-PARAMETRIC
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

ANCOVA
The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.

INCIDENCE
Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Indices:

- Pregnancy rate (%): (No. of pregnant females / No. of mated females) x 100

- Male Fetuses (%): (No. male fetuses / Total no. of fetuses) x 100

- Female Fetuses (%): (No. female fetuses / Total no. of fetuses) x 100

- Pre-Implantation Loss (%): [(No. of corpora lutea – no. of implantations)/ No. of corpora lutea] x 100

- Post Implantation Loss (%): [(No. of implantations – no. of live fetuses) / No. of implantations] x 100

- Litter % of Fetuses with Abnormalities: (No. of fetuses in litter with a given finding / No. of fetuses in litter examined) x 100

Historical control data:

Historical control data are reported in the attachment under section "Overall remarks, attachments" named "20293266 - Historical Control Data".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 300 and 1000 mg/kg bw/day, abnormal breathing sounds were recorded for 2/21 and 4/22 surviving animals, respectively, between Days 8 and 18 post-coitum. In general, this occurred on a single day of treatment only, except for one animal at 300 mg/kg bw/day which showed this symptom on four consecutive days.
Salivation was recorded after dosing at 300 and 1000 mg/kg bw/day in a dose-related manner. This sign was considered to be a physiological response rather than a sign of systemic toxicity, taking into account the nature and slight to moderate severity of the effect and its time of occurrence (i.e., within 1 hour after dosing).
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test material.
One female at 300 mg/kg bw/day was found dead on Day 12 post-coitum. Prior to death, this animal showed abnormal breathing sounds between Days 9 and 12 post-coitum and was cold to touch and presented with labored breathing, hunched posture, erected fur and decreased activity on Day 12 post-coitum. Additionally, slight weight loss (3%) was recorded between Days 9 and 12 post-coitum. At necropsy, red clear watery and frothy content was noted in the thoracic body cavity. Also, the lungs had failed to collapse, and dark red foci were observed on the lungs and thymus. This female was pregnant with twelve dead embryos (noted in the tables as dead fetuses) and one early resorption. Based on the recorded necropsy findings in the thoracic cavity, this death was considered gavage-related.
No further mortality occurred during the treatment period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Refer also to Table 3 under "Any other information on results incl. tables".
At 1000 mg/kg bw/day, slight weight loss was recorded for 3/22 animals between Days 6 and 9 post-coitum (2-6%), which recovered as treatment progressed.
At 300 mg/kg bw/day, slight weight loss was recorded for 2/21 surviving animals between Days 6 and 9 post-coitum (3 or 4%), which recovered as treatment progressed. These animals also showed a lower weight gain corrected for gravid uterus weight.
Mean body weight and body weight gain at 100 mg/kg/day as well as mean gravid uterus weight and mean body weight corrected for gravid uterus weight for all treated groups were considered not to be affected by treatment with the test material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Refer also to Table 4 under "Any other information on results incl. tables".
At 1000 mg/kg bw/day, mean food consumption was lower between Days 6 and 15 post-coitum (8-20% below control mean). Although mean food consumption showed recovery as treatment progressed, mean food consumption over the complete treatment duration remained 10% below the control mean. Between Days 6 and 9 post-coitum, lower food consumption was primarily recorded for two females.
At 300 mg/kg bw/day, mean food consumption was lower between Days 6 and 12 post-coitum (10% below control mean), followed by normal food consumption from Day 12 post-coitum onwards. Between Days 6 and 9 post-coitum, lower food consumption was primarily recorded for two females.
Food consumption at 100 mg/kg bw/day was considered not affected by treatment with the test material.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Refer also to Table 5 under "Any other information on results incl. tables".
At 1000 mg/kg bw/day, a lower mean Total T3 concentration was recorded (0.77x of control). Mean Total T3 remained within the historical control data range.
Serum levels of Total T3 at 100 and 300 mg/kg bw/day and Total T4 and TSH up to 1000 mg/kg bw/day were considered not to be affected by treatment with the test material.

Note: Lower limits of quantification were 0.008 uIU/mL, 0.1 ng/mL and 5.00 ng/mL for thyroid stimulating hormone (TSH), triiodothyronine (Total T3) and total thyroxine (Total T4), respectively.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test material-related alterations in thyroid gland weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, irregular surface of the non-glandular stomach was noted for 12/22 females, with dark red foci on the glandular stomach for one of these females.
All other macroscopic findings were considered not to be related to treatment with the test material. Macroscopic thyroid gland lesions (including the small size of the left thyroid gland for two females at 1000 mg/kg bw/day) occurred without microscopic correlates, and all macroscopic findings remained within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test material-related microscopic observations in the thyroid glands.
All the recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain. There was no test material related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

There were no abortions in any of the treated groups.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Refer also to Table 7 under "Any other information on results incl. tables".
Corpora lutea and implantation sites, and pre- and postimplantation loss in the control and test groups were similar and in the range of normal biological variation. The apparent higher mean pre-implantation loss at 100 mg/kg bw/day occurred in the absence of a dose-related trend and was therefore considered not related to treatment with the test material.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Refer also to Table 6 under "Any other information on results incl. tables".
No total resorption was reported for any of the females.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Refer also to Table 7 under "Any other information on results incl. tables".
Early and late resorptions incidences were comparable across all groups.

Dead fetuses:

no effects observed

Description (incidence and severity):

Refer also to Table 7 under "Any other information on results incl. tables".
No dead fetus was reported in any of the groups.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of pregnant females in the control and test groups were similar and in the range of normal biological variation.
A total of three females were non-gravid: one of the control group, one of the 100 mg/kg bw/day and one of the 300 mg/kg bw/day. In addition, one female at 300 mg/kg bw/day was found dead on Day 12 post-coitum. Therefore, the number of females with viable litters for evaluation was 21, 21, 20 and 22 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

other: not determined due to absence of adverse effects observed up to the highest tested dose.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Refer also to Table 7 under "Any other information on results incl. tables".
Fetal body weights were considered not to be affected by treatment with the test material.
Mean combined (male and female) fetal body weights were 5.1, 5.2, 5.2 and 5.1 gram for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Refer also to Table 7 under "Any other information on results incl. tables".
No reduction in the number of live fetuses was observed in any of the groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Refer also to Table 7 under "Any other information on results incl. tables".
The male:female ratio was unaffected by treatment with the test material up to 1000 mg/kg bw/day.
Mean sex ratios (males:females) were 48:52, 46:54, 52:48 and 49:51 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Refer also to Table 7 under "Any other information on results incl. tables".
There were no test material-related effects on litter size of any group.
Mean litter sizes were 11.9, 10.9, 11.8 and 12.0 fetuses/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Refer also to Table 7 under "Any other information on results incl. tables".
Fetal anogenital distance was considered not to be affected by treatment with the test material.
Mean male anogenital distance was 3.08, 3.06, 3.03 and 3.05 mm for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Mean female anogenital distance was 1.37, 1.37, 1.33 and 1.34 mm for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
The mean male normalized AGD was 1.774, 1.755, 1.730 and 1.759 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The mean female normalized AGD was 0.804, 0.798, 0.778 and 0.783 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Refer also to Table 8 under "Any other information on results incl. tables".
No test material-related external malformations or variations were recorded.
One fetus from the high-dose group (1000 mg/kg bw/day) presented with edema of the entire subcutis, which was related to a fluid-filled thorax during visceral examination. Based on its single occurrence this was considered not to be related to treatment with the test material.
No other external malformations occurred, and no external variations were recorded.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Refer also to Table 9 under "Any other information on results incl. tables".
No skeletal malformations were observed in this study.
All recorded skeletal variations occurred in a diverse array of skeletal structures across all groups. Also, these variations and any incidental findings were scored infrequently, were limited to control fetuses, were comparable to the control group and/or occurred in the absence of a dose-relationship. Therefore, these skeletal variations and incidental findings were considered not to be related to treatment with the test material.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related visceral malformations and variations were recorded.
Of the four visceral malformations observed in this study, three were found during cephalic examination. The affected control fetuses either had a small lens or small eye. One fetus at 100 mg/kg bw/day presented with situs inversus. One fetus at 300 mg/kg bw/day had a large lens. As these findings occurred for single fetuses only, were limited to control fetuses and/or occurred in the absence of a dose-related incidence, they were considered not to be related to treatment with the test material.
Visceral variations were limited to supernumerary liver lobes, convoluted ureters, and a fluid-filled thorax (same fetus as with edema of the entire subcutis; see previous section). The low incidences and/or group distribution of these findings and any incidental findings did not indicate a relationship to treatment with the test material.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: absence of adverse effects up to the highest tested dose.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Dose Formulation Analyses

Accuracy
The concentrations analyzed in the formulations of Group 2 (low-dose; 101%) and Group 3 (mid-dose; 101%) were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90-110% of target concentration).
No test material was detected in the Group 1 (vehicle) formulation.

Homogeneity
The formulations of Group 2 were homogeneous (i.e., coefficient of variation ≤ 10%; the result was 1.1%).

 

Table 3 - Summary of Gravid Uterine Weights and Gravid Uterus Adjusted Body Weights: Gestation (Excluding non-gravid animals)

		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Bodyweight on Day 6 (g)	Mean SD N %Diff	233.8 20.2 21 -	229.3 18.3 21 -1.9	227.2 14.2 20 -2.8	228.6 21.1 22 -2.2
Terminal bodyweight (g)	Mean SD N %Diff	340.1 31.1 21 -	329.8 28.3 21 -3.0	330.7 23.4 20 -2.8	335.3 34.0 22 -1.4
Gravid Uterus Weight (g)	Mean SD N %Diff	81.47 12.89 21 -	74.98 15.87 21 -7.97	81.77 10.88 20 0.37	82.22 17.15 22 0.92
Adjusted BWG (6-abw) (g)	Mean SD N %Diff	24.82 6.47 21 -	25.50 7.40 21 2.74	21.69 10.21 20 -12.63	24.46 8.34 22 -1.43

 

Table 4 - Summary of Food Consumption: Gestation

		Day(s) relative to mating
		6 to 9	9 to 12	12 to 15	15 to 18	18 to 21	6 to 21
0 mg/kg bw/day	Mean SD N	21.87 2.66 21	22.54 2.23 21	22.75 2.15 21	23.70 1.99 21	23.81 2.17 21	22.93 2.06 21
100 mg/kg bw/day	Mean SD N %Diff	20.94 2.23 21 -4.28	21.40 2.14 21 -5.07	21.68 2.09 21 -4.68	23.17 2.71 21 -2.21	22.86 2.02 21 -4.00	22.01 2.06 21 -4.03
300 mg/kg bw/day	Mean SD N %Diff	19.59* 2.85 21 -10.45	20.40** 1.69 21 -9.51	21.77 1.93 20 -4.31	23.50 1.83 20 -0.84	23.05 2.21 20 -3.19	21.69 1.67 20 -5.41
1000 mg/kg bw/day	Mean SD N %Diff	17.45** 3.63 22 -20.20	18.53** 2.42 22 -17.79	20.88* 1.98 22 -8.21	23.70 2.40 22 -0.01	22.89 2.87 22 -3.85	20.69** 2.01 22 -9.78

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

 

Table 5 - Summary of Thyroid Hormone Values

		T3 (ng/mL)	T4 (ng/mL)	TSH (mU/L)
0 mg/kg bw/day	Mean SD N	0.438 0.089 21	22.65 3.92 21	0.3176 0.1622 21
100 mg/kg bw/day	Mean SD N tCtrl	0.410 0.092 21 0.94	20.90 4.73 21 0.92	0.2963 0.1963 21 0.93
300 mg/kg bw/day	Mean SD N tCtrl	0.386 0.054 20 0.88	21.09 3.31 20 0.93	0.3573 0.1447 20 1.13
1000 mg/kg bw/day	Mean SD N tCtrl	0.337** 0.075 22 0.77	21.39 5.13 22 0.94	0.2852 0.1657 22 0.90

Anova & Dunnett: ** = p ≤ 0.01

Historical control data for pregnant Wistar Han rats (period 2020-2022):
Triiodothyronine (T3), Total (ng/mL): mean: 0.424, Range (Central 95%): 0.270-0.683; n=347
Thyroxin (T4), Total (ng/mL): mean: 23.40, Range (Central 95%): 15.07-45.67; n=347
Thyroid Stimulating Hormone (TSH; mU/L): mean: 0.3272, Range (Central 95%): 0.0838-0.9033; n=347

 

Table 6 - Summary of Maternal Performance and Mortality

		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Group size		22	22	22	22
Number of females pregnant	N %	21 95.5	21 95.5	21 95.5	22 100.0
Female with Live Fetuses	N %	21 100.0	21 100.0	20 95.2	22 100.0
Total Resorptions	N %	0 0.0	0 0.0	0 0.0	0 0.0
Female with all Nonviable	N %	0 0.0	0 0.0	1 4.8	0 0.0
Terminal Euthanasia	N %	22 100.0	22 100.0	21 95.5	22 100.0
Unscheduled Death/Euthanasia	N %	0 0.0	0 0.0	1 4.5	0 0.0
Found Dead	N %	0 0.0	0 0.0	1 4.5	0 0.0

 

 

Table 7 - Summary of Ovarian and Uterine Examinations and Litter Observations

		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Female with Live Fetuses	N %	21 100.0	21 100.0	20 100.0	22 100.0
Number of Corpora Lutea	Mean SD N %Diff	13.0 1.6 21 -	12.7 2.1 21 -2.2	12.9 1.4 20 -0.8	13.0 2.1 22 0.0
Number of Implantations	Mean SD N %Diff	12.2 1.8 21 -	11.2 2.3 21 -7.8	12.2 1.5 20 -0.3	12.4 2.4 22 1.4
Pre-implantation Loss (%)	Mean SD N %Diff	5.71 8.38 21 -	11.69 11.69 21 104.51	5.29 7.21 20 -7.39	4.78 9.22 22 -16.28
Total Number of Fetuses	Mean SD N %Diff	11.9 1.6 21 -	10.9 2.5 21 -8.4	11.8 1.6 20 -0.9	12.0 2.6 22 0.8
Number of Live Fetuses	Mean SD N %Diff	11.9 1.6 21 -	10.9 2.5 21 -8.4	11.8 1.6 20 -0.9	12.0 2.6 22 0.8
Number of Dead Fetuses	Mean SD N %Diff	0.0 0.0 21 -	0.0 0.0 21 -	0.0 0.0 20 -	0.0 0.0 22 -
Number of Early Resorptions	Mean SD N %Diff	0.3 0.7 21 -	0.4 0.8 21 14.3	0.4 0.5 20 20.0	0.4 1.1 22 22.7
Number of Late Resorptions	Mean SD N %Diff	0.0 0.0 21 -	0.0 0.0 21 -	0.0 0.0 20 -	0.0 0.0 22 -
Total Number of Resorptions	Mean SD N %Diff	0.3 0.7 21 -	0.4 0.8 21 14.3	0.4 0.5 20 20.0	0.4 1.1 22 22.7
Post-implantation Loss (%)	Mean SD N %Diff	2.46 4.75 21 -	3.73 8.24 21 51.65	3.29 4.15 20 33.43	3.39 9.85 22 37.75
Number of Live Male Fetuses	Mean SD N %Diff	5.8 2.1 21 -	5.2 2.1 21 -9.9	6.2 2.5 20 6.7	5.9 2.3 22 2.6
Number of Live Female Fetuses	Mean SD N %Diff	6.1 1.9 21 -	5.7 1.8 21 -7.0	5.6 2.1 20 -8.1	6.0 2.1 22 -0.8
Live Male Fetus/Litter (%)	Mean SD N %Diff	48.28 15.19 21 -	46.24 17.12 21 -4.23	51.93 19.25 20 7.55	48.74 16.06 22 0.95
Live Female Fetuses/Litter (%)	Mean SD N %Diff	51.72 15.19 21 -	53.76 17.12 21 3.95	48.07 19.25 20 -7.05	51.26 16.06 22 -0.89
Mean Fetal Weight males (g)	Mean SD N %Diff	5.275 0.290 21 -	5.334 0.323 20 1.118	5.378 0.179 20 1.954	5.246 0.272 22 -0.536
Mean Fetal Weight females (g)	Mean SD N %Diff	4.987 0.237 21 -	5.074 0.260 21 1.757	5.065 0.192 20 1.576	4.980 0.338 22 -0.132
Mean Fetal Weight all (g)	Mean SD N %Diff	5.116 0.242 21 -	5.201 0.283 21 1.664	5.222 0.141 20 2.088	5.102 0.292 22 -0.257
Mean Fetal AGD males (mm)	Mean SD N %Diff	3.08 0.16 21 -	3.06 0.19 20 -0.71	3.03 0.17 20 -1.76	3.05 0.18 22 -1.03
Mean Fetal AGD females (mm)	Mean SD N %Diff	1.37 0.15 21 -	1.37 0.15 21 -0.24	1.33 0.16 20 -2.81	1.34 0.14 22 -2.70
Mean Normalized Fetal AGD males	Mean SD N %Diff	1.774 0.093 21 -	1.755 0.113 20 -1.090	1.730 0.099 20 -2.458	1.759 0.109 22 -0.865
Mean Normalized Fetal AGD females	Mean SD N %Diff	0.804 0.090 21 -	0.798 0.089 21 -0.771	0.778 0.096 20 -3.258	0.783 0.080 22 -2.568

 

Table 8 - Summary of fetal abnormalities (External)

Exam type: External	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Number of fetuses examined	249	228	235	263
Number of fetuses evaluated	249	228	235	263
Number of litters examined	21	21	20	22
Number of litters evaluated	21	21	20	22
Malformation (number of fetuses)	0	0	0	1
Malformation (number of litters)	0	0	0	1

 

Table 9 - Summary of fetal abnormalities (skeletal)

Exam type: Skeletal	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Number of fetuses examined	125	113	116	131
Number of fetuses evaluated	249	228	235	263
Number of litters examined	21	21	20	22
Number of litters evaluated	21	21	20	22
Variation (number of fetuses)	94	80	89	88
Variation (number of littters)	21	21	20	22

 

Table 10 - Summary of fetal abnormalities (visceral body)

Exam type: Visceral Body	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Number of fetuses examined	124	115	119	132
Number of fetuses evaluated	249	228	235	263
Number of litters examined	21	21	20	22
Number of litters evaluated	21	21	20	22
Variation (number of fetuses)	6	17	14	21
Variation (number of littters)	5	11	9	12
Malformation (number of fetuses)	0	1	0	0
Malformation (number of litters)	0	1	0	0

 

Table 11 - Summary of fetal abnormalities (visceral head)

Exam type: Visceral Head	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Number of fetuses examined	124	115	119	132
Number of fetuses evaluated	249	228	235	263
Number of litters examined	21	21	20	22
Number of litters evaluated	21	21	20	22
Malformation (number of fetuses)	2	0	1	0
Malformation (number of litters)	2	0	1	0

 

Table 12 - Summary of relevant measured parameters

Dose level (mg/kg bw/day)	0	100	300	1000
Pregnant/total dams	21/22	21/22	21/22	22/22
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses	0	0	1*	0
Dams with live fetuses	21	21	20	22
Corpora lutea (mean number)	13.0	12.7	12.9	13.0
Implantations (mean number)	12.2	11.2	12.2	12.4
Resorptions (mean number) - Early - Late - Total (early + late)	0.3 0.0 0.3	0.4 0.0 0.4	0.4 0.0 0.4	0.4 0.0 0.4
Pre implantation loss (%)	5.71	11.69	5.29	4.78
Post implantation loss (%)	2.46	3.73	3.29	3.39
Dead fetuses (mean number)	0.0	0.0	0.0	0.0
Mean body weight on day 21 (g)	340.1	329.8	330.7	335.3
Mean body weight gain day 6-21 (g)	106.3	100.5	103.5	106.7
Gravid uterine weight (g)	81.47	74.98	81.77	82.22
Mean adjusted body weight gain (6-abw)	24.82	25.50	21.69	24.46
Live offspring (mean number)	11.9	10.9	11.8	12.0
Sex ratio (male%)	48.28	46.24	51.93	48.74
Mean fetal body weight males (g)	5.275	5.334	5.378	5.246
Mean fetal body weight females (g)	4.987	5.074	5.065	4.980
Mean fetal body weight combined (g)	5.116	5.201	5.222	5.102
Mean anogenital distance males (mm)	3.08	3.06	3.03	3.05
Mean anogenital distance females (mm)	1.37	1.37	1.33	1.34
Mean normalized anogenital distance males	1.774	1.755	1.730	1.759
Mean normalized anogenital distance females	0.804	0.798	0.778	0.783
Malformations number and % of fetuses number of litters - External - Visceral - Skeletal	0 (0.0%) 0 2 (1.59%) 2 0 (0.0%) 0	0 (0.0%) 0 1 (0.68%) 1 0 (0.0%) 0	0 (0.0%) 0 1 (0.71%) 1 0 (0.0%)	0 (0.35%) 1 0 (X.X%) 0 0 (0.0%) 0
Variations number and % of fetuses number of litters - External - Visceral - Skeletal	0 (0.0%) 21 6 (5.05%) 5 94 (75.44%) 21	0 (0.0%) 21 17 (16.19%) 11 80 (71.61%) 21	0 (0.0%) 20 14 (11.52%) 9 89 (77.68%) 20	0 (0.0%) 22 21 (14.64%) 12 88 (66.18%) 22

*, one gravid female of the 300 mg/mg bw/day was found dead on Day 12 post-coitum as a consequence of the gavage procedure

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for the test item were established:
NOAEL (maternal toxicity): At least 1000 mg/kg bw/day
NOAEL (developmental toxicity): At least 1000 mg/kg bw/day

Executive summary:

A developmental toxicity study in Wistar Han rats was conducted with the test item according to the OECD TG 414 (2018) and in accordance with GLP principles. The test item was administered daily by oral gavage to time-mated females during the critical period of organogenesis, i.e. from Day 6 to Day 20 post-coitum, in order to characterize the maternal toxicity and developmental toxicity likely caused by the exposure to the substance. The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg bw/day, based on the results of the Dose Range Finder. A number of 22 females were allocated in each dose group.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. Formulation analyses confirmed that formulations of test material in Water (Elix) were prepared accurately and homogenously.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (Total T3), thyroxine (Total T4), thyroid-stimulating hormone (TSH)), organ weights (thyroid gland), macroscopic examination, microscopic examination (thyroid gland), uterine contents, corpora lutea, implantation sites and pre- and postimplantation loss.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations and incidental findings.

An incidental death occurred in the 300 mg/kg bw/day as a result of the gavage procedure.

At 100 mg/kg bw/day no test material-related changes were noted.

At 300 and 1000 mg/kg bw/day abnormal breathing sounds were recorded after dosing but these were in general recorded incidentally and for a few surviving animals only. Salivation was also noted in these groups in a dose-related manner. However, this sign was considered to be a physiological response rather than a sign of systemic toxicity, taking into account the nature and slight to moderate severity of the effect and its time of occurrence (i.e., within 1 hour after dosing). 

Temporary slight weight loss and lower food consumption were also noted at 300 and 1000 mg/kg bw/day. The slight weight loss was recorded for some animals at these dose levels over the first three days of administration and coincided with lower food consumption over the first week of treatment. Both body weight and food intake however recovered as treatment progressed. Therefore, these observations were considered not to represent an adverse effect of the test material.

Thyroid hormone analysis revealed a statistically significant decrease of T3 blood concentration for the 1000 mg/kg bw/day group only but the value was within the historical control database of the laboratory. T4 and TSH measurements were comparable across groups and, thus, not affected by treatment. Thyroid weight measurement and pathology did not reveal any abnormalities that could have been related to treatment. It is therefore concluded that the reduction in T3 blood concentration occurred at 1000 mg/kg bw/day may be related to the test item administration but it is not considered as adverse since all the other thyroid-related parameters were not affected and in line with the values recorded in the control group.

At gross pathology, irregular surface of the non-glandular stomach was noted for 12/22 females of the 1000 mg/kg bw/day, with dark red foci on the glandular stomach for one of these females. These findings were considered to represent irritating properties of the test material following gavage administration. Considering that these macroscopic lesions were not accompained by any other relevant changes in health conditions, they were considered not to represent an adverse effect of the test material under the conditions of this study.

No test material-related changes were noted in any of the other maternal parameters investigated in this study (uterine contents, gravide uterus weight, corpora lutea, implantation sites, and pre- and post-implantation loss).

No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups. No test material-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations, developmental variations and incidental findings).

Overall, the conclusions of the study are agreed and the following NOAELs can be derived:

- NOAEL (maternal toxicity): At least 1000 mg/kg bw/day

- NOAEL (developmental toxicity): At least 1000 mg/kg bw/day