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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 May 2019 - 23 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was initiated after final decision of ECHA.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Amber liquid
Details on test material:
Storage Conditions: At room temperature
Test item handling: No specific handling conditions required
Irritant or corrosive: Yes
Volatile Vapour pressure: 2.3E-04 Pa.at 20°C
Specific gravity/density: 830 kg/m3 at 25°C
Specific details on test material used for the study:
Purity/Composition correction factor: No correction factor required.
Test item handling: No specific handling conditions required.
Stability for at least 5 hours at room temperature is confirmed over the concentration range 0.2 to 200 mg/mL and stability for 8 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL, Charles River Study No. 512048 and ABL Project 16059).

Test animals

Species:
rat
Strain:
other: Wistar Han
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 177-257 g
- Fasting period before study: no
- Housing: Individually in Macrolon plastic cages containing appropriate bedding
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: at least 5 days

No known contaminants were expected to be present in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed, and it was considered that there were no known contaminants in the water that would interfere with the objectives of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 45-62
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 May 2019 To: 23 May 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: the vehicle was chosen based on trial formulations performed at Charles River Den Bosch and on information provided by the sponsor.
- Concentration in vehicle: 0.3, 1.3 and 3.8 mg/mL for the low, mid and high dose, respectively.
- Amount of vehicle (if gavage): 4 mL/kg bw
Stability for at least 5 hours at room temperature was previously confirmed over the concentration range 0.2 to 200 mg/mL and stability for 8 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL, Charles River Study No. 512048 and ABL Project 16059.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure (Ardena Bioanalytical Laboratory Study No. ABL16059).
Dose formulation samples were collected for analysis during the first week of treatment for concentration analysis (all groups) and analysis of homogeneity (low and high dose groups only). In the second week of treatment, dose formulation samples were collected for concentration analysis and analysis of homogeneity (low dose group only).
All samples were shipped on dry ice to the analytical laboratory on the date of preparation.
Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was below or at 10%.
Details on mating procedure:
The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
Females were dosed on Day 6 to 20 post-coitum (15 days in total)
Frequency of treatment:
Once daily
Duration of test:
Females were sacrificed Day 21 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
1 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
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 the results of a previously performed 14-day dose range finder (Charles River Study No. 521047) and an extended OECD 422 study (Charles River Study No. 521048) in rats and in an attempt to produce graded responses to the test item. During the dose range finder, animals were dosed at 30 and 100 mg/kg bw/day by oral gavage. At 100 mg/kg bw/day, all animals were moribund after 11 days of treatment. At 30 mg/kg bw/day, effects were limited to body weight loss (-2 or -5% for all animals over Days 1-8, continuing for two animals to -5% and -6% over Days 1-14) and slightly reduced food intake. Based on these results, 1, 5 and 15 mg/kg/day were selected as dose levels for the extended OECD 422 study. During this study, animals received the test item for approximately 90 days. No adverse changes were noted in in-life parameters (i.e. mortality, clinical appearance, functional observations, body weights and food consumption) of females up to 15 mg/kg bw/day. As the effects on body weight at 30 mg/kg/day were considered unacceptable for the current study, the same dose levels as used in the extended OECD 422 study were selected.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Animals were observed for general health/mortality and moribundity twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Cage debris was examined to detect premature birth.

BODY WEIGHT: Yes
- Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

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

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Blood was collected on the day of scheduled necropsy (unfasted), concentration of Triiodothyronine (T3) Thyroxine (T4) and Thyroid-Stimulating Hormone (TSH) in serum was determined.
- All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the uterus and thyroid gland) were weighed.
- Thyroid glands were weighed at necropsy for all animals. Paired organs were weighed together. Organ to body weight ratio (using the body weight on Day 21 post coitum) were calculated.
- Thyroid glands of all animals of the control and the high dose group were histopathologically examined. Thyroids of all groups were collected, but it was considered of no added value to examine tissues of the low and mid dose group, since no test item-related changes were observed in the highest dose group.
Ovaries and uterine content:
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine: the number of corpora lutea, the weight of the gravid uterus, the number of implantation sites, the number and distribution of live and dead fetuses, the number and distribution of embryo-fetal deaths and the sex of each fetus based on the ano-genital distance.
Fetal examinations:
Live fetuses were euthanized. All litters were subjected to detailed external, visceral and skeletal examinations.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight.
The numbers of fetuses (litters) available for morphological examination were 230 (22), 263 (22), 243 (22) and 255 (22) in the control, 1, 5 and 15 mg/kg bw/day groups, respectively. External examination was done for all fetuses, visceral examination was done for approximately half of the fetuses of all groups, and skeletal examination was done for the other half of fetuses. Moreover, the visceral examination was added for two fetuses (one from the control group and one from the mid dose group) as during eviscerating prior to skeletal staining prominent visceral malformations were noticed.

Visceral Examinations:
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. As visceral malformations were suspected for two pups in the high dose group which were selected for skeletal examination, these fetuses were also subjected to visceral examination. All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations:
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson by a method similar to that described by Dawson and Inouye. Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.






Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose group vs. control group, mid dose group vs. control group, high dose group vs. control group. Datasets with at least 3 groups (the designated control group and at least 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.

Indices:
Maternal Variables
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum;
Corrected Body Weight Gains: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus;
Relative Food Consumption: Calculated against the body weight for scheduled intervals;
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum.

Reproduction and Developmental Variables
For each group, the following calculations were performed:
Pre-implantation loss (%): ((number of corpora lutea - number of implantation sites)/number of corpora lutea)*100
Post-implantation loss (%): ((number of implantation sites - number of live fetuses)/number of implantation sites)*100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%): ((number of viable fetuses affected/litter)/number of viable fetuses/litter)*100


Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were noted during the observation period.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights, body weight gain and weight gain corrected for gravid uterus were considered to have been unaffected by treatment up to 5 mg/kg bw/day. Mean body weight gain of females at 15 mg/kg bw/day was slightly reduced from start of treatment onwards. On Day 21 post coitum, mean body weight gain of these females was 7% lower than concurrent controls (statistical significance was achieved on Day 18 post coitum only). In addition, mean relative weight gain corrected for gravid uterus weight was statistically significantly lower at 15 mg/kg/day when compared with controls (7.7% vs 12.8%). The mean relative weight gain remained just within the historical control range. The correction for gravid uterus weight made clear that two pregnant females at 15 mg/kg bw/day actually lost weight over the treatment phase, i.e. Day 6 to Day 21 post coitum.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was considered unaffected by treatment up to 5 mg/kg bw/day.
At 15 mg/kg bw/day, mean food consumption was decreased throughout the treatment period when compared with concurrent control. Overall, relative food consumption was decreased with 8% when compared with concurrent control (mean of means). The differences in relative food consumption between females at 15 mg/kg bw/day and concurrent control were statistically significant and ranged from 7-11% on separate intervals.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum levels of total T3 and T4 and TSH were considered to be unaffected by treatment up to 15 mg/kg bw/day. All mean values remained within the historical control range. The statistically significantly lower mean TSH level in females at 1 mg/kg/day was considered to be a chance finding, in the absence of a dose-related response.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant higher thyroid gland weights (absolute and relative to body weights) were noted in the 15 mg/kg bw/day group females when compared with the control group females. All relative thyroid weights remained within normal historical control limits. Moreover, no treatment-related effects were noted in any of the thyroid hormones or during microscopic evaluation of the thyroid. The increase noted in thyroid organ weight was therefore considered a chance finding and unrelated to treatment with the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings in the thyroid gland of the control and high dose group animals.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group. Mean litter sizes were 10.5, 12.0, 11.0 and 11.6 fetuses/litter for the control, 1, 5 and 15 mg/kg bw/day groups, respectively.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were in the range of normal biological variation and considered unaffected by treatment up to 15 mg/kg bw/day. The statistically significant lower percentage of pre-implantation loss for females at 5 mg/kg bw/day was attributed to the relatively high concurrent control mean and in the absence of a dose related response considered unrelated to treatment.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The litter incidence of early and late resorptions and post-implantation loss was considered unaffected by treatment up to 15 mg/kg bw/day.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed up to and including the highest dose tested
Remarks on result:
other: 15 mg/kg bw/day was selected as highest dose because the body weight loss observed at 30 mg/kg bw/day in a 14-day dose range finder in non-pregnant rats was considered unacceptable for the current study.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The fetal weights (male, female and combined) in treatment groups were comparable with the controls and remained within the historical control range of the Test Facility. Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.3 and 5.1 gram for the control, 1, 5 and 15 mg/kg bw/day groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 15 mg/kg bw/day.
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment up to 15 mg/kg bw/day.
Two externally malformed fetuses were observed in this study. One fetus at 5 mg/kg/day had a meningocele, substantiated skeletally, and in addition appeared to have fusion of the mandibles. The other fetus was a control fetus that had microcephaly with according skeletal findings of the skull. Due to the single occurrence and/or occurrence in a control fetus, these malformations were considered spontaneous in origin.
External variations were not seen in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on skeletal morphology following treatment up to 15 mg/kg bw/day.
Aside from the underlying malformations of the two fetuses with an external skull finding (one in the control and one in the 5 mg/kg/day group, respectively), the latter fetus also had fusion of the mandibles. In addition, bent limb bones were observed in one fetus at 1 mg/kg bw/day. Both malformations were observed previously in historical controls and the group distribution did not indicate a treatment relationship. Therefore, they were considered chance findings.
Skeletal variations were observed in 75.0%, 77.3%, 81.0% and 80.8% of fetuses per litter in the control, 1, 5 and 15 mg/kg bw/day groups, respectively. All the ones noted occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were considered unrelated to treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 15 mg/kg bw/day.
Visceral malformations occurred in two fetuses at 1 and 15 mg/kg bw/day each. At the high dose, one fetus had a malpositioned atrium and ventricular septum defect and one fetus had a small eye and right-sided aorta. At the low dose, both affected fetuses had situs inversus. Due to the group distribution, single occurrence and/or occurrence in historical control fetuses, these malformations were considered unrelated to treatment.
Only two visceral variations (dilated ureter and convoluted ureter) were observed in this study and both occurred in one fetus at 15 mg/kg bw/day. The single occurrence did not indicate a treatment relationship. Moreover, both variations were observed previously among historical control fetuses.
Other effects:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal ano-genital distance (both sexes) noted after treatment up to 15 mg/kg bw/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to and including the highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of a prenatal developmental toxicity study performed in rats according to OECD guideline 414 and GLP principles, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Diamine methylated was established to be 15 mg/kg bw/day, as no adverse effects were seen at the highest dose tested (15 mg/kg bw/day).
Executive summary:

The objectives of this study were to determine the potential of Diamine methylated to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 1, 5 and 15 mg/kg/day, based on the results of a 14-day dose range finder (Test Facility Study No. 512047) and an extended OECD 422 study (Test Facility Study No. 512048). 15 mg/kg/day was selected as highest dose because the body weight loss observed at 30 mg/kg (described in a 14-day dose range finder in non-pregnant rats; Test Facility Study No. 512047) was considered unacceptable for the current study. Test substance was administered in corn oil at 4 mL/kg bw to 22 females per dose group.

 

Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity.

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 (T3, T4, and TSH), gross necropsy findings, number of corpora lutea, organ weights ((gravid) uterus and thyroid gland), uterine contents and histopathologic examination (thyroid gland).

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, ano-genital distance, external, visceral and skeletal malformations and developmental variations.

 

Results:

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels for Group 3 and 4 formulations. 

Formulations of Group 2 were below the target concentration, however as no adverse effects were noted in any of the study parameters up to 15 mg/kg/day, there was no impact on the study outcome. 

No test item-related effects were observed in the 1 and 5 mg/kg/day groups.

Mean food consumption and body weight gain was reduced throughout the treatment period in females at 15 mg/kg/day. In addition, body weight gain corrected for gravid uterus weight was statistically significantly lower for these females. Based on the magnitude of change, absence of any clinical symptoms and as values remained within the normal range, these effects were considered not adverse. 

No developmental toxicity was observed in the 1, 5 and 15 mg/kg/day groups.

 

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Diamine methylated was established as being 15 mg/kg/day.