Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
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 (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Den Bosch
- Age at study initiation: not specified
- Weight at study initiation: body weights within ± 20% of the mean for each set of animals
- Fasting period before study: not specified
- Housing: housed individually in Macrolon plastic cages
- Diet (e.g. ad libitum): Pelleted rodent diet, ad libitum. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22°C
- Humidity (%): 28 to 51%
- Air changes (per hr): 12 hour light/12 hour dark cycle
- Photoperiod (hrs dark / hrs light): Ten or greater air changes per hour with 100% fresh air

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Dehydrated and deacidified 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 1 hour after completion of the preparation of the formulation. 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 (0.92) and test item (1.0140). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis. During week 1 of treatment all groups were analysed for concentration and low dose and high dose groups were analysed for homogeneity. As the analytical results of low dose group were outside the acceptance criteria, a second concentration and homogeneity analysis was included during week 1. For concentration analysis duplicate sets of samples (approximately 500 mg) 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. For the homogeneity analysis duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was < 10%. Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
Not specified.
Duration of treatment / exposure:
single oral administration
Frequency of treatment:
once daily, 7 days a week
Duration of test:
Day 6 to Day 20 post-coitum, gestation days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 14-day dose range finder (DRF) in non-pregnant rats, and in an attempt to produce graded responses to the test item. In this DRF, females treated up to 1000 mg/kg were noted with flat gait and rales on single occasions. Body weight gain, food consumption and organ weights were normal, and in one female reddish isolated foci were noted on the glandular mucosa of the stomach. Based on these minor effects, a top dose level of 1000 mg/kg was selected for this prenatal developmental toxicity study in pregnant rats.
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked included: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 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: No
- 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
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy) 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 (gravid) uterus) were weighed. The nasal cavity including the nasopharynx, larynx incl. ventral pouch and lungs were collected at scheduled necropsy from all surviving animals and fixed in 10% buffered formalin for possible further examination of the respiratory difficulties. No histopathological examination was conducted.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the ano-genital distance.
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter ]
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% and 5% levels.
Indices:
Not calculated
Historical control data:
Charles River Den Bosch 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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of respiratory distress were observed among animals treated with test item surviving until scheduled necropsy as well as in those sacrificed in extremis. Reflux directly after dosing was seen for three surviving high-dose females (nos. 70, 72 and 88) on one or two occasions.
Rales (slight to moderate) was observed in 4, 8 and 9 females at 100, 300 and 1000 mg/kg, respectively, at a dose-related increased frequency and duration. Three out of 9 high-dose animals showing rales (nos. 70, 72 and 81) were additionally noted with other transient respiratory abnormalities, such as gasping and/or slight laboured and shallow respiration, discharge form nose and cough on one or three days during treatment. These breathing problems were considered to be related to the reflux of test item after administration via oral gavage.
Among animals showing rales, transient abnormal postures such as hunched or flat posture and flat gait were observed in one female at 300 mg/kg (no. 46) and in 5 females at 1000 mg/kg (nos. 70, 72, 79, 87 and 88) on one or two consecutive days during treatment.
Piloerection was noted in a few animals at 100 and 300 mg/kg, and in 8 females at 1000 mg/kg.
Transient and slight chromodacryorrhoea in the snout was observed in one female (no. 33) at 100 mg/kg and 2 females at 1000 mg/kg (nos. 72 and 88). Those two females at 1000 mg/kg presented with slight to moderate ptosis on one single day.
Salivation (slight to moderate) observed in one control female and in several females at 1000 mg/kg was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. 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.
Some of the clinical signs observed (respiratory effects, piloerection and abnormal posture), and body weight loss and reduced food consumption variations, may have been related to the respiratory distress and reflux caused by the administration of the test item via oral gavage; however, it cannot be excluded that some of these effects are related to systemic toxicity of the test item. Therefore, the conservative NOAEL for maternal effects is based on these findings.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study that was considered to represent a systemic test item effect.
A total of four females dosed at 1000 mg/kg spontaneously died or were sacrificed in extremis between Days 8 and 16 post-coitum:
Two females at 1000 mg/kg (nos. 86 and 77) spontaneously died on Day 8 and 14 post-coitum, respectively, without showing any signs of toxicity. At necropsy, a discoloured lung (dark red left lobe) was noted for female no. 86, which might be due to a bleeding and could be related to the oral gavage procedure. For female no. 77, no macroscopic abnormalities were observed at necropsy. Although no clear cause of mortality could be established for these two spontaneous deaths, they were considered unrelated to treatment since there were no relevant findings either during in-life or at necropsy that indicated a poor condition of these two females due to a systemic toxicological test item effect.
Moreover, two females at 1000 mg/kg (nos. 73 and 76) were sacrificed in extremis on Day 13 and 16 post-coitum, respectively for animal welfare reasons. The main clinical signs for these two females were related to respiratory distress. Female no. 73 was noted with slight rales, piloerection and gasping in the morning of Day 13 post-coitum (before dosing) and had an overnight body weight loss of about 17%. For female no. 76 reflux was observed directly after dosing on two occasions (i.e. Days 14 and 16 post-coitum) and this animal presented with hunched posture, slight rales and piloerection from Day 10 post-coitum onwards and slight laboured respiration from Day 14 post-coitum onwards. On one single day, flat gait, slight ptosis and moderate salivation was also noted. No body weight loss was observed for this female, but food consumption over Days 9-12 post-coitum was slightly lower than normal. No macroscopic findings were noted for these two females sacrificed in extremis. These premature decedents were considered to be related to reflux and respiratory distress, rather than a systemic effect of the test item.
No mortality occurred in the other groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls.
The slightly lower mean body weight and body weight gain (not statistically significant) observed in females at 300 mg/kg at the end of the gestation period (Days 18 and 21 post-coitum) were attributed to female no. 45 that presented with a body weight loss of 11% between Days 12 and 15 post-coitum and female no. 52 that presented with absence of body weight gain over Days 18 to 21 post-coitum. As these changes occurred in absence of a dose-related trend and were considered to be caused by individual animals, this was considered not to be toxicologically significant.
At 1000 mg/kg, some individual surviving animals (nos. 70 and 81) were noted with a body weight loss of 7% between Days 9 and 12 post-coitum followed by a normal to slightly reduced body weight gain until the end of the study period. In both females, this decrease in body weight was accompanied by a reduction in food intake. Another female at 1000 mg/kg (no. 72) was noted with a slight body weight loss of 2% between Days 6 and 9 post-coitum followed by normal body weight gain during the remaining days of treatment.
The slightly (not statistically significantly) lower mean body weight gain after correction for gravid uterus observed in females at 1000 mg/kg when compared to concurrent control mean (23.6 vs 30.9 gram) was mainly attributed to the 3 females (no. 70, 72 and 81) that presented with body weight loss during the study period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg, mean absolute and relative food consumption was statistically significantly decreased over Days 12-15 post-coitum and Days 12-18 post-coitum, respectively. Relative food consumption was reduced up to 11% when compared to control means. This reduction was mainly attributed to three females (nos. 70, 76 and 81) that presented with a decreased food intake from Day 12 post-coitum onwards. At the end of the treatment period both mean absolute and relative food consumption values were similar to those observed in the concurrent control group.
The statistically significantly higher food consumption at 300 mg/kg on Days 6-9 post-coitum occurred incidentally and in absence of a dose-related trend, and was therefore considered to be unrelated to treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at scheduled necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One female each at 100 and 300 mg/kg (nos. 34 and 58) was not pregnant. This finding was not considered to be test item-related as no dose-response was observed and as treatment started from Day 6 post-coitum onwards. All four females that did not survive until planned necropsy were pregnant and had normal implantation sites in development. All pregnant females surviving to scheduled necropsy had litters with viable fetuses.
Other effects:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in the numbers of corpora lutea.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
Remarks on result:
other: Clinical signs may have been due to the respiratory distress and reflux caused by oral gavage. It cannot be excluded that some of these effects are related to systemic toxicity.

Maternal abnormalities

Abnormalities:
effects observed, treatment-related

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 1000 mg/kg. Mean combined (male and female) fetal body weights were 5.3 gram for the 100 mg/kg group and 5.2 gram for the other groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.3, 10.8, 9.1 and 11.5 fetuses/litter for the control, 100, 300 and 1000 mg/kg groups, respectively.
The slightly lower mean litter size at 300 mg/kg was related to a slightly higher litter incidence of early resorptions and pre-implantation loss in this group when compared to controls. No statistical significance was reached and all values in the treatment group remained within the historical control range. As no dose-related response was observed, these changes were not considered to be test item-related.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment up to 1000 mg/kg.
Only one external malformation occurred in this study. A trunk omphalocele was observed in fetus from litter of a dam treated with 100 mg/kg. The single occurrence of this malformation in the low-dose group did not indicate a relationship to treatment.
External variations were not observed 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 1000 mg/kg.
Only one fetus (A038-06) at 100 mg/kg was observed with skeletal malformations; sternoschisis and rib anomaly. Due to the single occurrence of these malformations in one single fetus of the lowest dose group, they were considered to be unrelated to treatment.
Skeletal variations occurred at an incidence of 66.4%, 76.9%, 71.0% and 58.6% per litter in the control, 100, 300 and 1000 mg/kg groups, respectively.
There was a statistically significantly lower litter incidence of bent ribs at 1000 mg/kg. Litter incidences were 14.3, 14.5, 7.7 and 1.1% in the control, 100, 300 and 1000 mg/kg groups, respectively. Since the other chondrodystrophy (namely scapula bent) parameters were not affected, and as a similar low litter incidence of bent ribs occasionally occur in untreated controls, this skeletal variation was considered not to be toxicologically relevant.
Also noteworthy was the higher incidence of 7th cervical ossification sites at 1000 mg/kg. Litter incidences were 3.3, 6.5, 2.9 and 10.5% in the control, 100, 300 and 1000 mg/kg groups, respectively. Although the mean percentage was outside the historical control data, no dose-relationship could be established, and since it is known that these cervical ossification sites will be incorporated into the ventral arch of the 7th cervical vertebra during skeletal development, the increased incidence of this finding in the highest dose group was not considered to be toxicologically relevant.
All other variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 1000 mg/kg.
One fetus at 100 mg/kg was noted with visceral malformations. This fetus (A028-8) was observed with a total situs inversus and the presence of one lung lobe at both sides. Due to the single occurrence of these malformations, and as these were also earlier noted in the current historical control data, these findings were considered not to be related to treatment.
During the soft tissue cephalic examination on fixed heads, two control fetuses (A006-05 and A018-04), two fetuses of the 100 mg/kg group (A030-05 and A040-05), one fetus of the 300 mg/kg group (A051-11) and two fetuses of the 1000 mg/kg group (A085-06 and A085-10) were observed with small eyes. Since this malformation was equally distributed in all groups, including controls, and as this malformation was earlier noted among the current historical control data, it was considered that the occurrence of small eyes was not treatment-related and, therefore, was considered to be a chance finding.
Visceral variations were observed in 6.2%, 3.1%, 2.1% and 3.9% of fetuses per litter in the control, 100, 300 and 1000 mg/kg groups, respectively. All variations (i.e. small supernumerary liver lobe and convoluted ureter) occurred in the absence of a dose-related incidence, infrequently and/or at frequencies that were within the range of available historical control data.
Other effects:
not examined

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

See attachment for summary result tables and individual animal results tables.

Applicant's summary and conclusion

Conclusions:
In the prenatal developmental toxicity study, conducted according to the appropriate OECD 414 Test Guideline and in compliance with GLP in the rat, the reported NOAEL for maternal toxicity was 300 mg/kg bw/day based on clinical effects of systemic toxicity. The reported NOAEL for developmental toxicity was greater than 1000 mg/kg bw/day based on no adverse effects observed.