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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jan 2018 to ADD WHEN AVAILABLE
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
whole-body inhalation route of exposure used. Justification for route of exposure was given.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 10–11 weeks old
- Weight at study initiation: between 208 and 250 g on Gestation Day 0
- Fasting period before study: none
- Housing: Animals were individually housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was provided ad libitum throughout the study, except during inhalation exposure periods.The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during inhalation exposure periods. Periodic analysis of the water was performed.
- Acclimation period: 5 days CONFIRM WHEN AVAILABLE

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 26°C
- Humidity (%): 30% to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Remarks:
The route of administration was whole body inhalation exposure because the intended use of the test substance indicates that this would be a likely route of human exposure.
Vehicle:
air
Remarks:
humidified, filtered
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted using 500-L glass and stainless steel whole body exposure chambers.
- Method of holding animals in test chamber: whole-body exposure
- Source and rate of air: Air supplied to the whole-body chambers was provided from an in-house compressed nitrogen source or a HEPA- and charcoal-filtered, temperature and humidity-controlled supply air source.
- Method of conditioning air: not applicable
- System of generating particulates/aerosols: not applicable. Vapors of hexamethyldisiloxane were generated using a stainless steel bead column-type vaporization system. The column for each test substance chamber was filled with various sized glass beads and heated using heat tape controlled using a temperature controller and J-type thermocouple. A pump equipped with a piston head was used to deliver liquid test substance from the reservoir to the top of the bead column. Nitrogen was metered to the bottom of the bead column at a 90° angle using a regulator and rotameter-type flowmeter. Vaporization occurred as the test substance flowed over the surface of the heated beads while the nitrogen flowed up through the column.
- Temperature, humidity, pressure in air chamber: Temperature and humidity wre 19°C to 25°C and 30% to 70%, respectively. Oxygen content of the exposure atmospheres was measured during the method development phase and was 20.9% for all groups.
- Air flow rate: not applicable
- Air change rate: not applicable
- Method of particle size determination: not applicable
- Treatment of exhaust air: All chamber exhaust passed through the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.

TEST ATMOSPHERE
- Brief description of analytical method used: Nominal exposure concentrations were calculated for all test substance exposure chambers. The nominal concentration was calculated based on the total amount of test substance consumed during the exposure (as weighed prior to and at the termination of the generation) and the total volume of air passed through the chamber during exposure. Total air volume was calculated by multiplying the daily mean ventilation rate by the duration of generation. Exposure atmospheres were sampled and analyzed at approximately 45-minute intervals using a gas chromatograph (GC) with flame ionization detection (FID). Samples were collected from the approximate animal-breathing zone of each exposure chamber via heated stainless steel tubing. Sampling and analyses were performed as follows. An external multi-position valve permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop, the chromatograph was displayed, and the area under the sample peak was calculated and stored. The ln-quadratic equation based on the GC calibration curve was used to calculate the measured concentration in ppm.
- Samples taken from breathing zone: yes (see above)

VEHICLE (if applicable)
- Justification for use and choice of vehicle: humidified, filtered air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure atmospheres were sampled and analyzed at approximately 45-minute intervals using a gas chromatograph (GC) with flame ionization detection (FID). Samples were collected from the approximate animal-breathing zone of each exposure chamber via heated stainless steel tubing. Sampling and analyses were performed as follows. An external multi-position valve permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop, the chromatograph was displayed, and the area under the sample peak was calculated and stored. The ln-quadratic equation based on the GC calibration curve was used to calculate the measured concentration in ppm.
Details on mating procedure:
TO BE ADDED WHEN FINAL REPORT AVAILABLE
Duration of treatment / exposure:
6 hours
Frequency of treatment:
daily
Duration of test:
from Gestation Day 6 to 20, inclusively
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm
Remarks:
target concentration
Dose / conc.:
1 000 ppm
Remarks:
target concentration
Dose / conc.:
3 000 ppm
Remarks:
target concentration
Dose / conc.:
131 ppm (nominal)
Dose / conc.:
1 225 ppm (nominal)
Dose / conc.:
3 533 ppm (nominal)
Dose / conc.:
99 ppm (analytical)
Dose / conc.:
1 007 ppm (analytical)
Dose / conc.:
2 964 ppm (analytical)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a previously conducted two-generation reproduction study in which rats were exposed via whole-body vapor inhalation to target concentrations of 0, 100, 400, 1600, or 5000 ppm. The NOAEL for neonatal toxicity was considered to be 1600 ppm due to decreased offspring weights at 5000 ppm. Unlike the acute studies, there are no defined limit concentrations in 28-day subacute inhalation toxicity studies. The maximum concentration tested should consider: 1) the maximum attainable concentration, 2) the “worst case” human exposure level, 3) the need to maintain an adequate oxygen supply, and/or 4) animal welfare considerations. In the absence of data-based limits, the acute limits of the United Nations Globally Harmonized System of Classification and Labelling of Chemicals may be used (i.e., up to a maximum concentration of 5 mg/L for aerosols, 20 mg/L for vapors and 20,000 ppm for gases). The limit concentration should elicit unequivocal toxicity without causing undue stress to the animals or affecting their longevity. Therefore, a high exposure level of 3000 ppm was selected for this study.
- 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: once daily, beginning on the day of receipt and lasting through Gestation Day 21

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Gestation Days 0 (by supplier), 5, and 6–21 (daily).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was quantitatively measured on Gestation Days 5–21 (daily).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Animals were subjected to a complete necropsy examination, which included evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

OTHER: ORGAN WEIGHTS
- Kidneys, liver and lungs were weighed at necropsy for all scheduled euthanasia animals.

TISSUE COLLECTION AND PRESERVATION
- Representative samples of the kidneys, liver, lungs and all gross lesions were collected from all animals and preserved in 10% neutral-buffered formalin.
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
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [one-half of the fetuses in each litter ]
- Skeletal examinations: Yes: [one-half of the fetuses in each litter ]
- Head examinations: Yes: [all per litter ]
Statistics:
Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Data obtained from nongravid animals were excluded from statistical analyses.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance treated group to the control group by sex.
Maternal body weights and body weight changes (absolute and net), and food consumption, gravid uterine weights, organ weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance treated groups to the control group. Mean litter proportions of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre and postimplantation loss, and fetal sex distribution) 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 nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.
Indices:
Not calculated

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No test substance related clinical observations were noted at the daily examinations or 1–2 hours postexposure at any dosage level. Findings noted in the test substance exposed groups, including red material around the nose and hair loss on forelimb(s) and hindlimb(s), occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females in the control, 100, 1000, and 3000 ppm groups survived to the scheduled necropsy on Gestation Day 21.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100, 1000, and 3000 ppm groups were unaffected by test substance exposure.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 1000, and 3000 ppm groups was unaffected by test substance exposure.
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
Description (incidence and severity):
There were no test substance-related effects on organ weights in any exposure group. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on Gestation Day 21, no test substance-related internal findings were observed at exposure concentrations of 100, 1000, and 3000 ppm. Macroscopic findings observed in the test substance exposed groups occurred infrequently and/or in a manner that was not dose related.
Neuropathological findings:
no effects observed
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
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
>= 3 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: (equivalent to 19923.93 mg/m³ based on MW of 162.38 )

Maternal abnormalities

Abnormalities:
no effects observed

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): no effects observed
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
An external malformation, curly tail, was noted in one fetus in the 3000 ppm group. This malformation was noted in a single fetus, and therefore was not considered test substance related. No external developmental variations were observed in fetuses in this study.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations were observed in fetuses in this study.
The mean litter proportion of 14th rudimentary rib(s) in the 3000 ppm group (34.9% per litter) was statistically significantly higher than the concurrent control group (5.0% per litter) and the maximum mean value in the Charles River Ashland historical control data (14.57% per litter). This difference was considered test substance-related; however supernumerary ribs, such as 14th rudimentary ribs, have been found to resolve during maturation in rats.In the absence of other fetal effects, it is believed that a 14th rudimentary rib would not be considered a toxicologic concern or that these extra ribs would affect the quality of life of these animals, and therefore was considered to be nonadverse.
No other test substance-related skeletal developmental variations were noted. Findings observed in the test substance treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
No visceral developmental malformations were observed in fetuses in this study. No test substance-related visceral developmental variations were noted.
The only finding noted, renal papilla(e) not developed and/or distended ureter(s) was noted similarly in the control group and the difference in the mean litter proportion was not were not statistically significant compared to the concurrent control group, and the value was within the range of the Charles River Ashland historical control data.
Renal papilla(e) not fully developed was noted for 2, 1, and 3 fetuses in the control, 1000, and 3000 ppm groups, respectively. Dark red contents in the thoracic cavity, surrounding the lungs was noted for one fetus in the control group. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test article-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose related.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
>= 3 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Remarks on result:
other: (equivalent to 19923.93 mg/m³ based on MW of 162.38 )

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

ADD TABLES WHEN AVAILABLE

Applicant's summary and conclusion

Conclusions:
In the prenatal developmental toxicity study, conducted according to the appropriate OECD test guideline and in compliance with GLP, the reported NOAEC for developmental toxicity was greater than the highest dose tested of 3000 ppm (equivalent to 19923.93 mg/m³ based on MW of 162.38 ).