Registration Dossier

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:
2019-03-12 to 2019-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS870.6300
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Clear liquid
Content: 98.32%
Specific details on test material used for the study:
Physical state (at 20ºC): Neat Liquid
Purity / composition: ≥98.0 to ≤ 99.6%
Molecular weight: 138.20 g/mol
Colour: Clear
Storage conditions: Room temperature (ca. 23ºC); protected from light
Expiry date: July, 2019
Handling conditions: Personnel will wear gloves and auto-ventilated equipment equipped with filters appropriate vapours
Other: Sensitive to air, highly volatile, odour of peppermint.
Avoid inhalation and contact with eyes and skin. Test item may cause irritation of respiratory tract, eyes and skin
The neat substance without solvent will be tested

Test animals

Species:
rat
Strain:
other: Fischer 344 (F344/HanZtn Rj)
Remarks:
Although the F 344 strain is normally discouraged in reproduction studies (OPPTS 870.6300), the authorities specificaly asked for their use in order to compare the results with previous non-GLP reproduction toxicity studies, in which this strain was used
Details on test animals or test system and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Sex: Female
- Strain: Fischer 344 (F344/HanZtn Rj)
- Source Fischer 344: Janvier, Route des ChénesSecs, Le Genest Saint-Isle, F-53940 France
- Age: 8 - 13 weeks on arrival; 11 - 12 weeks on first item exposure
- Animals per cage (before / after distribution): Up to 5 (1 after distribution and before mating)
- Bedding material: Sodispan (SR-CHOPO-T) (autoclavable)
- Diet: ad libitum, Global diet, 2914C, Envigo Teklad
- Water: ad libitum, Tap water
- Acclimatisation period: at least 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 - 24.6° C
- Humidity (%): 28 - 61 %
- Light cycle: 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Remarks:
Sham filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Preparation of the exposure system
Previous to exposure onset, a complete setup of the system and flow/pressure characterization was performed according to the corresponding internal SOPs. The tasks were the following:
- Make an outline of each set up including identification of the corresponding equipment.
- Leak test through the atmosphere conveyor glass tubes.
- Identification of each group (name, dose, total number of animals, total flow of vapour/air).
- Check air temperature and relative humidity.
- Theoretical calculations regarding dilution.
- Check vacuum system in sampling ports: capillary installed, vacuum pressure measurement, sampling flow.
- Exposure test: determination of pressure inside the inhalation chamber versus room pressure and difference of pressure between both chamber compartments. Determination of atmosphere flow and vapour concentration at different positions of the chambers was obtained to confirm a homogenous distribution of the vapour.
- Six ports from the high level, six ports from the mid level and six ports from the low level. Calculation of coefficient of variation (%CV) within each level and across the three levels. Within each level, the CV was not higher than 5%, whereas the CV among the three levels will not exceed 10%.


Test item generation
- A vapour was generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).
- The test item was inside the generator vessel in liquid state.
- It was heated at a controlled temperature by means of a water bath equipped with a temperature probe to generate a vapour phase.
- At the same time, a source of compressed air at a controlled temperature was delivered into the VOC generator. This compressed air was mixed with the vapour phase of test item extracting the formulation of the product in a controlled amount of air (L/min) and concentration.
- The vapour was conveyed via glass tubing from the generator to the exposure chambers.
- The flow rate through the exposure chamber was adjusted as necessary.
- In order to prevent exposure of the animals to test item droplets from condensation, a filter was placed immediately before the exposure chamber.
- For sham exposure, filtered, compressed fresh air was provided to the exposure chamber.


Exposure conditions
- Inhalation exposure was performed using a flow-past, nose-only exposure system.
- The animals were confined separately in restraint glass tubes matching their size which are positioned radially around the exposure chamber.
- The exposure system ensures a uniform distribution and provides a constant flow of test material to each exposure tube.
- The flow of air at each tube was between 0.5 to 1.5 L/min, which was sufficient to minimize re-breathing of the test item vapour as it is more than twice the respiratory minute volume of rats.
- The position of the rats in each group within the exposure chamber was changed daily according to an insertion schedule.
- Sham-exposed rats were exposed to filtered, conditioned air, the exposure conditions being the same as those for the test item-exposed rats.
- Exposure chambers type EC-FPC-232 (anodized aluminium, volume inside compartment: approximately 3 L, equipped with glass exposure tubes) were used. The rats were individually exposed in glass tubes matching their size.
- Before starting the inhalation period, the animals were not adapted to the glass tubes to avoid pre-implantation losses due to the stress caused by the procedure itself.
- The temperature of the test atmosphere in the exposure chamber was maintained at 22 ± 5 °C and the relative humidity as required by experimental conditions.
- Air flow per animal was monitored regularly.
- No diet or drinking water was available during exposure.
- The time spent during animal shifting was not included within the exposure time.

Technical trials
- Technical trails were performed without animals and conducted before the animal phase of the study to establish the conditions for vapour generation and sampling.
- A vapour was generated for up to 6 hours for four days.
- During this period, the concentrations as well as the temperature and relative humidity of the chamber were monitored.
- The set-up conditions and results were similar to those obtained during the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Characterisation of test atmosphere
In order to characterize the test atmosphere and to check the reproducibility of aerosol generation, several parameters were determined at defined intervals.
- Determination of the nominal vapour concentration
The test item usage was determined daily by weighing the amount in the syringe pump and adjacent lines before and after each exposure to determine the quantity of test item used. The weight used was then be divided by the total air-flow volume to give the nominal concentration. The nominal concentration for different groups was calculated from the nominal concentration of high concentration chamber and the dilution factor for each group, respectively. These data were used for the purpose of monitoring the performance of the generation system.
- Analytical determination of the vapour concentration
Analytical determination of the vapour concentration was performed three times on each day of exposure for each test item group (groups B - D). Additional samples were analysed if considered necessary.
Vapour samples were captured in a solvent trap containing a defined volume of methanol (cooled with ice) positioned in an empty port of the inhalation chamber. Based on the results from the N-02130 study previously conducted at Vivotecnia, no additional solvent trap was necessary to place immediately after the first solvent trap, since it was demonstrated that all test item was expected to be captured during sampling. The sampling flow rate matched the air flow rate per port. The defined volume of methanol in the solvent trap was refilled to the start volume after sampling completion. Aliquots of samples of the resulting solution of test item in methanol were appropriately labelled and stored ca. 5°C until analysis.
Test item concentrations were analysed at Vivotecnia using an HPLC analytical method transferred by Swiss BioQuant AG during a previous study conducted at Vivotecnia. Samples of test item trapped in methanol were analysed within 3 days after sampling. The analytical method was validated at Vivotecnia in the study B-02791 .
Test item vapour concentration was determined three times a day on each day of exposure for each test item group by high performance liquid chromatography (HPLC) after collecting the vapour in a solvent trap containing 50 mL of methanol (cooled with ice) positioned in an empty port of the inhalation chamber. The analytical method was validated afterwards in Vivotecnia study B-02791. Sampling time was 5 minutes at a flow rate of approximately 1 L/min. Trap volume was determined before and after sampling in order to evaluate discrepancies in the final volume. Aliquots of the samples were analysed freshly immediately after the sampling process.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) to initiate the nightly mating period, until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day on which mating was detected was designated gestation day 0 (GD0).
Male rats of the same source and strain were used only for mating. These male rats were in the possession of Vivotecnia and were not considered part of the test system.
Duration of treatment / exposure:
Duration of dosing: Daily from day 5 post coitum (implantation, GD 05) to day 19 post coitum (GD 19)
Dosing regimen: 6h/day
Frequency of treatment:
6h/day
Duration of test:
Caesarean section on day 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Remarks:
Control group (Group A), Sham filtered air
Dose / conc.:
17 ppm (nominal)
Remarks:
Low dose group (Group B)
Dose / conc.:
53 ppm (nominal)
Remarks:
Mid dose group (Group C)
Dose / conc.:
150 ppm (nominal)
Remarks:
High dose group (Group D)
Dose / conc.:
0 ppm (nominal)
Remarks:
Control group (Group E), Pregnancy reference group
No. of animals per sex per dose:
24 pregnant female rats per dose
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
The present study was designed to assess the potential effects of the test item 3,5,5-trimethylcyclohex-2-enone (CAS 78-59-1) on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration through the inhalation route (nose-only) from day GD 05 to GD 19 (the day prior to Caesarean section). This study has helped to demonstrate any dose-related response and to establish the no-observed adverse effect level (NOAEL).
For this purpose, 120 pregnant female rats were distributed into 5 different experimental groups (A to E). Group E was a pregnancy reference group not exposed. Animals were exposed to diluted vapour from the test item (groups B to D) or to filtered compressed fresh air (Group A) 6 hours/day from GD05 to GD19 (the day prior to Caesarean section).
All animals were observed once daily for mortality and clinical observations. Body weight and food consumption were recorded at appropriate intervals. On gestational day 20, a caesarean section was performed on each female and selected organ were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Rationale for target concentration selection: T
he target concentrations were selected by the sponsor according to two previous dose range finding prenatal development toxicity studies (N-02669, VVT 2019 and N-02130, VVT 2016) available for this test item.
Within these dose range finding studies, mortality was observed at 1.25 mg/L (220 ppm), while exposure at 0.85 mg/L (150 ppm) did induce maternal toxicity (statistically significant body weight losses) in one of the dose range finding studies. Based on these results and due to the fact that the observed effects at 220 ppm shows a very steep dose-response curve of the test substance, 150 ppm was chosen as highest dose concentration for the OECD 414 main study.
This concentration level is slightly above 144 ppm, which was requested by ECHA in order to compare the results with the previous inhalation teratogenicity studies (Exxon, 1984 and Exxon, 1983). In these prior studies, isolated instances of exencephaly were noted in rat and mice foetuses at 144 ppm.

Inclusion criteria: All animals were subjected to individual veterinary examinations. Only animals in good health and with pregnancy status confirmed were included in the study
Stratification method: Animals were randomly distributed by means of the body weight stratification method at least 2 days before first administration

- Determination of the temperature
The temperature in the chamber was measured hourly during exposure using a thermohygrometer. The target range is 22±3ºC.
- Determination of the relative humidity
The relative humidity (%) in the vapour was measured hourly in each group during each day of exposure using a thermohygrometer.
- Determination of oxygen and CO2 concentration
The oxygen concentration of the test atmosphere was measured hourly during each day of exposure using an appropriate device. The oxygen and CO2 concentrations were maintained above 19% and below 1% respectively, during the exposure period.
- Airflow rate
The exposure airflow rate was adjusted as appropriate before the start of the exposure. The actual airflow rate was monitored hourly in each group during each exposure. Additional measurements were performed if considered necessary.

Examinations

Maternal examinations:
Experimental data concerning, body weight, food consumption, general clinical signs, thyroid weight, gross necropsy findings (dams) and ovaries and uteri examination were recorded through Provantis 8.5.2.1TM (Instem LSS Ltd., Staffordshire, United Kingdom) preclinical software.

CAGE SIDE OBSERVATIONS: Yes
The animals were examined once daily by cage side observation for mortality and morbidity. Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.
Where the condition of an animal required premature sacrifice, it was euthanized by a sodium pentobarbital overdose. Any decision regarding premature sacrifice were made after consultation with the Study Director (or a nominated person) and with the study monitor, if possible.

DETAILED CLINICAL OBSERVATIONS: Yes
On exposure days, clinical observations were recorded before exposure, just after exposure and 1h after exposure. Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.
Any visible clinical signs, discomfort and mortality was recorded in accordance with the humane endpoints guidance document of the OECD (ENV/JM/MONO(2000)7). Observations included changes in skin, eyes, and mucous membranes. Alterations in respiratory pattern or behaviour as well as changes in posture and response to handling and the presence of abnormal movements were also recorded.

BODY WEIGHT: Yes
Body weight were recorded on gestation day 0 (GD0), at least every 3 days and on GD20. In addition, body weight after sacrifice was also recorded.
Additional body weights were obtained as necessary for animal welfare reasons.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food was weighed on GD 0, 3, 5, 8, 11, 14, 17 and 20. Food consumption was calculated on these defined intervals by cage, estimated the daily food intake for each animal.
Data was presented in terms of consumption per animal per day for the periods (days 0 – 3), (3 – 5), (5 – 8), (8 – 11), (11 – 14), (14 – 17) and (17 – 20).

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

POST-MORTEM EXAMINATIONS: Yes
- Gross necropsy
At the scheduled necropsy (GD20) females were sacrificed by CO2 asphyxiation and the foetuses were removed by caesarean section. For all animals the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs examined. Organs or tissues showing any macroscopic abnormalities were removed and fixed in appropriate fixative. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to accentuate possible haemorrhagic areas of implantation sites and position of foetuses in the uterus.
- Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight were recorded. From all dams, the weight of the thyroid gland was recorded and preserved in 4 % neutral-buffered formaldehyde for histopathological assessment. Weight of gravid uteri including the cervix was recorded.
Any organ with gross lesions was collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant.
Additionally, lungs from all dams will be instilled via trachea with formalin at approximately 30 cm H2O pressure collected and fixed in neutral phosphate buffered 4% formaldehyde solution for possible further examinations.

- Endocrine disrupter relevant endpoints
Concentration of Thyroid hormones, including T4, T3 and thyroid-stimulating hormone (TSH), were measured by the appropriate analytical method from all animals. Blood samples were collected from the abdominal aorta at the end of the study in non-treated tubes and serum samples were processed following the appropriate SOP.
Serum samples of all animals were examined at the end of treatment for an evaluation of test item-related effects on thyroid hormones at BSL BIOSERVICE Scientific Laboratories Munich GmbH (delegated phase). After arrival of serum tubes (a total of 360 tubes = 3 aliquots per animal and sampling a two-times 30µl and one-time 100µl) shipped on dry ice, tubes was stored at < -70°C.
Parameter Units
T3 pg/mL or ng/mL
T4 pg/mL or ng/mL
TSH pg/mL or ng/mL
Hormone determination was performed on the MagPIX, Luminex or on the DRG:HYBRID-XL Analyzer, DRG.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
For pregnant females the following observations were made:

- Number of corpora lutea
- Number and distribution of implantations in each uterine horn, classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses. The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix.
- Gravid uterus weight: The uteri (and contents) of all females with live foetuses were weighed during necropsy on day GD 20 to enable the calculation of the corrected body weight

- Placental weight
The live foetuses and their placentae were removed.


Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight were recorded. From all dams, the weight of the thyroid gland was recorded and preserved in 4 % neutral-buffered formaldehyde for histopathological assessment. Weight of gravid uteri including the cervix was recorded.
Fetal examinations:
Foetal Pathology
Foetuses were removed from the uterus by Caesarean section (C-section), sexed, weighed individually and examined for gross external abnormalities. External examination of foetuses was performed at Vivotecnia. The anogenital distance (AGD) was measured in all live rodent foetus. After external examination, foetuses were assigned with a number, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique)]. Approximately one half of the foetuses (foetuses with odd numbers) from each litter will be fixed in Bouin’s fixative. Then foetuses were sent in the same fixative medium to the test site AnaPath GmbH within 3 weeks. At the test site the foetuses were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissues were preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol (ca. 94%), foetuses were sent in the same fixative medium to the test site AnaPath GmbH for further skeletal evaluation. Carcasses were processed through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined, and all abnormal findings and variations were recorded. The assessment included but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.
Foetuses with abnormalities were photographed, when considered appropriate.

The foetal pathology phase report is attached to this endpoint study record.

Terminology Used in the Assessment of the Data
Term Description
Empty Implantation Site: Very early resorption or aborted implantation
Early Resorption: Amorphous mass being resorbed
Late Resorption: Clearly defined foetal body being resorbed
Dead Foetus: Appearance of live foetus at C-section but without induced respiration or movement
Live Foetus: Breathing and/or moving foetus
Malformation: A structural change in a foetus that would probably impair its health or development
Variation: A foetal change that is unlikely to adversely affect survival or health. This includes a delay in growth or morphogenesis that has otherwise followed a normal pattern of development


Calculations
Comparison Group A and E, against Groups B, C and D.
Mean foetal body weights were calculated separately by sex for each litter; group mean body weights were calculated (separately by sex) from the litter means.
The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages were calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.

Pre-implantation loss (%) = (# of corpora lutea –# of implantation sites) x 100/ no. corpora lutea

Post-implantation loss (%) = (# of implantation sites – # of live foetuses) x 100/ #. of implantation sites

Statistics:
Provided that the sample size was considered adequate, the following parameters will be subject to statistical analysis:
- Body weight
- Body weight change
- Gravid uterine weight
- Body weight change corrected for gravid uterine weight
- Food consumption
- Absolute and relative organ weights
- T3, T4 and TSH levels
- Anogenital distance statistically evaluated by gender

Analysis of data from two groups:
Test of normal distribution Yes Un-paired t-test
No Mann-Whitney test
Test of equal variance No Un-paired t-test with Welch’s correction

Analysis of data from more than two groups
Test of normal distribution Yes One-way ANOVA Dunnett’s post test (comparison versus control group)
Tukey post test (comparison of all groups)
No Kruska-Wallis Dunn's post test
Test of equal variance No Kruska-Wallis Dunn’s post test
A value of P < 0.05 will be considered statistically significant
Indices:
Fertility Index, Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter
Historical control data:
Although the Fisher 344 strain is normally discouraged in reproduction studies (OPPTS 870.6300), the authorities specifically asked for their use in both the DRF and main studies in order to compare the results with previous non-GLP reproduction toxicity studies, in which the Fischer 344 strain was used.
Due to the very limited number of historical control data on Fischer 344 rats this OECD 414 study was performed using two control groups and three dose-level groups in order to receive scientifically meaningful results.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The most frequent clinical signs present in animals belonging to all experimental groups, including sham, were chromorrhinorrea, chromodacryorrhea and wet fur. These signs are commonly observed in nose-only inhalation studies and, since it was equally present in the control group, it can be considered not to be caused by the test item. In addition, it was observed loss of stability at the beginning of the studio in animals exposed to the high dose, however this sign disappeared after the first days of exposure.
Additionally, incidental bleeding through the vagina was observed from days 12 to 16 of gestation in animals belonging to groups A, B, C and D. Even though the number of females with occasional bleeding was higher in the high dose group, these signs were single observations (< 2 and not in consecutive days) and there was not a dose-dependent relationship. Therefore, these observations were considered incidental and probably not test item related.
Mortality:
no mortality observed
Description (incidence):
None of the animals died during exposure. All animals survived until the schedule necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The exposure regimen resulted in only transient reductions in maternal weight in group D,151 ppm (approximately 3.5% reduced from Control Group A and 12.3% reduced from Pregnancy Reference Group E) on day 17 of Gestation.
No statistically significant differences were found in mean body weight gain among the four experimental groups neither during the exposure period nor at the end of the study. Body weight gain was reduced at GD 14-17, but this difference was not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was decreased between the days 5 and 8 of the exposure period in Group D (151 ppm) when compared to Group Control A (up to approximately 15%) and Pregnancy Reference Group E (30.9%), p<0.01. Even though it cannot be ruled out a test item effect on food consumption at the beginning of the study, after these transient reductions there were a similar consumption among the five experimental groups (no significant differences) at the end of the study. Therefore, the variation on food consumption at the beginning of the exposure was considered incidental and devoid of any toxicology effect.
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):
No statistical differences were recorded neither in terminal body weight nor in gravid uterus weight when compare the Group Control A with the exposed groups. The adjusted body weight to gravid uterus was also similar among exposed groups at concentrations of 18, 55 and 151 ppm, respectively. However, when compared Group E pregnancy reference group with both Control A and exposed Groups, there were some statistically significant increased. Nevertheless, these differences were considered to be related with the procedure itself rather than with the exposure.
Uterine weight in Group C (55 ppm) and D (151 ppm) was reduced (96% of the control group value) but this reduction was not considered adverse because the differences were slight, within normal animal variation and there were no statistically significant differences between the treated groups and control group.

Absolute and relative organ weights of thyroid gland were similar among all experimental groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No relevant macroscopic abnormalities related to the exposure to the test item were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Under the conditions of this study all animals survived their scheduled period and there were no necropsy findings or histology changes in the thyroid glands that could be related to the treatment with the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Endocrine disrupter relevant endpoints
The serum levels of total T3 were elevated in dosed groups C and D (exposed to 55 and 151 ppm 0.86 mg/L of test Item) when compared to Control Group A. This elevation was not due to enhanced T4 to T3 conversion. In addition, when compared with Reference Group E, only difference with Group D became significant. Individual data showed a great variability between animals from the same group. Taking into consideration that not differences were observed in TSH and T4 levels, the values were considered incidental findings and devoid of toxicological significance or not treatment-related. When compared with historic control data from BSL Bioservice, thyroid hormone levels were within the normal range.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in number of corpora lutea, implantation sites or litter size.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A slight increase on post-implantation loss (not statistically significant in any case) was recorded for dams of groups C (55 ppm) and D (151 ppm). In addition, 2 dams from group D (151 ppm) presented total resorption. These differences were not significant and were considered not test item related. In addition, the post-implantation losses (%) where within the observed range (6.86%-18.46%) from vivotecnia historical control data in Fischer 344 sham filtered exposed.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
A total of two pregnant dams with total resorption were recorded in the high dose group (151 ppm ).
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
A total of two pregnant dams with total resorption were recorded in the high dose group (151 ppm ).
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no significant decrease in the number of foetuses alive in any of the dose groups.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Caesarean section (C-section) on day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
From the 120 dams enrolled in the study, 117 were pregnant.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 151 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: no adverse and statistically significant maternal toxicity was observed, in addition no differences on reproductive parameters were observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were some marginal differences of the exposures on foetal weight at GD 20 post coitum between control group A and exposed groups B (18 ppm) and C (55 ppm). The very marginal (3-5%) difference while achieving statistical significance in total foetal, male and female weights, was considered to be within the normal biological variation and therefore, devoid of toxicological significance or not treatment-related. On the other hand, the statistical difference in foetal body weight when compared to controls at the high dose-level showed a decrease of 13.6%, consequently a test item effect on foetal weight in the high dose group D cannot be excluded.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of foetuses alive were similar among dose groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentage of male foetuses were also similar between all dose groups.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
No statistical differences were found in litter weight, although a decreased of 9% was observed in group D (151 ppm) compared with Control group A.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related findings were noted during external examination of foetuses at any exposure level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No malformation was observed during the skeletal examination at any dose level and no test item-related findings were noted.
The alterations observed during the skeletal examination were assigned to
• bone ossification stage/supernumerary ribs, which were related to a change from the expected ossification state of rat bones in this stage.
• bone or cartilage variations, which were used to describe structural changes (as changes in shape or size).
The type and frequencies of recorded variations were similar at all dose levels, in the control groups, group A (Sham filtered air) and group E (pregnancy reference).

- Ossification stage/supernumerary ribs
Ossification stage of foetal skeleton and number of supernumerary ribs showed no indication of any test item-related effect at any dose level.
At dose levels of 55 ppm (group C) and 151 ppm (group D), a higher number of incomplete ossified supraoccipital bone was observed when calculated either on a foetus or a litter basis. At dose levels of 18 ppm and 151 ppm a higher number of non-ossified caudal vertebrae was observed when calculated either on a foetus or a litter basis. For both alteration no dose dependency was noted, therefore these findings were considered not to be test item related but a result of biological variability.

- Bone and cartilage variations:
Bone and cartilage variations showed no indication of any test item-related effect at any dose level.
Among bone and cartilage variations, an increase in the incidence of sternebrae offset ossification sites was noted at 18 ppm and 55 ppm when compared to the incidence in pregnancy reference (group E) (20% and 22% respectively, versus 4% in the pregnancy reference group). Whereas, no significant difference was observed when compared to the concurrent control (Sham filtered air). Furthermore, this finding was noted only in 2% of foetuses of group D (151 ppm) when compared to the concurrent control (Sham filtered air), as no dose-dependency was noted, this finding was considered to be incidental and not test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During visceral examination of the foetuses, findings were noted in:

33% examined foetuses in 78% litters in Sham filtered air (Group A)
41% examined foetuses in 88% litters in 3,5,5-trimethylcyclohex-2-enone (Group B,18 ppm)
35% examined foetuses in 96% litters in 3,5,5-trimethylcyclohex-2-enone ( Group C, 55 ppm)
35% examined foetuses in 75% litters in 3,5,5-trimethylcyclohex-2-enone ( Group D 151 ppm)
53% examined foetuses in 96% litters in Pregnancy Reference (Group E)

No test item related findings were noted in any treated group.

Malformation such as renal pelvis dilated severe was observed in group B (litter 31, foetus 356) in one of 143 foetuses examined. Since this alteration occurred only in one foetus, it was considered to be incidental and not test item related.
The overall incidences of foetuses or litters with any remaining finding were isolated incidences and did not indicate any test item effect. Thus, they were considered not to be test item-related. The incidence of one single variations in form of thymus long and testis malpositioned were increased on foetal and litter basis when compared to group A (Sham filtered air) and they were comparable to those observed in group E (pregnancy reference). These specific findings were known to be normally associated with a slight immaturity of development; therefore, they were considered to be not related to treatment.
Other effects:
no effects observed
Description (incidence and severity):
No differences were observed in anogenital distance for both females and males.
Details on embryotoxic / teratogenic effects:
Taking into account the test item effect on foetal body weight, a NOAEL for embryo-foetal effects was established at 55 ppm (0.31 mg/L). However, under the conditions of the study the test item did not reveal any teratogenic potential up to and including the highest dose level (NOAEL, teratogenic > 151 ppm).

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 151 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Dose descriptor:
NOAEL
Effect level:
55 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
Under the condition of this inhalation OECD 414 study in Fischer F344 rats the test item did not reveal any teratogenic potential up to and including the highest dose level of 151 ppm (0.86 mg/L).

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Summary Foetal External examinations

No treatment-related findings were noted during external examination of foetuses at any exposure level.

The table that summarizes the macroscopic findings observed at necropsy (only foetuses with findings are included in the table) is attached to this endpoint study record.

Inhalation Technical Data: Tables see attached document to this endpoint study record

In-Life Data: Tables: see attached document to this endpoint study record

Test atmosphere related parameters

 Test item concentration in vapour

Throughout the study period, the variation of the mean test item concentrations with respect to the target values was within the ± 3% and the Coefficient of Variation (CV) on study means was 8%, 10% and 7% in groups B, C and D, respectively.

The deviations from target concentration on the daily individual atmosphere determinations in Groups B, C and D were <± 20% accepted criteria according the OECD guidelines except for 2 not consecutive days, in group C, when deviation from target was always < 22%.

Therefore, even though the deviations from target concentration on the daily individual atmosphere determinations were punctually above the ± 20% accepted criteria (according the OECD guidelines), the results obtained did not impact the objective of the study.

Summary of the mean test item concentrations (mg/L air) in test atmosphere:

Group

Exposure

Target test item conc.

Test item vapour concentration (mg/L air)

(mg/L air)

Mean

SD

RDS (%)

% Deviation from target

A

Air

-

-

-

-

-

B

Low dose

0.1

0.10

0.01

8

2

C

Mid dose

0.3

0.31

0.03

10

3

D

High dose

0.85

0.86

0.06

7

1

 

Nominal concentration

The nominal concentration was calculated from the daily consumed test item and the air flow feeding the vapour generator and thus it was determined for the overall inhalation system and for each exposure concentration. Overall, this value is within the expected range for this type of atmosphere generation, especially taking into account the actual concentrations determined at the representative sampling ports for every group.

 

Temperature and relative humidity

Mean values for exposure temperature were between 21.78 and 21.55 °C for all groups. Mean relative humidity was in range 6.25 - 7.23 % for the different groups. These values are below the target range for husbrandy. The difference was considered due to technical reasons. However, it was considered not to have affected the outcome of the study.

   

Oxygen and carbon dioxide (CO2)

Mean oxygen concentration was 20.9 % for all groups. Carbon dioxide was always < 1 % (mean concentration of 0.04 % for all groups).

Flow per animal

The flow rate per animal was between 0.5 and 1.5 L/min, which is sufficient to avoid re-breathing as it was more than twice the minute volume of rats.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the inhalation OECD 414 study with Fischer rats a no-observed-adverse-effect level (NOAEL) for maternal effects may be established at 151 ppm (0.86 mg/L).
The test item did not reveal any teratogenic potential up to and including the dose level of 151 ppm (0.86 mg/L) and therefore, the NOAEL for developmental effects was determined to be >= 151 ppm (0.86 mg/L). As an effect on foetal body weight at 151 ppm (0.86 mg/L), a NOAEL for embryo-fetal effects was established at 55 ppm (0.31 mg/L).
Executive summary:

Introduction and study outline

The objective of this developmental toxicity study was to assess the potential effects of the test item on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration of the test item through the inhalation route (nose-only) during the period of organogenesis, from gestation day GD05 to day GD 19 (the day prior to Caesarean section).

Therefore, this study aimed to characterize maternal and foetal toxicity at the exposure levels tested and todetermine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

 

For this purpose, 120 pregnant female rats were distributed into 5 different experimental groups (A to E). Group E was a pregnancy reference group not exposed. Group Ewas included to be able to identify the possible findings related with the inhalation procedure itself, as the stress of the procedure could affect the pregnancy outcome and related variables. Therefore, differences between group E and groups A, B, C and D are just related with the procedure itself and not with the test item exposure. Furthermore, Group E served as second control group. Animals were exposed to diluted vapour from the test item (groups B to D) at nominal concentrations of 17, 53 and 150 ppm (0.1, 0.3 and 0.85 mg/L air, respectively), respectively, or to filtered compressed fresh air (Group A) 6 hours/day from GD05 to GD19 (the day prior to Caesarean section).

The vapourwas generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).

In order to characterize the test atmosphere and to check the reproducibility of the vapour generation and dilution, the following analytical parameters were determined at defined intervals: test item concentrations, temperature, relative humidity as well as oxygen and carbon dioxide concentrations through exposure chamber.

 

All animals were observed three times daily for mortality and clinical observations before, just after and within 1 hour after exposure. Body weight and food consumption were recorded at appropriate intervals.

On gestational day 20, a caesarean section was performed on each female. The animals were subjected to a macroscopic postmortem evaluation and corpora lutea/implantation data were recorded and the gravid uterus and thyroid gland were weighed. The foetuses were examined for externally visible abnormalities, sexed and weighed. Approximately one-half of the fetuses (alternating fetuses) in each litter were examined for soft-tissue abnormalities using a microdissection procedure. The other half of the fetuses were then stained with Alizarin Red S and examined for skeletal abnormalities and ossification variations.

 

Results

The composition and characteristics of test atmospheres were stable throughout the study period in Groups A, B, C and D. The mean concentrations obtained deviated 3% from the respective target. The overall mean (± standard deviation) analytical exposure concentrations for the respective exposure groups were as follows: 0.10 ± 0.1, 0.31 ± 0.031, and 0.86 ± 0.05 mg/L corresponding to 18, 55 and 151 ppm.

Maternal toxicity

None of the animals died during exposure.

The most frequent clinical signs present in animals belonging to all experimental groups, including sham, were chromorrhinorrea, chromodacryorrhea and wet fur. These signs are commonly observed in nose-only inhalation studies and, since it was equally present in the control group, it can be considered not to be caused by the test item. In addition, it was observed loss of stability at the beginning of the study in animals exposed to the high dose, however this sign disappeared after the first days of exposure.

The exposure regimen resulted in only transient reductions in maternal weight in high dose group D (approximately 3.5% reduced from Control Group A and 12.3% reduced from Pregnancy Reference Group E).Body weight gain was also reducedat GD 14-17, but this difference was not statistically significant. Food consumption was decreased at the beginning of the exposure in Group D (151 ppm) when compared to Group Control A (up to approximately 15%) and Pregnancy Reference Group (30.9%) between the day 5 and 8 of the exposure period. However, the Pregnancy Reference Group E, had a significantly increased in food consumption when compare with all the exposed groups including sham filtered control group A. Consequently, the difference with group E were considered to be related with the exposure procedure and not with the test item.

The Reference Pregnancy Group when compared with the test item exposure groups, showed differences in Body weight, Bodyweight gain and food consumption, however there were also differences when compare the Control Group A with the Pregnancy Reference Group E. Therefore, it was considered that the differences in Body weight, Bodyweight gain and food consumption with group E were related with the stress of the procedure itself and not with the exposure to the test item.

The serum levels of total T3 were elevated in dams from groups C and D (exposed to 55 and 151 ppm of test Item), this elevation was not due to enhanced T4 to T3 conversion. In addition, no differences were observed in thyroid weight and after the histopathology evaluation and when compare with historic control data from BSL Bioservice the T3 levels were within the normal range.

No relevant necropsy findings related to the test item exposure were recorded for the dams. There were no effects of the exposure on the pregnancies and/or gravid uterus weight. There were no statistically significant differences in pre- and post-implantation losses, even though, post-implantation loss (%) was higher for mid and high-dose group compared to control group, where a 2-fold increase was recorded for dams of group D (high dose, 151 ppm) compared to control group. These differences were not significant and were considered not test item related. In addition, two dams from group D had complete resorption which increased the total percentage of post-implantation losses for the group. In addition, the post-implantation losses (%) where within the observed range (6.86%-18.46%) in Fischer 344 sham filtered exposed from vivotecnia historical control data.

Foetal toxicity

There was no foetal death recorded during the study and the sex ratio for live fetuses were unaffected by treatment.

The number of total and live foetuses was similar among all groups. With regards to litter size, it was similar in all the groups. Even though litter weight was smaller in the group D (151 ppm), approximately 9% smaller than the control group A, this difference was not statistically significant. On the other hand, Fetal body weight per litter (male, female, or total) was significantly reduced in all the exposed groups, these differences were below 5% for groups B (18 ppm) and C (55 ppm) and considered devoid of any toxicological significance, but a reduction of 13.6% on foetal body weight, when exposed at 151 ppm (Group D), was both statistically and biologically significant.

Finally, no external, visceral or skeletal abnormalities or variations were noted, and it was concluded that no test item related findings were noted in any treated group, neither during skeletal examination, nor during visceral examination. Therefore, the test item did not reveal any teratogenic potential up to and including the dose level of 151 ppm (0.86 mg/L). As an effect on foetal body weight at 151 ppm (0.86 mg/L), a NOAEL for embryo-foetal effects was established at 55 ppm (0.31 mg/L).

Conclusion

    

-   No adverse and statistically significant maternal toxicity was observed during the study, in addition no differences on reproductive parameters were observed. Only transient reductions in maternal weight gain on GD17 and food consumption between day 5 and 8 of gestation and an incidental increased on T3 serum levels. It was considered that a no-observed-adverse-effect level (NOAEL) for maternal effects may be established at 151 ppm (0.86 mg/L).

 

-        There was no effect of the exposures at any level on pregnancy outcome, in terms of pre- or post-implantation loss or the incidence of fetal abnormalities and variants. Therefore, under the conditions of this study, the test item did not reveal teratogenic potential up to and including the dose level and the NOAEL for developmental effects was determined to be >= 151 ppm (0.86 mg/L).

Nevertheless, taking into account the test item effect on foetal body weight, a NOAEL for embryo-foetal effects was established at 55 ppm (0.31 mg/L).