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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:
31 October 2019 to 6 November 2019
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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF, Guideline 2-1-18, Teratogenicity Study
Version / remarks:
2000
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:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (CRL) (Raleigh, North Carolina)
- Age at study initiation: Sexually mature adults.
- Weight at study initiation: 200-246 g
- Fasting period before study: No
- Housing: Upon arrival and after assignment, animals were housed one per cage in plastic cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated Lixit ® valve-type watering system. Animals were housed in the same room; however, cages were contained in separate racks by dose group.
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 in meal form, ad libitum except during the six-hour exposure period
- Water (e.g. ad libitum): municipal water, ad libitum except during the six-hour exposure period
- Acclimation period: at least four days
- Feed and water analysis: There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study. Copies of these analyses are maintained in the study file.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-24°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Remarks:
Inhalation was selected as the route of administration since it is a potential route for human exposure.
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2 cubic meter stainless steel and glass Rochester-type inhalation whole-body exposure chambers [1.3 meters (m) x 1.2 m wide x 1.2 m deep with pyramidal top and bottom] under dynamic airflow conditions. The D5 exposure chambers were operated at a slightly negative pressure relative to the surrounding area. The control (0 ppm) chamber was operated at a pressure that was slightly positive to the surrounding area to minimize the potential for accidental exposure of the control animals to the test material in the case of fugitive emissions.
- Method of holding animals in test chamber: Test animals were housed singly in wire mesh cages to minimize crowding during the exposure.
- Source and rate of air: D5 vapors were generated using a glass J-tube method (Miller et al., 1980). Liquid test material was metered into the glass J-tube(s) assembly and vaporized by passing a preheated stream of nitrogen gas, approximately 30 - 50 litres per minute, through a bead bed in the glass J-tube. Glass wool filters were placed at the exit of the J-tubes and HEPA filters were placed at the inlet of each whole-body exposure chamber. The control chamber (filtered air) received the same volumetric flow of nitrogen as the exposure chambers. The nitrogen was heated with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. The generation system was electrically grounded and the J-tubes were changed as needed. Chamber airflow was maintained at rate of approximately 450-600 L/min, which was sufficient to provide the normal concentration of oxygen to the animals.
- Method of conditioning air: The vaporized D5 in nitrogen gas was rapidly diluted and mixed with filtered chamber supply air prior to passing through the HEPA filters and entering the chambers to achieve approximately 450-550 litres per minute total flow rate and the desired D5 chamber concentration. On the day of the first exposure, the chambers were monitored to ensure sufficient CO2 and O2 levels.
- System of generating particulates/aerosols: not applicable
- Temperature, humidity, pressure in air chamber: Chamber temperature and relative humidity were displayed and monitored continuously, and recorded approximately every hour using a resistance temperature device (RTD) and a humidity sensor (Omega HX94C, Omega Engineering Inc., Norwalk, Connecticut), respectively. Calibration of the temperature and relative humidity sensors were conducted pre-exposure. Chamber temperature and relative humidity were maintained at approximately 22 ± 2 °C and 40-60%, respectively.
- Air flow rate: Airflow through each exposure chamber was displayed and monitored continuously, and recorded approximately every hour using a calibrated differential pressure transducer (Model 264, Setra System, Boxborough, Massachusetts) coupled to a calibrated orifice plate.
- Air change rate: approximately 12 - 18 calculated air changes per hour.
- Method of particle size determination: not applicable
- Treatment of exhaust air: not applicable

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of D5 vapor were measured with a sampling probe located approximately in the centre of the chamber within the breathing zone of the animals and were determined at least twice per hour during each exposure period using an Agilent Gas Chromatograph 6890A with flame ionization detector (GC/FID). D5 peak area (height) was determined using integration by ChemStation software (Agilent Technologies, Inc., Palo Alto, California). The exposure chambers were monitored with a real-time laser aerosol monitor (Casella CEL-712 Microdust Pro, Casella Inc., Buffalo, NY) for the presence of aerosol throughout the study and the concentration of aerosol was measured at least three times per exposure day for the treated chambers and once/week for the control chamber. During the pre-inlife phase of the study, prior to exposure, the chambers were checked to ensure that a uniform distribution of D5 vapor was present from at least five sample points in the breathing zone of the animals. The GC/FID was calibrated, and a standard curve compiled pre-exposure using air standards of D5 vapor by vaporizing measured volumes of D5 vapor into Tedlar sampling bags (SKC, Eighty Four, Pennsylvania) along with metered volumes of HEPA-filtered room air. The analytical (D5) vapor concentration during each exposure was interpolated from the standard curve and was reported as the definitive exposure concentration. The analytical system was checked prior to and after each exposure with a D5 vapor standard of known concentration. The nominal concentration of test material in each chamber was calculated based on the amount of test material fed into the inhalation apparatus each day and the total chamber airflow through the chamber for each exposure period.
- Samples taken from breathing zone: Yes. Chamber concentrations of D5 vapor were measured with a sampling probe located approximately in the center of the chamber within the breathing zone of the animals and were determined at least twice per hour during each exposure period . During the pre-inlife phase of the study, prior to exposure, the chambers were checked to ensure that a uniform distribution of D5 vapor was present from at least five sample points in the breathing zone of the animals.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of D5 vapor were measured with a sampling probe located approximately in the centre of the chamber within the breathing zone of the animals and were determined at least twice per hour during each exposure period using an Agilent Gas Chromatograph 6890A with flame ionization detector (GC/FID).
Details on mating procedure:
- Impregnation procedure: Sexually mature virgin females were naturally mated with males of the same strain (one male:one female) at CRL
- M/F ratio per cage: one male:one female
- Length of cohabitation: Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males' cages.
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males' cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier, and maintained in the study file. Rats arrived in the test laboratory on GD 1 or 2.
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
7 days/week
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Filtered air, control group
Dose / conc.:
32 ppm (analytical)
Remarks:
32 ± 1.1 ppm (analytically-determined mean chamber concentrations over the course of the study)
Dose / conc.:
74 ppm (analytical)
Remarks:
74 ± 2.0 ppm (analytically-determined mean chamber concentrations over the course of the study)
Dose / conc.:
161 ppm (analytical)
Remarks:
161 ± 3.8 ppm (analytically-determined mean chamber concentrations over the course of the study)
Dose / conc.:
29 ppm (nominal)
Remarks:
29.1 ± 0.6 ppm (nominal, based on amount of test material fed into inhalation apparatus each day and total chamber airflow for exposure period); equivalent to 455 mg/m3
Dose / conc.:
81.2 ppm (nominal)
Remarks:
81.2 ± 2.1 ppm (nominal, based on amount of test material fed into inhalation apparatus each day and total chamber airflow for exposure period); equivalent to 1062 mg/m3
Dose / conc.:
169.7 ppm (nominal)
Remarks:
169.7 ± 5.6 ppm (nominal, based on amount of test material fed into inhalation apparatus each day and total chamber airflow for exposure period); equivalent to 2427 mg/m3
No. of animals per sex per dose:
24
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Exposure levels for this study were selected on the basis of the method development study (Bell and Hotchkiss, 2020) and the two-generation reproduction study (Stump, 1999). The high exposure concentration was the highest attainable, stable, vapor concentration without the presence of a significant amount of aerosol (~ 0.1 mg/m3) in the test atmosphere. The lower exposure levels were selected to provide exposure response data for any toxicity that may have been observed among the high exposure level rats.
- Rationale for animal assignment (if not random): randomised

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted twice daily, approximately at the same time each day.
- Cage side observations checked: significant clinical abnormalities that are clearly visible upon a limited examination, and to monitor the general health of the animals. Significant abnormalities that could have been observed included, but were not limited to: decreased/increased activity, repetitive behaviour, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency, and faecal/urine quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Upon arrival, clinical observations were conducted on all animals at least once daily at approximately the same time each day (prior to exposure period). Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, faeces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behaviour, injuries or palpable mass/swellings.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 0 by the supplier, on GD 3, and prior to exposure on GD 6, 9, 12, 15, 18, and prior to necropsy (GD 21). Statistical analysis of body weights was performed using data collected on GD 0, 3, 6, 9, 12, 15, 18, and 21. Statistical analysis of body weight gains was conducted for the following intervals: GD 0-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6-21, and 0-21.

FOOD CONSUMPTION: Yes
The amount of feed consumed was recorded and statistically analyzed for all animals every 3 days from GD 3-21 by weighing feed containers at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed container - final weight of feed container) / (# of days in measurement cycle)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. The liver and kidneys were dissected from the carcass and were incised. The thyroid with parathyroids were also dissected from the carcass. Any obvious gross pathologic alterations were recorded, and the weight of the liver, kidneys, thyroid with parathyroids (weighed after fixation) and gravid uterus (the gravid uterus for dam #341 was inadvertently not recorded) were recorded. The ratios of organ weights to terminal body weight were calculated (only absolute weight for gravid uterus). The thyroid with parathyroids and representative portions of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of liver, kidneys, and gross lesions were not conducted. Transponders were removed and saved with the preserved tissues.

OTHER:
Serum for Thyroid Hormone: Blood samples were obtained from the orbital sinus following anaesthesia with a mixture of isoflurane vapours and medical oxygen at the scheduled necropsy. Terminal blood samples were collected from all females. Approximately 2.5 mL of blood was collected for hormone analysis (T3, T4, and TSH) in tubes with no anticoagulant, and allowed to clot at room temperature for ~ 30 minutes. Analyses of blood for T3, T4, and TSH was performed from all pregnant animals on study (n=95 animals; all dams were pregnant with the exception of one animal (#420) in the 161 ppm (analytical) group.). Histopathological examination of the thyroid was conducted on all pregnant rats.
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 fetuses were given an external examination which included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail.
- Soft tissue examinations: Yes: At least one half of all the fetuses in each litter were chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations. The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder, and reproductive organs.
- Skeletal examinations: Yes: The second half of all the fetuses in each litter not selected for visceral examination were skinned, eviscerated, preserved in alcohol, and double stained with Alcian Blue and Alizarin Red S for cartilage and bone examination. A thorough evaluation of the fetal skeleton was conducted on the stained fetuses with the exception of one fetus from dam #421 that could not be evaluated for a skeletal examination due to an error during the fetal staining process.
- Head examinations: Yes: The heads of the fetuses used for visceral examinations were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages, and tongue.
- Anogenital distance: Anogenital distance (AGD; absolute and relative to the cube root of fetal body weight) was measured on all live fetuses prior to euthanasia using a digital calliper. The sequence of the data collection was conducted without knowledge of treatment group and counterbalanced across groups to the extent possible on each day to control for potential confounding influences of collection timing.
Statistics:
The litter, rather than the pup, was considered as the experimental unit. Maternal body weights, corrected by body weight, maternal body weight gains, organ weights, T3, T4, TSH, anogenital distance, fetal body weights and feed consumption were evaluated by Bartlett’s test (alpha = 0.01) for homogeneity of variance. Based on the outcome of Bartlett's test, a parametric or non-parametric analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05) or the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction were performed, respectively. Feed consumption values were excluded from analysis if the feed was spilled or scratched.
Frequency of pre- and post-implantation loss, and fetal alterations were analyzed using a censored Wilcoxon test with Bonferroni’s correction applied when the incidence was greater than 5%. The number of corpora lutea, implantations and litter size were evaluated using a non-parametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (alpha = 0.05) with Bonferroni’s correction. Fetal sex ratios - using a binomial distribution test. Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method (alpha = 0.02) and, if excluded, were excluded for sound scientific reasons. Dunnett’s test and Bonferroni’s correction corrected for multiple comparisons to the control and were reported at the corrected alpha level. Means and standard deviations were calculated for descriptive purposes for chamber concentration, chamber aerosol concentration, chamber temperature, humidity, and airflow.
Indices:
Not specified
Historical control data:
Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Examinations performed on all animals revealed no treatment-related findings.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant differences in the body weights, body weights corrected for gravid uterine weight, or body weight gains of any treated groups when compared to their respective controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant differences in the amount of feed consumed by any treated groups when compared to their respective controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Dams in the 161 ppm (analytical) group had a slight statistically significant increase in absolute (10.9%) and relative (9.3%) liver weights compared to controls. These were considered treatment related but non-adverse due to the small magnitude of change compared to controls. Kidney and thyroid gland weights for all treated groups and liver weights for the 32 and 74 ppm (analytical) groups were similar to their respective controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no maternal gross observations.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Sprague Dawley rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to exposure of rats to D5.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant differences on T3, T4, or TSH serum concentrations in any treated groups when compared to controls.

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
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
There were no treatment-related or statistically significant effects on pregnancy rates, resorption rates, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss or gravid uterine weights at any exposure concentration level. All dams were pregnant with the exception of one animal (#420) in the 161 ppm (analytical) group.

Effect levels (maternal animals)

Dose descriptor:
NOAEC
Effect level:
>= 161 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or statistically significant external alterations in any treated group. Incidental findings bearing no relationship to treatment included the malformations anasarca, domed skull, macroglossia, shortened muzzle, short trunk, polydactyly, and ankylodactyly. These observations were considered unrelated to treatment as they were all confined to one litter (Dam #392) in the 74 ppm (analytical) group and lack of an exposure-response relationship.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related skeletal alterations in any treated group. Incidental findings bearing no relationship to treatment included the variations supernumerary skull bone, delayed ossification (DO) parietal, DO cervical centra, DO sternebrae, DO thoracic centra, irregular pattern of ossification sternebrae, wavy ribs, calloused ribs, extra 1st lumbar rib, DO lumbar centra and the malformations fused ribs, missing lumbar centra, missing lumbar vertebrae, extra metacarpals, extra phalanges, and extra metatarsals. These observations were considered unrelated to treatment due to the low incidence, occurrence in the control group, and/or lack of a dose response relationship. Fetuses in the 161 ppm (analytical) treatment group had statistically-identified lower incidence of DO thoracic centra which was considered unrelated to treatment as a treatment-related effect in this parameter is expected to be a higher value than control.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or statistically significant visceral alterations in any treated group. Incidental findings bearing no relationship to treatment included the variations fused lung, missing caudal lung lobe, supernumerary hepatic liver lobule, petechial haemorrhage adrenal, and bifurcated renal vein, and the malformations situs inversus, hydronephrosis, and hypoplastic testis. These observations were considered unrelated to treatment due to the low incidence, occurrence in the control group, and/or lack of a dose response relationship.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or statistically significant craniofacial alterations in any treated group. An incidental finding bearing no relationship to treatment included the malformation hydrocephaly in the 74 ppm (analytical) group (Dam #392). This observation was considered unrelated to treatment due to the isolated occurrence and lack of a dose-response relationship.
Details on embryotoxic / teratogenic effects:
There were no treatment-related or statistically significant effects on fetal sex ratios, fetal body weights absolute and relative anogenital distance at any exposure concentration level. All dams were pregnant with the exception of one animal (#420) in the 161 ppm (analytical) group. There were no treatment-related differences in the incidence of any fetal alteration in any of the treated groups compared to controls. The small number of alterations observed in fetuses from dams either occurred at low frequencies, in the control group, and/or were not dose related.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
>= 161 ppm (analytical)
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

- Concentration analysis: Analytically-determined mean chamber D5 vapor concentration values were 0.0 ± 0.0, 32 ± 1.1, 74 ± 2.0, and 161 ± 3.8 ppm over the course of the study.  

The nominal concentration of test material in each chamber was calculated based on the amount of test material fed into the inhalation apparatus each day and the total chamber airflow through the chamber for each exposure period. The nominal concentrations were 29.1 ± 0.6, 81.2 ± 2.1, and 169.7 ± 5.6 for D5 exposure chambers. 

Chamber aerosol concentrations were 0.000 ± 0.0000, 0.004 ± 0.006, 0.036 ± 0.011, and 0.083 ± 0.021 mg/m3 for the 0, 32, 74, and 161 ppm (analytical) D5 exposure chambers, respectively.  

- Chamber temperature: Mean chamber temperature values for all chambers were maintained between 22.4 and 24.4 °C.  

- Chamber humidity: Mean chamber relative humidity was maintained in the range of 35.3 - 38.7% for all exposure chambers.  

- Chamber airflow: The mean chamber airflow in all four exposure chambers was maintained between 463 and 569 L/min.

See attachments for detailed result tables.

Applicant's summary and conclusion

Conclusions:
In the inhalation prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the concluded NOAEC for maternal and developmental effects was greater than 161 ppm (analytical mean measured, equivalent to 2427 mg/m3), the highest achievable vapour concentration. There were no adverse effects reported in the study.