Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Kanagawa, Japan
- Age at study initiation: 10 weeks old
- Weight at study initiation: 234.0g to 299.3g
- Fasting period before study: Not applicable
- Housing: Mating period: 1 male and 1 female per cage, other period: 1 or 2 rats/cage (same sex)
- Diet (e.g. ad libitum): Pellet feed for experimental animals (CRF-1: Oriental Yeast Co., Ltd., Tokyo, Japan, radiation sterilized)
- Water (e.g. ad libitum):
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1°C to 24.3°C
- Humidity (%): 37.7% to 66.3%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours/day, 7:00 to 19:00

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Details on exposure:
The test substance supplied from the supplier was used as is. The test substance was gasified by passing through pressurized air at a specified flow rate into the test substance in a stainless-steel cylinder held into a constant temperature water bath set at 15°C. The generated gas was passed through a heat exchanger set at 15°C. Then, the test atmosphere at the intended concentration was prepared by mixing the test substance gas with pressurized air at specified flow rate. The test atmosphere was exposed to the rats by continuously supplying it to the chamber. The air from the chamber was exhausted to outside air after passing through a filter and scrubber. The rats held in the restraint tubes (Muenster Ltd.) were connected to the chamber to start exposure, when hydrocarbon concentration in the test atmosphere continuously monitored by a hydrocarbon monitor reached temporally equilibrium after the start of generating the test atmosphere containing the test substance. The rats were disconnected from the chamber to terminate exposure 6 hours after the start of exposure. In the control group, the rats held in the restraint tubes were connected to the chamber supplied air at a specified flow rate to start exposure. The rats were disconnected from the chamber to terminate exposure 6 hours after the start of exposure. Hydrocarbon concentration in the test atmosphere was continuously monitored with the hydrocarbon monitor (HCM-1B: Shimadzu Corp., Kyoto, Japan) every exposure. Temporal variability of the concentration was confirmed by recording the output voltage of the hydrocarbon monitor. The amount of test substance used during the inhalation exposure was calculated from the weight difference of the cylinder containing the test substance between before and after the test atmosphere generation. The total air volume supplied to the chamber was obtained by multiplying the elapsed time of the test atmosphere generation to the air flow rate of the test atmosphere supply (minimum unit: 0.1 L/min). The nominal concentration (minimum unit: 10 ppm) was calculated with the following equation from the amount of the test substance used and the total air volume supplied to the chamber. The test substance in the test atmosphere was quantified by a gas chromatography (hereinafter abbreviated as “GC”) according to the method validated in the acute inhalation toxicity study of the test substance (study No. B130803), then, the exposure concentration was calculated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Equipments
(1) GC: GC-14B (Shimadzu Corp.)
Recorder: D-7500 (Hitachi, Ltd., Tokyo, Japan)
Syringe: 1 mL gastight syringe (#1001: Hamilton Company, NV, USA) Sampling bag: 1.55 L of inner volume (actual inner volume, Tedlar bag: GL Sciences Inc., Tokyo, Japan)
(2) GC conditions
Detector: hydrogen flame ionization detector
Columm: 5% SE-30, Uniport HP (100 - 120 mesh),
inside diameter 3.2 mm, length 2 m, glass column
Injection temperature: 150°C
Detector temperature: 150°C
Column temperature: 60°C
Carrier gas: nitrogen
Carrier gas flow: 40 mL/min
Injection volume: 0.5 mL
(3) Preparation of standard gas
The standard gases were prepared according to the following table. A specified quantity of the test substance gas was accurately injected into the sampling bag with the 1 mL gastight syringe. Then, standard gases (minimum unit: 10 ppm) were prepared by filling up the sampling bag with the pressurized air (0.01 MPa or less).

INSERT TABLE

(4) Confirmation of specificity
Air in the experimental room was injected to the GC with the 1 mL gastight syringe and analyzed. It was confirmed that there were no peaks considered to interrupting quantification at the retention time of the derived peak from the test substance on the obtained chromatogram. Acceptable range for the specificity was decided the peak area of the interference peak to be less than 5% of peak area derived from the test substance of ST-1. The analytical results met the criterion of this study.

(5) Preparation of calibration curve
Each standard gas was injected once to GC and analyzed. The calibration curve was constructed by a linear regression formula obtained from the concentrations and peak areas of the standard gases by the least square method. The acceptable range to the linearity of the calibration curve was decided the coefficient of correlation of the calibration curve to be more than 0.995. The analytical results met the criterion of this
study.

Sampling and analysis of the test atmosphere
(1) Equipments
Sampling bag: 1.55 L of inner volume (actual inner volume, Tedlar bag: GL Sciences Inc.)
Vacuum pump: general-purpose pump
Vacuum sampling container: a container for sampling the test atmosphere to the sampling bag inside it by reducing inner pressure (in-house made)
50 mL syringe: SS-50LZ (Terumo Corp., Tokyo, Japan)

(2) Sampling
Sampling position: exposure port of the chamber
Date and time of sampling: 30 minutes, 3 hours, and 5 hours 30 minutes after the start of exposure on the day of start of exposure and thereafter 7-days intervals 30 minutes after the start of exposure on other day.
Number of sample: 30 minutes after the start of exposure on the day of start of exposure and 7 days intervals: 3 samples (simultaneous sampling from 3 exposure ports)
others: 1 sample/time point
Sampling method: The sampling bag was put into the vacuum sampling container, and connected to the sampling port. The test atmosphere was sampled to the sampling bag by reducing inner pressure of the container with a vacuum pump.

(3) Preparation of analytical sample
(a) Sampling the collected samples at 200 mL into another sampling bag with the 50 mL
syringe
(b) Filling up the sampling bag with pressurized air (0.01 MPa or less)

(4) Analysis and exposure concentration calculation
Analysis subject: analytical sample Number of analysis: 1 analysis/sample Exposure concentration calculation:
calculating of exposure concentration from the calibration curve and dilution factor of the analytical sample (minimum unit: 10 ppm)

(5) Evaluation of exposure concentration
(a) Calculated items
Exposure concentration: mean concentration of exposure concentration
(representative value on 3 samples/time point: mean value, representative value on the day of 3 times sampling: mean concentration on the day)
Concentration homogeneity: coefficient variation of exposure concentration of simultaneous collected 3 samples on 30 minutes after the start of exposure (minimum unit: 0.1%)
Temporal concentration stability: coefficient variation of exposure concentration (on the day of 3 times sampling, minimum unit: 0.1%)
relative error of each measuring value to mean concentration (on the day of 3 times sampling, minimum unit: 0.1%) (b) Criteria values of temporal concentration stability
Coefficient variation: 10% or less
Relative error: ±10% or less

Air flow rate of air-supply to the chamber (minimum unit: 0.1 L/min) was measured 9 times: at the start of the test atmosphere supply, start of the exposure, 1 hour intervals from start of exposure, end of the exposure, and end of the test substance supply.
Details on mating procedure:
A mating pair constructed from 1 male and 1 female was housed in 1 cage from 16:00 on the start day of mating. Vaginal smear was collected every morning to examine presence of sperm. The female, which was confirmed a vaginal plug or presence of sperm in vaginal smear, was defined as success of copulation. The day of copulation was defined as “gestation day” (GD 0). The copulated female was defined as pregnant animal (hereinafter dam). Mating was continued until obtaining necessary number of dams to conduct the study.
Doses / concentrationsopen allclose all
Dose / conc.:
31 639 mg/m³ air (analytical)
Dose / conc.:
5 210 ppm (analytical)
Dose / conc.:
62 489 mg/m³ air (analytical)
Dose / conc.:
10 290 ppm (analytical)
Dose / conc.:
124 553 mg/m³ air (analytical)
Dose / conc.:
20 510 ppm (analytical)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal findings were observed in any dams.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Description (incidence):
There were no deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistical significant differences were noted in body weight or body weight gain of the dams between the control group and groups exposed to the test substance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistical significant differences were noted in food consumption of the dams between the control group and groups exposed to the test substance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormalities were observed in any dams on the necropsy conducted on GD 20.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Percentage of pre-implantation loss in the HCFO-1224yd(Z) 30,364 mg/m3) (5000 ppm) group was lower than it in the control group. There was no relationship with exposure concentration, and therefore the differences were considered to be incidental.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
>= 20 510 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: No effects observed
Key result
Dose descriptor:
NOEC
Effect level:
>= 124 553 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: 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:
no effects observed
Description (incidence and severity):
Several abnormalities were found on external examination of fetuses and placental examination. No statistically significant differences were noted in the incidence of these abnormalities among the groups, and therefore the abnormalities were considered to be incidental.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Several anomalies and variations were observed in the control and HCFO-1224yd(Z) 121,456 mg/m3 (20000 ppm) groups. These anomalies and variations did not have statistically significant differences between the control and HCFO-1224yd(Z) 121,456 mg/m3 (20000 ppm) groups, and therefore the anomalies and variations were considered to be incidental. No statistically significant differences were observed in progress of ossification between the control and HCFO-1224yd(Z) 121,456 mg/m3 (20000 ppm) groups.
Visceral malformations:
no effects observed
Description (incidence and severity):
Several anomalies were observed in the control and HCFO-1224yd(Z) 121,456 mg/m3 (20000 ppm) groups. No statistically significant differences were observed in the number and incidence of anomalies between the control and HCFO-1224yd(Z) 121,456 mg/m3 (20000 ppm) groups, and therefore the anomalies were considered to be incidental.
Other effects:
not specified

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOEC
Effect level:
> 20 510 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: No effects observed
Key result
Dose descriptor:
NOEC
Effect level:
124 553 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: No effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Executive summary:
Prenatal developmental toxicity of HCFO-1224yd(Z) was evaluated by nose-only inhalation exposure in rat.
The target exposure concentrations of this study were set at 0 (control), 30,364, 60,728 and 121,456 mg/m3 (5000, 10000, and 20000 ppm respectively). The rats
(20 animals per group) were exposed to each test atmosphere from gestation day (GD) 6 to GD 19 for 6 hours a day.
On GD 20, the animals were subjected to necropsy and cesarean section, and the embryo-fetus and placenta were examined.
The actual exposure concentrations were 31,639, 62,489 and 124553 mg/m3 (5210, 10290, and 20510 ppm, respectively).
In the examination of effects of the test substance to dams, the test substance exposure resulted in no noteworthy changes in clinical sign, body weight,
and food consumption.
No abnormal findings were found in necropsy.
Moreover, the test substance exposure did not affect the maintenance of pregnancy or reproductive function of dams.
In the examination of the effects to embryo-fetus, there were no changes attributable to the test substance in the observation on cesarea, visceral or skeletal examination.
In conclusion, it was concluded that the no observed effect level (NOEL) of prenatal developmental toxicity of HCFO-1224yd(Z) is more than 124,553 mg/m3 (20510 ppm) in the condition of this study.