[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07/11/2018 - 12/06/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Discussed in "Principles of Method if Other Than Guideline"

Principles of method if other than guideline:

1 - Molecular weight of the test substance

The molecular weight of the test substance was described in the information obtained from industry group as 294.0. The test atmosphere was generated during 1st–8th exposure based on this information. The correct molecular weight was 200.05. Exposure concentration was calculated by the correct molecular weight in all exposure. The study results were evaluated by the correct exposure concentration and it was concluded that the study reliability was secured.

2 - Number of animals in the cage during the quarantine period

Since one female was euthanised, one female (animal No. 59033) housed in the same cage was individually housed in a cage from the day after the animal receipt to the end of the quarantine period. This female was allocated to the study group after the acclimatisation period for 2 weeks and it was concluded that the individual housing did not affect the animal health condition.

3 - Exposure concentration

On the exposure concentration analysis, the peak retention time shifted on the analysis of the sample at 3 hours after the start of exposure in the 1000 ppm group on Day 57. The GC was used in another study with different column temperature. The setting of the temperature was returned for this study, and the analysis was carried out after confirmation the temperature indication to the prescribed level. However, it was estimated that the actual temperature did not reach the prescribed level, this resulted in the shift of the peak retention time. It was judged that the analysis was not reliable as a result. Since other time points were reliable and concentrations were stable over time, it was concluded that this deviation did not affect the study reliability.

4 - Subject pups on blood chemistry

In the study protocol, it was described that 1 male and 1 female per litter would be subjected to blood chemistry evaluation. It was defined that the eliminated pups were subjected to blood chemistry in the section for litter size in the study protocol. Inconsistent description resulted in the deviation from the study protocol. Since the subject pups were selected depending on the number of sex on day 4 of lactation, the pups with one sex only were subjected to the examination in a few animals. The study reliability was not affected because the subject of examination could be identified from overall description of the study protocol.

5 - Sample volume of blood chemistry

The blood sampling was conducted in one pup and in two parental females (animal Nos.50204 and 50404) - the blood sample volumes were less than 0.4 mL. Though the volume was inconsistent with the study protocol, measurement of blood chemistry could be conducted in these animals and reliability of study was secured.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

EC Number:

609-858-6

Cas Number:

406-78-0

Molecular formula:

C4H3F7O

IUPAC Name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

Test material form:

liquid

Specific details on test material used for the study:

Lot No. 51807130
Purity 99.98%

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

This strain is widely used in reproductive and developmental toxicity study using rodents, and there are abundant historical data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were acclimatised from receipt until the beginning of the testing. At the beginning of this time, they were quarantined for 5 days. Observations taken during this period included a clinical observation, body weight measurements and estrous cycle measurements.

Animals were housed in a temperature range of 21.8 - 25.0°C; a relative humidity range

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Rats were individually housed in a wire-mesh cage. The cage was put into the inhalation chamber, which ensured air ventilation. The start of exposure in the test group was defined as the point at which the test substance reached equilibrium. The start of exposure in the control group was defined at 18 minutes after the door closing, which was equivalent to the time taken to reach 95% atmosphere equilibrium (t95). End of exposure was defined as 6 hours after the start of exposure.

Details on mating procedure:

1:1 (1 male and 1 female) mating was used in this study. The female was placed with the same male day and night from the evening on day 15 (the first day of mating) for a maximum of 14 days except during exposure. Every morning, the females were examined via vaginal smear. Day 0 of gestation was defined as the day a vaginal plug or sperm was found. The following parameters were calculated:

(1) Number of days until copulation: Days from start of mating to copulation confirmation
(2) Copulation index (%): (Number of copulated males or females/number of pairs) × 100
(3) Fertility index (%): (Number of pregnant males or females/number of copulated males or females) × 100

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification of doses was performed using a Shimadzu Corp. GC-14B Gas Chromatography machine. The injection temperature was 150°C, and column temperature was 60°C.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

35 days total

Details on study schedule:

Exposure started approximately 14 days before the start of the mating period, and lasted until the day before necropsy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

500 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

4 089.5 mg/m³ air (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 ppm (nominal)

Doses / concentrations 5

Dose / conc.:

8 179 mg/m³ air (nominal)

Doses / concentrations 6

Dose / conc.:

1 500 ppm (nominal)

Doses / concentrations 7

Dose / conc.:

12 269 mg/m³ air (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

1:1 (1 male and 1 female) mating was used in this study. The female was placed with the same male day and night from the evening on day 15 (the first day of mating) for a maximum of 14 days except during exposure. Every morning, the females were examined via vaginal smear. Day 0 of gestation was defined as the day a vaginal plug or sperm was found. The following parameters were calculated:

(1) Number of days until copulation: Days from start of mating to copulation confirmation
(2) Copulation index (%): (Number of copulated males or females/number of pairs) × 100
(3) Fertility index (%): (Number of pregnant males or females/number of copulated males or females) × 100

Day 0 of lactation was defined as the day of delivery. The parental females were observed for maternal behavior, including lactation, nest building, and cannibalism until 13 days after delivery (day 13 of lactation).

Oestrous cyclicity (parental animals):

Estrous cycle of the females in each group was examined by vaginal smear sampling in the morning from the day of start dosing to the day confirmed copulation or end of mating period in the females not confirmed mating. Estrous cycle was classified the stage of diestrus (D), proestrus (P), estrus (E), and metestrus (M). The count of estrous and estrous cycle were calculated. Moreover, estrous cycle was determined at day 13 of lactation by vaginal smear sampling.

Sperm parameters (parental animals):

The testes were fixed and preserved in 10vol% phosphate-buffered formalin, fixed with Bouin’s solution and preserved in 10vol% phosphate-buffered formalin. In the dead animal, all organs and tissues were fixed and preserved in 10vol% phosphate-buffered formalin. (2) Preparation of specimen and microscopic examination. In the control group and high concentration exposure group, hematoxylin and eosin stained specimens were prepared according to the routine method. Moreover, the testes were processed to PAS stained specimen. The histopathological specimens were microscopically examined. The testes were microscopically examined with identification of stages of seminiferous tubule based on spermatogenesis cycle.

Litter observations:

All live pups were individually weighed on postnatal days 0, 4, and 13 after birth with an electrical balance (PB3002-S: Mettler-Toledo K.K.).
Anogenital distance (AGD) of the pups after litter size adjustment was measured with a vernier caliper on postnatal day 4 after birth. The AGD was normalized by the cube root
of body weight on the day of AGD measurement.
The number of nipple/areola was counted in all male pups on day 12 after birth, and the following parameter was calculated.
Nipple development index: (Number of abnormal male pups retaining nipples/number of examined male pups) × 100

Postmortem examinations (parental animals):

The following organs/tissues were collected and weighted:
Thyroids/parathyroids, testes, epididymides, levator ani/bulbocavenosus muscle complex, cowper's glands, glans penis, ovaries

They were weighed on the scheduled necropsy with an electrical balance (AW120: Shimadzu Corp.). The bilateral organs were weighed simultaneously for both sides. The thyroid/parathyroid was weighed after fixation with formalin. Moreover, relative organ weight (body weight-relative) was calculated from body weight measured on the day of necropsy. No measurement was conducted in the dead animal.

The following organs/tissues were examined via histopathology:
Testes, epididymides, ovaries, uterus

The collected organs and tissues were fixed and preserved in 10vol% phosphate-buffered formalin. The testes and epididymis were fixed with Bouin’s solution and preserved in 10vol% phosphate-buffered formalin. In the dead animal, all organs and tissues were fixed and preserved in 10vol%
phosphate-buffered formalin. (2) Preparation of specimen and microscopic examination. In the control group and high concentration exposure group, hematoxylin and eosin stained specimens were prepared according to the routine method. Moreover, the testes were processed to PAS stained specimen. The histopathological specimens were microscopically examined. The testes were microscopically examined with identification of stages of seminiferous tubule based on spermatogenesis cycle.

Postmortem examinations (offspring):

All offspring were examined externally for gross abnormalities including oral cavity on postnatal day 13. Thereafter, head, organs and tissues in the thorax and abdominal cavities were observed for gross abnormalities after euthanisation according to the same method as parental animals. The thyroids of 1 male and 1 female pups per litter were fixed and preserved in 10 vol% phosphate-buffered formalin. External anomalies were examined in dead offspring. The following indexes for external anomalies were calculated. External anomaly by type retention index was not calculated according to the study results.
(1) External anomalies retention index: (Number of pups indicating external anomaly/number of pups examined) × 100
(2) External anomalies by type retention index: (Number of pups indicating external anomaly by type/number of pups examined) × 100

Statistics:

The following statistical analyses were conducted. Safety Study System (tsPharma LabSite: Fujitsu Ltd.) was used in the statistical analyses. The statistical analysis system (EXSUS: CAC Croit Corp.) and SAS system (SAS Institute Inc.) were used in the analysis which did not use the Safety Study System. In the data on offspring, the data calculated in each litter were used in the analysis.

Multiple comparison test
In the following parameters, variance was assessed using Bartlett’s test (significant level: 5%). When the variance was homogeneous, multiple comparison test was conducted according to Dunnett’s test. Otherwise, multiple comparison test was conducted according to Steel’s test. Significance levels were set at 1% and 5%. Body weight, body weight gain, food consumption, organ weight, body weight-relative organ weight, estrous cycle, number of estrous cycle, days required for copulation, duration of gestation, number of implantation, number of offspring (live pups and stillbirth pups), body weight of pups in each sex, AGD, blood chemistry

X2 test
In the following parameters, statistical analysis was performed between the control group and groups exposed the test substance. Copulation index, fertility index, delivery index, sex ratio

Wilcoxon rank-sum test
In the following parameters, statistical analysis was performed between the control group and groups exposed the test substance.
Stillborn index, external anomalies retention index, delivery index, birth index, viability on days 4 and 13, male nipple development index

Reproductive indices:

Numbers of implantations were examined in the ovaries and uterus obtained from the parental females at necropsy. The following parameters were calculated from the examination results.
(1) Duration of gestation: From day 0 of gestation to completion of delivery
(2) Gestation index (%): (Total number of delivered females/total number of pregnant) ×
100
(3) Delivery index (%): (Number of pups/number of implantation) × 100

Offspring viability indices:

Number of pups (stillbirth and live birth), sex, and external abnormalities (including runts) were examined on day 0 of lactation. Thereafter, they were observed daily in clinical signs and mortality. The following parameters were calculated from the observation results.

(1) Birth index: (Number of live pups/number of implantation) × 100
(2) Stillborn index: (Number of stillborn pups/total number of pups) × 100
(3) Viability on day 4:
(number of live pups on day4/total number of pups at birth) × 100
(4) Viability on day 13:
(number of live pups on day 13/ number of live pups on day 4) × 100
(5) Sex ratio: (Male pups/total number of pups) × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Mass on chest in 1 female at 4089.5 mg/m3 (500 ppm), no dose-response observed suggesting no test substance related finding

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1 male died day 9 at 12268.5 mg/m3 (1500 ppm)

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decreased bodyweight gain in females day 7 of lactation period at 4089.5 mg/m3 (500ppm), in males day 8 at 8179 mg/m3 (1000ppm), in males day 8 and females day 7 of gestation, and day 7 of lactation at 12268.5 mg/m3 (1500ppm)

Increased bodyweight gain in females day 8 at 12268.5 mg/m3 (1500ppm)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant low value in males day 1 at 12268.5 mgm3 (1500ppm)

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:

no effects observed

Description (incidence and severity):

No statistically significant effect on blood T4 levels in males or pups

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No effect on organs

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic cutaneous mass (adenocarcinoma) of mammary gland in 1 female at 4089.5 mg/m3 (500 ppm), considered spontaneous change

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decrease in count of estrus in females at 12268.5 mg/m3 (1500ppm)
Statistically significant increase in estrus cycle (day) in females at 12268.5 mg/m3 (1500ppm)

The actual concentration in the 12269 mg/m3 (1500ppm) group was more than 16358 mg/m3, or 2000 ppm (16930.53-20202.13 mg/m3, or 2070–2470 ppm). No differences were noted between the control group and 8179 mg/m3 (1000 ppm) group exposed to the actual concentration of 11123.44-13086.4 mg/m3 (1360-1600 ppm)

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

All females showed evidence of copulation
No statistically significant effect in reproductive parameters, eg, number of days until copulation, copulation index, and fertility index

Statistically significant decreased delivery index and birth index values at 4089.5 mg/3 (500ppm)- no exposure concentration-dependencies observed, not attributable to test substance
No statistically significant effect on parameters to do with results of delivery and observation of lactation, eg, duration of gestation, delievery index, or birth index.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEC

Effect level:

> 1 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOEC

Effect level:

> 12 268.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No effect on clinical signs or abnormalities
No effect on external anomalies

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effect on indices such as stillborn index or viability index

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant effect on body weight

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

no effects observed

Description (incidence and severity):

No change observed in necropsy

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOEC

Generation:

F1

Effect level:

> 1 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOEC

Generation:

F1

Effect level:

> 12 268.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

1

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 500 ppm

Treatment related:

no

2

Reproductive effects observed:

no

Lowest effective dose / conc.:

12 268.5 mg/m³ air

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

It was concluded that the no observed adverse effect level (NOAEL) of reproduction/developmental toxicity of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether is greater than 12268.5 mg/m3 (1500 ppm) for males and females.

Executive summary:

A reproduction/developmental toxicity screening test of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether (HFE-347pc-f) was conducted using Crl:CD(SD) rats in accordance with the OECD guidelines for the testing chemicals (No. 421) by inhalation exposure.

The actual exposure concentrations were 4580.24, 9078.69, and 13822.51 mg/m3 (560, 1110, and 1690 ppm).

The test substance exposure to the parental animals resulted in no toxicological significance changes. No abnormalities were found in clinical observation, no toxicological significant differences among the groups in body weight, body weight gain, food consumption, or plasma T4 level. Moreover, the test substance exposure did not result in changes in the reproductive functions. No differences among the groups were noted in estrous cycle, days required for copulation, copulation index, fertility index, duration of gestation, delivery index, or birth index. Moreover, no visible histopathological changes were found in any animals.

The test substance exposure did not show developmental toxicity. Body weight of offspring did not show differences among the groups. No abnormalities were noted in the external or clinical observation of offspring. No differences among the groups were noted in birth index, stillborn index, viability index, or sex ratio. Anogenital distance was not change among the groups. No nipple was observed in any male pups. There were no changes in the necropsy of offspring conducted after 4 days of lactation. From the above results, it was concluded that the no observed adverse effect level (NOAEL) of reproduction/developmental toxicity of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether is greater than 12268.5 mg/m3 (1500 ppm) for males and females.