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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24/09/2019 - 21/01/2020
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(1Z)-1-chloro-2,3,3,3-tetrafluoroprop-1-ene
EC Number:
813-937-2
Cas Number:
111512-60-8
Molecular formula:
C3HClF4
IUPAC Name:
(1Z)-1-chloro-2,3,3,3-tetrafluoroprop-1-ene
Test material form:
gas
Specific details on test material used for the study:
Purity ranged between 99.10 - 99.19%, and the substance stability was confirmed by CoA from the sponsor.

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:
The quarantine period was set for 5 days from animal receipt. The following examinations were conducted during the quarantine period.

· Clinical observation: Once a day

· Body weight measurement: The day of animal receipt and the day of end of quarantine Crust formation was observed in 1 male as spontaneous finding and 1 female indicated body weight loss within normal range. It was concluded that evaluation of the health condition of animals was not affected by these spontaneous changes during the quarantine period.

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
nose only
Vehicle:
unchanged (no vehicle)
Details on exposure:
Rats held in the restraint tubes were connected to the inhalation chamber at 5 minutes or more elapsed from the start of the test atmosphere generation, which allowed sufficient time for the test substance to come to equilibrium prior to exposure.

The time to concentration equilibrium (t95) was estimated to be approximately 30 seconds, which is calculated according to the OECD guideline with an inner volume of the chamber (approx. 2.5 L) and air flow ratio of test atmosphere supply (16 L/min.). In consideration of the time required to the concentration equilibrium in the air over time, the time point to start of exposure was defined at 5 minutes or more after the start of the test atmosphere generation. The rats were removed from the chamber to terminate exposure 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 day of start mating) for maximally 14 days except during exposure. On every morning, the females were examined by 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:
Sampling was performed accordingly: 30 minutes, 3 hours, and 5 hours 30 minutes after the start of exposure on the day of start of exposure and thereafter at 7-day intervals 30 minutes after the start of exposure on the other days Sampling at the 3 times was conducted at 7-day intervals until Day 29 and for the exposure concentration of 40,000 ppm on 29th exposure.

Thereafter, sampling for the 3 times was conducted every exposure day. Re-measurement was conducted at 20,000 ppm on the 35th exposure, because it was expected that there had been an operational failure during the sampling injection to GC. Since reliability was confirmed on re-measurement, the re-measurement value was used as the exposure data.
Duration of treatment / exposure:
Treatment per day was 6 hours.

The males were subjected to the exposure for 35 days in total from 14 days before mating until the day before necropsy via the mating period.

The females were subjected to the exposure from 14 days before mating to day 19 of gestation (the day of confirmation of copulation designated as day 0 of gestation) via mating period, and day 3 of lactation to day 12 of lactation (the day of completion of delivery designated as day 0 of lactation). A single non-delivered female was exposed until day 19 of gestation. A single non-mating female was exposed until the day before necropsy.
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
10 410 ppm
Dose / conc.:
1 366 711.66 mg/m³ air
Dose / conc.:
20 920 ppm
Dose / conc.:
2 746 552.15 mg/m³ air
Dose / conc.:
41 640 ppm
Dose / conc.:
5 466 846.63 mg/m³ air
No. of animals per sex per dose:
12
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
Daily observation was conducted from the first day of exposure to the day of necropsy.
Oestrous cyclicity (parental animals):
Oestrous cycle of females in each group was examined by vaginal smear sampling in the morning from the first day of exposure to the day of confirmed copulation or end of mating period in the females that were not confirmed as mating. Oestrous cycle was classified according to the stage of dioestrus (D), proestrus (P), oestrus (E), and metestrus (M). The count of oestrous and oestrous cycle was calculated. Moreover, oestrous cycle was determined at day 13 of lactation by vaginal smear sampling.
Sperm parameters (parental animals):
The testes were microscopically examined with identification of stages of seminiferous tubule based on spermatogenesis cycle.
Litter observations:
On day 4 after birth, the size of litter was adjusted by eliminating extra pups by random selection to yield 4 pups per sex per litter. If the number of pups in one sex was below 4, the size of litter was adjusted to 8 pups per litter. The eliminated pups were euthanized by an overdose of intraperitoneal injection of thiopental sodium (Ravonal: Nipro ES Pharma Inc.). Two extra pups per litter were subjected to blood chemistry analysis.
Postmortem examinations (parental animals):
Organ Weights

The organs and tissues were weighed on the scheduled necropsy using an electronic 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 ratio) was calculated from body weight measured on the day of necropsy.

In 1 female of the 40,000 ppm group, thyroid weight was removed from evaluation due to congenital unilateral thyroid defect.
Preservation of organs and tissues

The organs and tissues listed in Section 6.6.5.1 were collected. The collected organs and tissues were fixed and preserved in 10 vol% phosphate-buffered formalin. The testes and epididymis were fixed with Bouin’s solution and preserved in 10 vol% phosphate-buffered formalin.

Preparation of specimen and microscopic examination.

In the control group and high concentration exposure group, hematoxylin and eosin (H.E.) stained specimens were prepared from the organs and tissues according to the routine method. Moreover, the testes were processed to PAS stained specimen. The ovaries of all females were processed into H.E. stained specimen before microscopic examination.
The histopathological specimens were microscopically examined. The testes were microscopically examined with identification of stages of seminiferous tubule based on spermatogenesis cycle. The organ/tissue with gross abnormality of all animals was processed into H.E. stained specimen and subjected to microscopic examination.
Postmortem examinations (offspring):
All offspring were examined externally for gross abnormalities including the 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 for the parental animals. The thyroids of 1 male and 1 female pups per litter, excluding those from the parental animal with only female pup, were fixed and preserved in 10 vol% phosphate-buffered formalin. The following indices for external anomalies were calculated. The “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:
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. For the data on offspring, the data calculated in each litter were used in the analysis.

The following data was excluded from evaluation: body weight and food consumption after confirmation of mating on non-pregnant female, body weight of non-copulated female.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No dead animals were found in this study.

“Wound on hindlimb” was observed in 1 female exposed to 10,000 ppm as incidental change.

No exposure concentration-dependency was noted in the incidence of this finding, suggesting that it was not related to exposure to the test substance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In the group exposed to 5,466,846.63 mg/m3, there were statistically significant lower body weight gains compared to the control group on Days 15 and 29 in males and Day 7 of gestation in females. Since there were no statistically significant differences in body weight or continuities in the differences for body weight gains, these differences were considered to have no toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In the group exposed to 5,466,846.63 mg/m3, there were statistically significant lower values compared to the control group on Days 14, and Day 6 of gestation in females. The lack of continuities in these low food consumption data suggested that they were of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the parental males, which were sampled on Day 36, there were no statistically significant differences of plasma total T4 and TSH levels between the control group and groups exposed to the test substance. In the offspring, which were sampled on Day 13 of lactation, statistically significant lower levels of total T4 were observed s in the group exposed to 2,746,552.15 mg/m3 but not the other concentrations, when compared to the control group. The mean levels of TSH in all exposed groups were lower than the control value, but only reached statistical significance in those exposed to 2,746,552.15 mg/m3.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes related to exposure to the test substance upon histopathological
examination.

In the testis, atrophy of seminiferous tubules was observed in one male exposed to 2,746,552.15 mg/m3 and one male exposed to 5,466,846.63 mg/m3, and degeneration of seminiferous tubular epithelium was observed in one male exposed to 5,466,846.63 mg/m3. Atrophy of seminiferous tubules was considered to be attributable to the test substance in the previous study conducted in the test facility (study No. B160417) because the incidence was high those exposed to 2,746,552.15 mg/m3. However, in the current study, the incidences of atrophy of seminiferous tubules and degeneration of seminiferous tubular epithelium observed in males exposed to either 20,000 and/or 5,466,846.63 mg/m3 were low, and they are known to occur spontaneously in rats of this strain and age. In addition, atrophy of seminiferous tubules was observed unilaterally. Therefore, these changes were considered to be spontaneous changes. Other microscopic findings were considered to be spontaneous changes due to their incidence, pathological nature, or being common occurrences in rats of this strain and age.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in count of estrus or estrous cycle in any groups exposed to the test substance in comparison with those in the control group.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
One female exposed to 1,366,711.66 mg/m3 was not pregnant. One female exposed to 2,746,552.15 mg/m3 did not copulate. These occurrences were considered to be within the normal ranges. There were no statistical significant differences in the reproductive functions between the control group and groups exposed to the test substance, such as number of days until copulation, copulation index, or fertility index.

Results: P1 (second parental generation)

Effect levels (P1)

Key result
Dose descriptor:
NOEC
Effect level:
> 5 466 846.63 mg/m³ air
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no statistical significant differences in the observation results of offspring between the control and the groups exposed to the test substance, such as birth index, stillborn index, viability index on days 4 or 13, or sex ratio. No external anomalies or clinical abnormalities were observed in any pups.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistical significant differences in body weight between the control group and the groups exposed to the test substance.
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant low value in AGD normalized value was observed in males exposed to 5,466,846.63 mg/m3 in comparison with the control group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No abnormal nipple development was observed in any of the exposed male pups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no changes in any animals.

Effect levels (F1)

Key result
Dose descriptor:
NOEC
Generation:
F1
Effect level:
> 5 466 846.63 mg/m³ air
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Total T4 and TSH levels in females sampled on Day 13 of lactation were statistically significantly reduced when compared to the levels in the controls in those exposed to 2,746,552.15 mg/m3, but not in those exposed to 1,366,711.66 mg/m3 or 5,466,846.63 mg/m3. The levels in those exposed to 5,466,846.63 mg/m3tended to be low, when compared to levels in the controls. The effect was not dose-dependent.
 
The normalised anogenital distance in male offspring exposed to 5,466,846.63 mg/m3were statistically significantly reduced when compared to the distance in controls. The effect was slight in magnitude.
 
Males exposed to 5,466,846.63 mg/m3showed reduced absolute weights of epididymides, seminal vesicles and prostate glands when compared to the weights of the controls. Females exposed to 5,466,846.63 mg/m3showed increased absolute and relative weights of the thyroid gland when compared to the weights of the controls. Atrophy of the seminiferous tubule, as seen in males exposed to 2,746,552.15 mg/m3the 90-day study, was not observed in this study. The difference might be due to the difference in the duration of exposure. There were no functional consequences of the adverse pathology in males in the current study.
 
The effects seen in the current study, taking into account the findings of the 90-day study, are insufficient to warrant the classification of HCFO 1224yd(Z) for reproductive toxicity.
Executive summary:

A reproduction/developmental toxicity screening test of HCFO-1224yd(Z) was conducted in Crl:CD(SD) rats by nose-only inhalation exposure in accordance with the OECD guideline No. 421 (2016).

 

The actual exposure concentrations were 1,366,711.66 mg/m3(10,410 ppm), 2,746,552.15 mg/m3(20,920 ppm), and 5,466,846.6 mg/m3(41,640 ppm). There were no environmental changes considered to be affecting the study results during the exposure.

Total T4 and TSH levels in females sampled on Day 13 of lactation were statistically significantly reduced when compared to the levels in the controls in those exposed to 2,746,552.15 mg/m3, but not in those exposed to 1,366,711.66 mg/m3or 5,466,846.63 mg/m3. The levels in those exposed to 5,466,846.63 mg/m3tended to be low, when compared to levels in the controls. The effect was not dose-dependent.

 

The normalised anogenital distance in male offspring exposed to 5,466,846.63 mg/m3were statistically significantly reduced when compared to the distance in controls. The effect was slight in magnitude.

 

Males exposed to 5,466,846.63 mg/m3showed reduced absolute weights of epididymides, seminal vesicles and prostate glands when compared to the weights of the controls. Females exposed to 5,466,846.63 mg/m3showed increased absolute and relative weights of the thyroid gland when compared to the weights of the controls. Atrophy of the seminiferous tubule, as seen in males exposed to 2,746,552.15 mg/m3the 90-day study, was not observed in this study. The difference might be due to the difference in the duration of exposure. There were no functional consequences of the adverse pathology in males in the current study.

 

The effects seen in the current study, taking into account the findings of the 90-day study, are insufficient to warrant the classification of HCFO 1224yd(Z) for reproductive toxicity.