Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21/06/2019 - 12/03/2020
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:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See "Principles of Method" section.
Principles of method if other than guideline:
The following deviations from the study plan occurred during the study but were not considered to have compromised the validity or integrity of the study:

1. Due to a technician oversight, some T4 serum samples at termination and PND 4 (4 total samples) were not processed within 1 hour of collection, the longest total processing time was 1 hour and 9 minutes.
2. On Day 1, all Set 1 animals were inadvertently not observed for signs of toxic or pharmacologic effects prior to exposure.
3. Post-exposure observations for signs of toxic or pharmacologic effects on various days during the Treatment and Cohabitation Phases were inadvertently performed up to 48 minutes late for some animals.
4. Physical examinations were to be completed post-exposure, however, some animals were examined pre-exposure on various days during the Treatment and Gestation Phases.
5. The presence of milk was inadvertently recorded during the unscheduled necropsy of Pup 0815 from Dam No. 4573
6. Exsanguination of Animal Nos. 2536, 2537, 2538 and 2540 following euthanasia via isoflurane inhalation cannot be confirmed because it was inadvertently not documented.
7. Animal Nos. 1517, 2534, 4571 and 4576 did not have a physical examination on LD 14 due to the fact that these animals were inadvertently marked dead in the Pristima compter system on that day.
8. By oversight, a terminal body weight was not taken for Animal No. 4577 on GD 25.
9. Paired organs for the F0 and F1 animals at Terminal necropsy were inadvertently not weighed separately
10. Due to lack of documentation, it cannot be confirmed if the thyroid (parathyroid) gland was trimmed for Pup No. 4573-1M.
11. Terminal hormone (T4) serum samples were harvested 1 to 17 minutes late for Animal Nos. 1001, 1002, 2022, 2023, 2024, 3042, 3043, 3045, 4066 and 3551. The terminal TSH serum samples were harvested 10 minutes late for Animal Nos. 1001 and 1002.
12. The wrong tube type (plain/1.1) was documented on the terminal hormone blood sample collection record on 28 August 2019.
13. The day of mating (GD 0) was missed for Animal No. 2534; therefore, GD 14 and GD 20 body weight and food consumption could not be evaluated.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test item: HCFO-1233yd(Z), also known as Amolea AS-300
Intended use: Cleaning fluid
Supplier: AGC Inc., 10, Goikaigan, Ichihara-shi, Chiba 290-8566, Japan
Description: Colorless liquid
Storage conditions: Room temperature, protected from light
Lot number: 190125
Purity: 91.531%
Retest date: 25 January 2020

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
SD rats were used due to the extensive library of historical control data.
Sex:
male/female
Details on test animals and environmental conditions:
Animals underwent a routine pre-test period for 2 weeks, and no work was conducted for the first 5 days. A twelve-hour light/dark cycle was provided and controlled via an automatic timer. Monitored and maintained within the range of 20 to 26 °C and 30 to 70%, respectively.

From arrival until one day prior to treatment, animals were pair or group housed (2 or 3 rats of the same sex per cage, respectively) in solid bottom cages with cellulose-based contact bedding.
From the initiation of treatment (pre-cohabitation phase until termination of the study), the F0 males were pair-housed [same sex and treatment group per cage (except during the cohabitation phase)] in solid bottom cages with cellulose-based contact bedding. F0 Females were pair housed during precohabitation until mating (presumed gestation) in solid bottom cages with cellulose-based contact bedding. In accordance with the OECD 421 Test Guideline, each F0 female was individually housed during presumed gestation and housed with her litter after delivery.
During cohabitation, the male and female rats were co-housed (1:1) within each treatment group in solid bottom cages with cellulose-based contact bedding.

Test animals were fed a Teklad Global 18% Protein Rodent Diet, which was freely available (as was water).

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose only
Vehicle:
unchanged (no vehicle)
Details on exposure:
Animals were exposed for 6 hours daily.
Details on mating procedure:
Within each treatment group, the F0 male and female rats were co-housed (1:1 in the male’s cage) until evidence of mating was seen or until 14 consecutive days of cohabitation had elapsed. Female rats were observed each morning for the presence of a vaginal plug or sperm in the vaginal smear. If not mated, the stage of the estrous cycle was recorded. The day on which evidence of mating was observed was defined as Day 0 of presumed gestation (GD 0). Once mated, the female rat was removed from the mating cage and housed individually until delivery. Presumed pregnant dams that did not deliver litters were euthanized on presumed GD 25.
In the absence of positive signs of mating, the morning of the last day of cohabitation was considered “presumed GD 0” for that female. When a female had no positive signs of mating (copulatory plug and/or sperm), but appeared visibly/palpably pregnant, she was allowed to deliver.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 Hours
Frequency of treatment:
2 weeks pre-cohabitation, during cohabitation (up to 2 weeks) and continuing until termination (approximately 37 days) for male animals and for 2 weeks pre-cohabitation, during cohabitation (up to 3 weeks), continuing up to GD 19 and from LD 3 (re-initiation of exposure) until LD 13 (approximately 64 days) for female animals.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
509 ppm
Dose / conc.:
1 737 ppm
Dose / conc.:
5 104 ppm
Dose / conc.:
2 964.06 mg/m³ air
Dose / conc.:
10 115.1 mg/m³ air
Dose / conc.:
29 722.1 mg/m³ air
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
Animals were observed in their cages twice daily for mortality and general condition.
Oestrous cyclicity (parental animals):
Vaginal smears were obtained and the stage of estrous cycle was determined daily for each F0 female pretest (to determine suitability for study), during pre-cohabitation, during cohabitation until mating was confirmed and at necropsy.
Sperm parameters (parental animals):
During the microscopic examination of the testes, special emphasis was placed on the stage of spermatogenesis and the interstitial testicular cell structure. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
Litters were observed as soon as possible after completion of parturition for the number of live and dead pups, runts and pup abnormalities and the sex of each pup. Thereafter, litters were observed twice daily (morning and afternoon); all pups in the litter were uniquely identified by tattoo after parturition was completed. The litters were not culled; the full number of pups remained in each litter. The presence of dead pups was recorded and these were removed from the litter as found and necropsied. Unusual observations and the absence of milk in the stomach were noted. Maternal behavior, particularly relating to maternal care of the litter, was monitored.
Postmortem examinations (parental animals):
Macroscopic postmortem examinations were performed on all F0 rats. Postmortem examinations included an external examination as well as a detailed internal examination. Special attention was paid to the organs of the reproductive system. The number of implantation sites, scars and corpora lutea was recorded for each female rat. For apparently non-pregnant animals, and for apparently empty horns, the number of uterine implantation sites was checked after staining with ammonium sulfide.
Postmortem examinations (offspring):
Macroscopic post-mortem examinations (external only) were performed on all surviving F1 pups on PND 13. Particular attention was paid to the external reproductive organs, which were examined for signs of altered development. Unusual observations, including gross abnormalities, were noted and then the carcasses were discarded.
F1 pups that died during the study were given a macroscopic postmortem examination. Particular attention was paid to the external reproductive organs which were examined for signs of altered development. Unusual observations, including gross abnormalities and the absence of milk in the stomach (up to PND 2) were noted. All carcasses were discarded following examination.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were two test item-related clinical observations in males and females exposed to 29722.10 mg/m3 HCFO 1233yd(Z). Specifically, abnormal gait and decreased activity were noted for 50% of each sex beginning on Days 1 and 2, respectively, of the pre-cohabitation phase. Although these signs did not persist past Day 4 of exposure, they were considered to be adverse due to the total number of affected animals as well as their association with previous studies with this test item.
There were no test item-related clinical observations in males or females exposed to 29722.10 mg/m3 HCFO 1233yd(Z) during the cohabitation, post cohabitation, gestation, or lactation phases or when exposed to concentrations ≤ 10115.10 mg/m3 during any phase. Any clinical observations noted were not considered to be test item-related due to their lack of relation to dose, infrequent occurrence, small magnitude, and/or common occurrence in this laboratory animal species.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no test item-related mortality over the course of this study.
Two females at 29722.10 mg/m3 (Animal Nos. 4577 and 4580) were euthanized on presumed Gestation Day 25 as they failed to litter. During the necropsy, it was confirmed that these females were not pregnant. All remaining males and females survived until scheduled termination.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on body weight for males or females over the course of this study.
Any changes noted were not considered to be related to treatment due to their small magnitude sporadic occurrence and/or lack of relation to dose.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item-related effects on food consumption for males or females over the course of this study.
Any changes noted were not considered to be related to treatment due to their small magnitude and/or lack of relation to dose.
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:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Test item-related microscopic findings were not present in the tissues examined.
All other microscopic findings occurred sporadically or at similar incidence and severity in the control and test item-exposed groups and were considered incidental and due to biological variability.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related effects on estrous cycling in females exposed to 2964.06 mg/m3 HCFO 1233yd(Z).
Although the total numbers of cycles were comparable across all groups, the number of regular cycles for females exposed to 2964.06 mg/m3 was 42.9% of the control value and the cycle length was significantly increased. Despite the test item-related effect on estrus, there was no impact on mating and fertility and the effect was therefore considered to be non-adverse.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no test item-related effects on mating and fertility.
There were 10, 9, 10, and 10 males and 10, 9, 10, and 10 females that mated following exposure to 0, 2964.06, 10115.10, and 29722.10 mg/m3 HCFO 1233yd(Z), respectively. Of the respective females, 10, 10, 10 and 8 were confirmed to be pregnant. The number of days to mating was comparable across all exposed groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
10 115.1 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was a test item-related observation in litters from dams exposed to 29722.10 mg/m3 HCFO 1233yd(Z). The litter observation, little or no milk present in the stomach, was noted from 50% (4 of 8) of the litters on 17 different occasions. As there were no test item-related effects on the body weights or viability indices for the F1 pups at this exposure level, the observation was considered to be non-adverse.

Any additional observations noted were considered not to be treatment-related due to their common occurrence in this age and species of laboratory animal.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no test item-related effects on the following indices: post-implantation survival, live birth, and live litter size on PNDs 1, 4, 7, 11, and 13.
Although the viability index at PND 4 in pups from dams exposed to 29722.10 mg/m3 HCFO 1233yd(Z) was statistically significantly decreased compared to the control value, the difference is considered to be minimal. Additionally, the viability index was within historical control data for the Testing Facility. Any additional differences were considered not to be test item-related due to their small magnitude and/or a lack of relation to the level of exposure.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on pup sex or body weight.
Any differences noted were considered not to be test item-related as they were within the range of the control values and/or due to their small magnitude.
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:
no effects observed
Description (incidence and severity):
There were no test item-related thyroid hormone changes.
All differences between control and treated mean values, including those that were statistically significant, were considered not to be test item-related as they lacked a dose relationship and/or there was general overlap between individual control and treated values.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no test item-related effects on anogenital distance (F1 males and females)
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no test item-related effects on nipple retention
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Test item-related differences in organ weights did not occur. All organ weight changes, including those that were statistically significant, were considered not to be test item-related because they were sporadic, independent of exposure level, small in magnitude and/or did not have macroscopic or microscopic correlates.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related macroscopic findings in the F1 pups that survived until scheduled termination or the unscheduled decedents; all animals were within normal limits.
The macroscopic findings for one unscheduled decedent exposed to 29722.10 mg/m3 HCFO 1233yd(Z) (Animal No. 4575-0740) was undetermined due to severe autolysis.
Histopathological findings:
no effects observed
Description (incidence and severity):
Test item-related microscopic findings were not present in the tissues examined.
All other microscopic findings occurred sporadically or at similar incidence and severity in the control and test item-exposed groups and were considered incidental and due to biological variability.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
29 722.1 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:
Two test item-related clinical observations (abnormal gait and decreased activity) were noted for 50% of both males and females exposed to 29722.10 mg/m3 HCFO 1233yd(Z) during the precohabitation phase. Although these signs did not persist past Day 4 of exposure, they were considered to be adverse due to the total number of affected animals as well as their association with previous studies with this test item.

There were test item-related decreases in the number of regular estrous cycles in dams exposed to 29722.10 mg/m3 HCFO 1233yd(Z) as well as an increase in cycle length. Specifically, the number of regular cycles was 42.9% of the control value. Despite the test item-related effect on estrous, there was no impact on mating and fertility. There was a test item-related observation in the litters from dams exposed to 29722.10 mg/m3 HCFO 1233yd(Z). The litter observation, little or no milk present in the stomach, was noted in 50% (4 of 8) of the litters on 17 different occasions. As there were no test item-related effects on the body weights or viability indices for the F1 pups exposure at this level, the observation was considered to be non-adverse.

There was no test item-related mortality in F0 males or females over the course of this study. Additionally, there were no test item-related effects on body weight, food consumption, mating and fertility, organ weights, macroscopic or microscopic findings.

There were 10, 9, 10, and 10 males and 10, 9, 10, and 10 females that mated following exposure to 0, 2964.06, 10115.10, and 29722.10 mg/m3 HCFO 1233yd(Z), respectively. Of the respective females, 10, 10, 10 and 8 were confirmed to be pregnant. There were no test item-related effects on the following indices: post-implantation survival, live birth, and live litter size on PNDs 1, 4, 7, 11, and 13. Also, there were no test item-related effects on the length of gestation, total corpora lutea, number of implantations, pre- or post-implantation loss, average number of viable pups on PNDs 1, 4, 7, 11 and 13, pup sex or body weight, anogenital distance (F1 males and females) or nipple retention (F1 males only) or macroscopic findings.

In conclusion, due to the adverse clinical signs noted at the highest exposure level for the F0 generation and the lack of adverse test item-related findings in the F1 generation, the F0 systemic no observable adverse effect level (NOAEL) was 10115.10 mg/m3 while the reproductive and developmental NOAEL was 29722.10 mg/m3.
Executive summary:

Sprague-Dawley CD rats (10 animals/sex/group) were exposed via nose-only inhalation to 0 (air control), 2964.06, 10115.10 or 29722.10 mg/m3of HCFO-1233yd(Z). Exposures were 360 minutes in duration for 2 weeks pre-cohabitation, during cohabitation (up to 2 weeks) and continuing until termination (approximately 37 days) for male animals and for 2 weeks pre-cohabitation, during cohabitation (up to 3 weeks), continuing up to GD 19 and from LD 3 (re-initiation of exposure) until LD 13 (approximately 64 days) for female animals. At the end of the treatment period, animals were euthanized and necropsied. Parameters evaluated during the study for the F0animals were: viability, clinical observations, body weights, food consumption, estrous cycling, mating and fertility, parturition and littering, hormone analysis, organ weights, macroscopic observations and microscopic pathology. Parameters evaluated for the F1 pups were: viability, clinical observations, sex, body weights, ano-genital distance, nipple retention, hormone analysis and macroscopic observations.

Two adverse test item-related clinical observations (abnormal gait and decreased activity) were noted for 50% of both males and females at 29722.10 mg/m3during the precohabitation phase. Although these signs did not persist past Day 4 of exposure, they were considered to be adverse due to the total number of affected animals as well as their association with previous studies with this test item.

There were non-adverse test item-related decreases in the number of regular estrous cycles in dams exposed to 29722.10 mg/m3as well as an increase in cycle length. Specifically, the number of regular cycles was 42.9% of the control value. Despite the test item-related effect on estrous cycling, there was no impact on mating and fertility. There was a test item-related litter observation at 29722.10 mg/m3. The litter observation, little or no milk present in the stomach, was noted in 50% (4 of 8) of the litters on 17 different occasions. As there were no test item-related effects on the body weights or viability indices for the F1pups exposed at this exposure level, the observation was considered to be non-adverse.

There was no test item-related mortality in F0males or females over the course of this study. Additionally, there were no test item-related effects on body weight, food consumption, mating and fertility, organ weights, macroscopic or microscopic findings.

There were 10, 9, 10, and 10 males and 10, 9, 10, and 10 females that mated following exposure to 0, 2964.06, 10115.10, and 29722.10 mg/m3, respectively. Of the respective females, 10, 10, 10 and 8 were confirmed to be pregnant. There were no test item-related effects on the following indices: post-implantation survival, live birth, and live litter size on PNDs 1, 4, 7, 11, and 13.Also, there were no test item-related effects on the length of gestation, total corpora lutea, number of implantations, pre- or post-implantation loss, average number of viable pups on PNDs 1, 4, 7, 11 and 13, pup sex or body weight, anogenital distance (F1males and females) or nipple retention (F1males only) or macroscopic findings.

In conclusion, due to the adverse clinical signs noted at the highest exposure level for the F0 generation and the lack of adverse test item-related findings in the F1generation, the F0 systemic no observable adverse effect level (NOAEL) was 10115.10 mg/m3while the reproductive and developmental NOAEL was 29722.10 mg/m3.