(E)-1,2-difluoroethylene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive/Developmental Toxicity Screening Study in the Rat by Inhalation Administration (OECD 421)

The purpose of this study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, with administration of the test item, TKN1, by inhalation (snout only) administration for at least 4 weeks.

 

Three groups of 10 male and 10 female rats received TKN1 at target exposure concentrations of 3000, 9000 or 27000 ppm by inhalation administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the air control.

 

During the study, clinical condition, body weight, food consumption, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

 

Results

 

F0 responses

Body weight gain in animals exposed to 26900 ppm was lower in males over the four weeks of exposure and females over the two weeks of exposure prior to pairing.

There were no test item-related effects on clinical observations, food consumption, organ weights or macroscopic and microscopic pathology.

Estrous cyclicity, pre-coital interval, gestation length, mating performance and fertility were unaffected by the test item.

There was no effect on circulating levels of thyroxine (T4) in adult males at termination.

 

F1 responses

The clinical condition, litter size, sex ratio, post birth survival indices and growth of offspring were unaffected by parental exposure.

The ano-genital distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

No macroscopic findings considered to be related to paternal treatment were recorded.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

 

Conclusion

It was concluded that inhalation administration of TKN1 to parental Sprague Dawley rats at concentrations of 3080, 9490 or 26900 ppm for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment was associated with signs of parental toxicity at 26900 ppm, manifest as lower body weight gain of males and females.  

In the context of this study, TKN1 showed no effects on reproductive/developmental performance.

In the context of this study, TKN1 showed no evidence of being an endocrine disrupter.  There were no effects on thyroxine levels in adults or offspring, organ weights, external examination of offspring, and nipple counts in male offspring.

Within the constraints of this screening study, the no-observed-adverse-effect level (NOAEL) of TKN1 for local/systemic toxicity and reproductive/developmental toxicity was considered to be 26900 ppm.

 

 

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (pre-study chemistry): 02 July 2020; Experimental completion date (pathology): 07 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Remarks:

None that were considered to have affected the integrity or validity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The purpose of this study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints

Specific details on test material used for the study:

Test item: TKN1
Test item identity (including alternative names): (E)-1,2-difluoroethylene
Appearance: Liquefied gas
Storage conditions: At ambient temperature (15 to 25°C)
Supplier: Sponsor
Lot numbers: N1200114, N1200115, N1200116, N1200215, N1200302
Expiry date: 13 August 2022 for Lots N1200114, N1200115, N1200116 and N1200215 and 09 September 2022 for Lot N1200302
Purity: >99.999%

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain/Species Crl:CD(SD) rat.

Supplier Charles River (UK) Ltd.

Number of animals ordered 44 males and 48 females.

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Males: 19 days before commencement of treatment.
Females: 5 days before commencement of estrous cycle evaluation and 19 days before commencement of treatment.

Age of the animals at the start of treatment Males 10 to 12 weeks old.
Females 10 to 13 weeks old.

Weight range of the animals at the start of treatment Males 311 to 384 g.
Females 209 to 289 g.

Allocation and Identification
Allocation On arrival and non-selective allocation to cages.

Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.

On Day 1 of study all animals were weighed and body weights were reviewed by Study Management. Body weight of animals did not exceed 20% of the mean for each sex. Allocation may have been adjusted to reduce inter /intra-group variation.

Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Replacement
Before the commencement of treatment, study allocation was revised to replace females with atypical estrous cycles with spare females showing normal estrous cycles of suitable weight from the same batch.

Replacement before treatment Atypical estrous cycles 2 females

Animal Care and Husbandry
Environmental Control
Rodent facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24C and 40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.
Nesting material Paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding.

Diet Supply
Diet SDS VRF1 Certified pelleted.
A sample (250 g) of the first batch of diet used was retained within Pharmacy (frozen -10 to -30°C) until finalization of the report. The samples was discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted (except during exposure).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted (except during exposure).

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding, nesting material, plastic shelter and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

snout only

Vehicle:

air

Details on exposure:

The inhalation route of administration was chosen to simulate the conditions of potential human exposure.

Exposure System
Components of the system:
• Exposure System:
• Flow through nose-only chamber
• Aluminum alloy construction comprising a base unit, an animal exposure section, a top section and a pre-chamber

• Animal Restraint:
• Plastic nose-only restraint tube

• Aerosol Generation:
• Pressurized cylinder
• Flow rate control was achieved by using a Porter flowmeter fitted with a needle valve

• Extract Airflow:
• Drawn by in-house vacuum system
• Filtered locally
• Extract flow: 20-24 L/minute

• Airflow Monitoring:
• High quality tapered tube flowmeters - calibrated daily
• In-line flowmeters monitored continuously

• System Containment:
• Systems housed in separate ventilated cabinets

Details on mating procedure:

Pairing commenced After a minimum of two weeks of exposure.
Male/female ratio 1:1 from within the same exposure groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Atmosphere Concentration:
Atmosphere samples collected as follows:
-Sample collection media: All plastic syringe fitted with a Luer-lok
-Sample frequency: 1 sample from Group 1/day; 3 samples/test group/day
-Sample location: Animal exposure port

Analytical Method: GC

GC conditions:
Column: ZB-5, 30 m x 0.32 mm ID, 0.25 µm film thickness
Column temperature: 360 °C
Injection mode: Split
Injector temperature: 100 °C
Injection volume: 500 µL
Detector type: Flame Ionisation
Detector temperature: 250 °C
Oven temperature programme: 30°C
Run time: 1 minute
Approximate retention time: 0.64 minutes.
Gases:
Carrier: Helium at 4 mL/min
Purge flow Helium at 3.0 mL/min (split ratio 1:20)
Make up: Helium at 30 mL/min
Oxidant: Air at 400 mL/min
Fuel: Hydrogen at 40 mL/min

The GC system was calibrated using external standards. Peak area data acquired by the data capture software using a 1st order fit was subjected to least squares regression analysis with 1/x weighting.
GC Conditions were established using a Shimadzu GC-2010 Plus Gas Chromatograph (including Shimadzu AOC-20i Autosampler and FID detector).

The mean achieved atmosphere concentrations were 103, 105 and 100% of target for Groups 2, 3 and 4, respectively.
The variation between samples was greater than anticipated with a number of samples excluded due to suspected leaks from the syringes. This is considered to be related to the attached luer-lok fittings malfunctioning or being mis-handled during sample processing. However, the mean reported atmosphere concentrations are considered to be an accurate reflection of the atmospheres the rats were exposed to. This is supported by the nominal atmosphere concentration which was very close to the mean total achieved concentration.

Duration of treatment / exposure:

Duration of daily exposure 360 minutes.

Frequency of treatment:

Daily

Details on study schedule:

Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the air control.

Animals of the F1 generation were not dosed.

Dose / conc.:

0 ppm

Remarks:

Air control

Dose / conc.:

3 000 ppm

Remarks:

Achieved: 3080 ppm

Dose / conc.:

9 000 ppm

Remarks:

Achieved: 9490 ppm

Dose / conc.:

27 000 ppm

Remarks:

Achieved 26900 ppm

No. of animals per sex per dose:

10 males/ 10 females

Control animals:

yes, concurrent no treatment

Details on study design:

Rationale for Dose Level Selection
The target exposure concentrations used in this study (0, 3000, 9000 and 27000 ppm) were selected in conjunction with the Sponsor.

Exposure concentrations were selected in conjunction with the Sponsor based on the results of a 14-Day study in the rat in which TKN1 at achieved concentrations of 4903, 14218 or 47942 ppm were tolerated in life but were associated with adverse microscopic pathology findings in the heart (left ventricular cardiomegaly) at target concentrations ≥15000 ppm and in the nose/turbinates (degeneration of the vomeronasal organ) at target concentrations ≥15000 ppm. In a 4-Week toxicity study (Study Number NT27RG), inhalation exposures for 5 days/week were well tolerated at target concentrations of 3000, 10000 and 15000 ppm, with lower body weight gains noted in females. Microscopic pathology findings after 4 weeks included neuronal cell degeneration in the vomeronasal organ at ≥ 3,000 ppm.

Target exposure concentrations of 3000, 9000 and 27000 ppm were selected for this study. The high exposure concentration should have elicited a body weight effect and could have caused impaired mating performance and the low exposure concentration was expected to be a no adverse effect concentration

Positive control:

None

Parental animals: Observations and examinations:

Serial Observations:
Clinical Observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Exposure:
Detailed observations were performed to establish and confirm a pattern of signs in association with exposure according to the following schedule:

F0 males:
Week 1 - daily
Week 2 onwards - Once each week

F0 females:
Week 1 - daily
Week 2 onwards - Once each week
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 6 and 12

Detailed observations were recorded at the following times in relation to exposure:
• Pre-exposure observation
• As each animal is returned to its home cage
• As late as possible in the working day

Clinical Signs:
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
F0 males Once each week
F0 females Once each week
Gestation phase - Days 0, 7, 14 and 20 after mating
Lactation phase - Days 1, 7 and 13 of lactation

Body Weight:
The weight of animals was recorded as follows:
F0 males: Before exposure on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.
F0 females: Weekly during acclimatization
Before exposure on the day that treatment commenced (Week 0) and weekly before mating.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 6, and 13 of lactation.
On the day of necropsy.

Food Consumption:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 animals: Weekly, before pairing. For males in Week 2, food consumption was recorded over six not seven days for logistical reasons.
Food consumption was not recorded for males and females during the period when paired for mating, but recommenced for males once pairing of all the animals was completed.

For females after mating food consumption was performed to match the body weight recording:
Days 0-7, 7-14 and 14-20 after mating
Days 1-4, 4-6 and 6-13 of lactation.

From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.


Parturition Observations and Gestation Length:
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.

Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears: Using cotton swabs during the following phases:
For 14 days before start of exposure (all females including spares); animals that failed to exhibit regular 4-5 day cycles were not allocated to study.
For 15 days before pairing.
For four days before scheduled termination (Days 10 to 13 of lactation).

Wet smears: Using pipette lavage during the following phases:
After pairing until mating (for maximum of 14 days).

Estrous Cycle:
The incidence and percentage females showing the following classifications of estrous cycles before treatment commenced are presented:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 5 days)
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus (beginning before pairing)

Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination.

Sperm parameters (parental animals):

See light microscopy

Litter observations:

Records Made During Littering Phase:
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.

Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.

Individual offspring body weights: Days 1, 4, 7 and 13 of age.

Ano-genital distance: Day 1 - all F1 offspring.

Nipple/areolae count: Day 13 of age - male offspring.

Postmortem examinations (parental animals):

Terminal Investigations:
Method of Kill:
All adult animals: Carbon dioxide asphyxiation (no carbon dioxide exposure until after completion of blood sampling).

Necropsy:
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of Necropsy:
F0 males: After at least 4 weeks of treatment.
F0 females: Day 13 of lactation.


Females:
The following were recorded:
Each uterine horn Number of implantation sites was counted.

Organ Weights:
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation:
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:

Testes: Initially in modified Davidson’s fluid.

Histology:
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List: All F0 animals in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 animals.

Routine staining: Sections were stained with hematoxylin and eosin; except testes which are also stained with Periodic Acid Schiffs (PAS).

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Scheduled kill All adult animals in Groups 1 and 4
All adult animals of all groups.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell or stage-specificity of testicular findings was noted.

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.


PLEASE SEE TABLE IN "ANY OTHER INFORMATION ON METHODS"; "PATHLOGY PROCEDURES FOR ALL F0 PARENTAL ANIMALS.

Postmortem examinations (offspring):

Time of Necropsy:
F1 offspring Day 13 of age.

Offspring
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination: All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.#

Thyroid glands were preserved from one male and one female in each litter, where possible.

Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 and 13 of age. Group mean litter size and SD were calculated from the individual litter values.

Statistics:

PLEASE REFER TO ANY OTHER INFORMATION ON METHODS

Reproductive indices:

Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) x 100

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born / number pregnant) x 100

Offspring viability indices:

Survival Indices
The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 / Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering/ Number of live offspring on Day 4 (after selection for thyroid hormone bleed) x 100

Group mean values were calculated from individual litter values

Offspring Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on
Days 1, 4 (before and after selection for thyroid hormone bleed) and 13 of age.

Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item-related clinical signs noted at the regular physical examination or in association with exposure.

Signs associated with the administration procedure included wet fur and red staining on the head, nose and/or eyes on return to the home cage. These were seen in animals from all groups, including controls, generally disappeared by the end of the working day and are considered to be due to the method and duration of restraint.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

After four weeks of exposure, group mean bodyweight gain of males exposed to 26900 ppm was slightly lower than the concurrent control (0.85X Control). The magnitude of response was greater in Week 1.

Group mean bodyweight gain of females exposed to 26900 ppm was slightly lower than the concurrent control (0.44X Control respectively) over the two weeks prior to pairing. Group mean body weight change of females exposed to 26900 ppm was similar to controls after mating and during lactation.

There were no test item related effects on body weight change in animals exposed to 3080 or 9490 ppm

PLEASE REFER TO THE FOLLOWING ATTACHED TABLES: "BODY WEIGHT - GROUP MEAN VALUES FOR MALES (F0)" and "BODY WEIGHT - GROUP MEAN VALUES FOR FEMALES (F0)"

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related effects on group mean food consumption of males throughout the study or of females, prior to mating, after mating or during lactation.

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid Hormone Sampling T4 Analysis:
There were no statistically significant or test item-related effects on the group mean serum concentration of thyroxine (T4) at termination of adult males, or male and female offspring on Day 13 of age.

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):

There were no test item-related microscopic changes in females killed on Day 13 post partum and in males killed after 4 weeks of exposure.

In males killed after 4 weeks of exposure, the testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

All microscopic findings were considered spontaneous and incidental.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females assigned to the study had regular estrous cycles prior to the start of treatment.

There were no test item related findings on estrous cycles in the first 2 weeks of treatment prior to pairing.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In males killed after 4 weeks of exposure, the testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item related effects on mating performance or fertility.

The pre-coital interval was 1-4 days for the majority of parings, indicating mating occurred at the first estrous opportunity. One pairing at 26900 ppm did not result in pregnancy until the last day of the pairing period (Day 14), the female was noted as acyclic during pairing. In isolation this late mating is considered to be coincidental and not test-item related.

All pairings showed evidence of mating. The percentage mating and the conception and fertility indices were all 100%.

There were no test item-related effects on gestation length or the gestation index. All females produced a live litter.

Prior to termination all females smeared were in diestrus. There were no test item related effects.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicty

Effect level:

26 900 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: There were no test item-related effects on clinical observations, food consumption, organ weights or macroscopic and microscopic pathology.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive/developmental toxicity

Effect level:

26 900 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on reproductive/developmental performance.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item related effects related to parental treatment in the few clinical signs noted in the offspring from birth to Day 13 of age.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on group mean body weight on Day 1 of age or group mean body weight gain to Day 13 of age.

PLEASE REFER TO THE ATTACHED TABLES: "BODY WEIGHT - GROUP MEAN VALUES FOR MALE OFFSPRING (F1)" and "BODY WEIGHT - GROUP MEAN VALUES FOR FEMALE OFFPSRING (F1)"

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 statistically significant or test item-related effects on the group mean serum concentration of thyroxine (T4) of male and female offspring on Day 13 of age.

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 related to parental treatment on the ano-genital distance on Day 1 of age, corrected for the body weight.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no nipples/areolae noted in any male offspring on Day 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic changes at necropsy of offspring.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Litter size, Sex Ratio and Survival Indices:
There were no treatment related effects on the number of implantations, litter size or sex ratio. The offspring survival indices were similar in all groups with no adverse effects.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

26 900 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on reproductive/developmental performance.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Atmosphere Analysis

Summary data are presented below:

Group	Chamber Concentration (ppm)
	Target	Achieved
2	3000	3080
3	9000	9490
4	27000	26900

 

The achieved chamber concentrations were 103, 105 and 100% of the target concentrations for Groups 2, 3 and 4 respectively.

Conclusions:

It was concluded that inhalation administration of TKN1 to parental Sprague Dawley rats at concentrations of 3080, 9490 or 26900 ppm for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment was associated with signs of parental toxicity at 26900 ppm, manifest as lower body weight gain of males and females.
In the context of this study, TKN1 showed no effects on reproductive/developmental performance.
In the context of this study, TKN1 showed no evidence of being an endocrine disrupter. There were no effects on thyroxine levels in adults or offspring, organ weights, external examination of offspring, and nipple counts in male offspring.
Within the constraints of this screening study, the no-observed-adverse-effect level (NOAEL) of TKN1 for local/systemic toxicity and reproductive/developmental toxicity was considered to be 26900 ppm.

Executive summary:

The purpose of this study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, with administration of the test item, TKN1, by inhalation (snout only) administration for at least 4 weeks.

 

Three groups of 10 male and 10 female rats received TKN1 at target exposure concentrations of 3000, 9000 or 27000 ppm by inhalation administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the air control.

 

During the study, clinical condition, body weight, food consumption, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

 

Results

 

F0 responses

Summary data for chamber concentrations is presented below:

Group	Chamber Concentration (ppm)
	Target	Achieved
2	3000	3080
3	9000	9490
4	27000	26900

 

The achieved chamber concentrations were 103, 105 and 100% of the target concentrations for Groups 2, 3 and 4 respectively.

Body weight gain in animals exposed to 26900 ppm was lower in males over the four weeks of exposure and females over the two weeks of exposure prior to pairing.

There were no test item-related effects on clinical observations, food consumption, organ weights or macroscopic and microscopic pathology. Estrous cyclicity, pre-coital interval, gestation length, mating performance and fertility were unaffected by the test item.

There was no effect on circulating levels of thyroxine (T4) in adult males at termination.

 

F1 responses

The clinical condition, litter size, sex ratio, post birth survival indices and growth of offspring were unaffected by parental exposure.

The ano-genital distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

No macroscopic findings considered to be related to paternal treatment were recorded.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

 

Conclusion

It was concluded that inhalation administration of TKN1 to parental Sprague Dawley rats at concentrations of 3080, 9490 or 26900 ppm for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment was associated with signs of parental toxicity at 26900 ppm, manifest as lower body weight gain of males and females.  

In the context of this study, TKN1 showed no effects on reproductive/developmental performance.

In the context of this study, TKN1 showed no evidence of being an endocrine disrupter.  There were no effects on thyroxine levels in adults or offspring, organ weights, external examination of offspring, and nipple counts in male offspring.

Within the constraints of this screening study, the no-observed-adverse-effect level (NOAEL) of TKN1 for local/systemic toxicity and reproductive/developmental toxicity was considered to be 26900 ppm.

 

 

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

26 900 ppm

Study duration:

subacute

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Refer to effects of fertility section.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

screening study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (pre-study chemistry) 02 July 2020; Experimental completion date (pathology) 07 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Organization for Economic Co-operation and Development: Testing of Chemicals (Guideline 421; Reproduction/Developmental Toxicity Screening Test; 29 July 2016).

Deviations:

no

Remarks:

none that impacted integrity or validity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item: TKN1
Test item identity (including alternative names): (E)-1,2-difluoroethylene
Appearance: Liquefied gas
Storage conditions: At ambient temperature (15 to 25°C)
Supplier: Sponsor
Lot numbers: N1200114, N1200115, N1200116, N1200215, N1200302
Expiry date: 13 August 2022 for Lots N1200114, N1200115, N1200116 and N1200215 and 09 September 2022 for Lot N1200302
Purity: >99.999%

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

Supplier Charles River (UK) Ltd.

Number of animals ordered 44 males and 48 females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Males: 19 days before commencement of treatment.
Females: 5 days before commencement of estrous cycle evaluation and 19 days before commencement of treatment.

Age of the animals at the start of treatment Males 10 to 12 weeks old.
Females 10 to 13 weeks old.

Weight range of the animals at the start of treatment Males 311 to 384 g.
Females 209 to 289 g.

Allocation and Identification
Allocation On arrival and non-selective allocation to cages.

Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.

On Day 1 of study all animals were weighed and body weights were reviewed by Study Management. Body weight of animals did not exceed 20% of the mean for each sex. Allocation may have been adjusted to reduce inter /intra-group variation.

Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Replacement
Before the commencement of treatment, study allocation was revised to replace females with atypical estrous cycles with spare females showing normal estrous cycles of suitable weight from the same batch.

Replacement before treatment Atypical estrous cycles 2 females

Animal Care and Husbandry
Environmental Control
Rodent facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used throughout the study except during pairing.

Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.

Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.
Nesting material Paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding.

Diet Supply
Diet SDS VRF1 Certified pelleted.
A sample (250 g) of the first batch of diet used was retained within Pharmacy (frozen -10 to -30°C) until finalization of the report. The samples was discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted (except during exposure).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted (except during exposure).

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding, nesting material, plastic shelter and Aspen chew blocks.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

snout only

Vehicle:

air

Details on exposure:

Exposure System

Components of the system:
• Exposure System:
• Flow through nose-only chamber
• Aluminum alloy construction comprising a base unit, an animal exposure section, a top section and a pre-chamber

• Animal Restraint:
• Plastic nose-only restraint tube

• Aerosol Generation:
• Pressurized cylinder
• Flow rate control was achieved by using a Porter flowmeter fitted with a needle valve

• Extract Airflow:
• Drawn by in-house vacuum system
• Filtered locally
• Extract flow: 20-24 L/minute

• Airflow Monitoring:
• High quality tapered tube flowmeters - calibrated daily
• In-line flowmeters monitored continuously

• System Containment:
• Systems housed in separate ventilated cabinets

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Atmosphere Concentration:
Atmosphere samples collected as follows:
-Sample collection media: All plastic syringe fitted with a Luer-lok
-Sample frequency: 1 sample from Group 1/day; 3 samples/test group/day
-Sample location: Animal exposure port

Analytical Method: GC

GC conditions:
Column: ZB-5, 30 m x 0.32 mm ID, 0.25 µm film thickness
Column temperature: 360 °C
Injection mode: Split
Injector temperature: 100 °C
Injection volume: 500 µL
Detector type: Flame Ionisation
Detector temperature: 250 °C
Oven temperature programme: 30°C
Run time: 1 minute
Approximate retention time: 0.64 minutes.
Gases:
Carrier: Helium at 4 mL/min
Purge flow Helium at 3.0 mL/min (split ratio 1:20)
Make up: Helium at 30 mL/min
Oxidant: Air at 400 mL/min
Fuel: Hydrogen at 40 mL/min

The GC system was calibrated using external standards. Peak area data acquired by the data capture software using a 1st order fit was subjected to least squares regression analysis with 1/x weighting.
GC Conditions were established using a Shimadzu GC-2010 Plus Gas Chromatograph (including Shimadzu AOC-20i Autosampler and FID detector).

The mean achieved atmosphere concentrations were 103, 105 and 100% of target for Groups 2, 3 and 4, respectively.

Details on mating procedure:

Pairing commenced After a minimum of two weeks of texposure.
Male/female ratio 1:1 from within the same exposure groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Duration of treatment / exposure:

Males Two weeks pre-pairing up to necropsy after minimum of four weeks.
Females Two weeks before pairing, then throughout pairing and gestation to Day 20 of gestation inclusive, and Day 4 to 12 of lactation.
Treatment withdrawn after Day 20 of gestation to avoid littering during exposure, and up to Day 4 of lactation to avoid separation of dam and litter in early lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Duration of daily exposure 360 minutes.

Duration of test:

approximately six weeks (males) and up to nine weeks (females) (including a two week pre-pairing
phase, pairing, gestation and early lactation for females)

Dose / conc.:

3 000 ppm

Remarks:

Achieved: 3080 ppm

Dose / conc.:

9 000 ppm

Remarks:

Achieved: 9490 ppm

Dose / conc.:

27 000 ppm

Remarks:

Achieved: 26900 ppm

No. of animals per sex per dose:

10 males, 10 females

Control animals:

yes, concurrent no treatment

Details on study design:

The target exposure concentrations used in this study (0, 3000, 9000 and 27000 ppm) were selected in conjunction with the Sponsor.
Exposure concentrations were selected in conjunction with the Sponsor based on the results of a 14-Day study in the rat in which TKN1 at achieved concentrations of 4903, 14218 or 47942 ppm were tolerated in life but were associated with adverse microscopic pathology findings in the heart (left ventricular cardiomegaly) at target concentrations ≥15000 ppm and in the nose/turbinates (degeneration of the vomeronasal organ) at target concentrations ≥15000 ppm. In a 4-Week toxicity study (Study Number NT27RG), inhalation exposures for 5 days/week were well tolerated at target concentrations of 3000, 10000 and 15000 ppm, with lower body weight gains noted in females. Microscopic pathology findings after 4 weeks included neuronal cell degeneration in the vomeronasal organ at ≥ 3,000 ppm.
Target exposure concentrations of 3000, 9000 and 27000 ppm were selected for this study. The high exposure concentration should have elicited a body weight effect and could have caused impaired mating performance and the low exposure concentration was expected to be a no adverse effect concentration.

Maternal examinations:

Clinical Observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Exposure:
Detailed observations were performed to establish and confirm a pattern of signs in association with exposure according to the following schedule:

F0 males: Week 1 - daily
Week 2 onwards - Once each week
F0 females: Week 1 - daily
Week 2 onwards - Once each week
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 6 and 12

Detailed observations were recorded at the following times in relation to exposure:
• Pre-exposure observation
• As each animal is returned to its home cage
• As late as possible in the working day

Clinical Signs:
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:

F0 males: Once each week
F0 females: Once each week
Gestation phase - Days 0, 7, 14 and 20 after mating
Lactation phase - Days 1, 7 and 13 of lactation

Body Weight:
The weight of animals was recorded as follows:

F0 males: Before exposure on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.

F0 females: Weekly during acclimatization
Before exposure on the day that treatment commenced (Week 0) and weekly before mating.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 6, and 13 of lactation.
On the day of necropsy.

Food Consumption:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 animals Weekly, before pairing.
Food consumption was not recorded during the period when paired for mating, but recommenced for males once pairing of all the animals was completed.

For females after mating food consumption was performed to match the body weight recording:
Days 0-7, 7-14 and 14-20 after mating
Days 1-4, 4-6 and 6-13 of lactation.

From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

Thyroid Hormone Analysis (T4/TSH)

Samples from offspring on Day 13 of age and adult males were assessed for levels of Thyroxine (T4).

Necropsy:

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of Necropsy: F0 females- Day 13 of lactation.

PLEASE REFER TO TABLE IN ANY OTHER INFORMATION ON METHODS: "PATHOLOGY PROCEDURES FOR ALL F0 PARENTAL ANIMALS

Females
The following were recorded:
Each uterine horn Number of implantation sites was counted.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes Initially in modified Davidson’s fluid.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List All F0 animals in Groups 1 and 4 at scheduled termination.
Abnormalities only All F0 animals.
Routine staining Sections were stained with hematoxylin and eosin; except testes which are also stained with Periodic Acid Schiffs (PAS).

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Scheduled kill All adult animals in Groups 1 and 4

All adult animals of all groups. Abnormalities.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell or stage-specificity of testicular findings was noted.

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Ovaries and uterine content:

Estrous Cycle:
Dry and wet smears were taken as follows:
Dry smears:
Using cotton swabs during the following phases:
For 14 days before start of exposure (all females including spares); animals that failed to exhibit regular 4-5 day cycles were not allocated to study.
For 15 days before pairing.
For four days before scheduled termination (Days 10 to 13 of lactation).

Wet smears:
Using pipette lavage during the following phases:
After pairing until mating (for maximum of 14 days).

Parturition Observations and Gestation Length:
Duration of gestation Time elapsing between the detection of mating and commencement of parturition.

Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Fetal examinations:

Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination: All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.

Thyroid glands were preserved from one male and one female in each litter, where possible.

Records Made During Littering Phase
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.

Statistics:

PLEASE REFER TO ANY OTHER INFORMATION ON MATERIALS AND METHODS

Indices:

PLEASE REFER TO ANY OTHER INFORMATION ON MATERIALS AND METHODS

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item-related clinical signs noted at the regular physical examination or in association with exposure.
Signs associated with the administration procedure included wet fur and red staining on the head, nose and/or eyes on return to the home cage. These were seen in animals from all groups, including controls, generally disappeared by the end of the working day and are considered to be due to the method and duration of restraint.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

After four weeks of exposure, group mean bodyweight gain of males exposed to 26900 ppm was slightly lower than the concurrent control (0.85X Control). The magnitude of response was greater in Week 1.

Group mean bodyweight gain of females exposed to 26900 ppm was slightly lower than the concurrent control (0.44X Control respectively) over the two weeks prior to pairing. Group mean body weight change of females exposed to 26900 ppm was similar to controls after mating and during lactation.

There were no test item related effects on body weight change in animals exposed to 3080 or 9490 ppm.

PLEASE REFER TO THE ATTACHED TABLES: "BODY WEIGHT - GROUP MEAN VALUES FOR MALES (F0)" and "BODY WEIGHT - GROUP MEAN VALUES FOR FEMALES BEFORE PAIRING (F0)"

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related effects on group mean food consumption of males throughout the study or of females, prior to mating, after mating or during lactation.

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid Hormone Sampling T4 Analysis
There were no statistically significant or test item-related effects on the group mean serum concentration of thyroxine (T4) at termination of adult males, or male and female offspring on Day 13 of age.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related changes in organ weight parameters in males killed after 4 weeks of exposure.
All differences in organ weight parameters in males were consistent with normal variation and considered incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic changes in females killed on Day 13 post partum.

All macroscopic findings were considered spontaneous and/or incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic changes in females killed on Day 13 post partum and in males killed after 4 weeks of exposure.

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no test item-related effects on gestation length or the gestation index. All females produced a live litter.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All pairings showed evidence of mating. The percentage mating and the conception and fertility indices were all 100%.

Other effects:

no effects observed

Description (incidence and severity):

All females assigned to the study had regular estrous cycles prior to the start of treatment.

There were no test item related findings on estrous cycles in the first 2 weeks of treatment prior to pairing.

There were no test item related effects on mating performance or fertility.

The pre-coital interval was 1-4 days for the majority of parings, indicating mating occurred at the first estrous opportunity. One pairing at 26900 ppm did not result in pregnancy until the last day of the pairing period (Day 14), the female was noted as acyclic during pairing. In isolation this late mating is considered to be coincidental and not test-item related.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

26 900 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: There were no test item-related effects on clinical observations, food consumption, organ weights or macroscopic and microscopic pathology.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive/developmental toxicity

Effect level:

26 900 ppm

Based on:

test mat.

Basis for effect level:

other: no effects on reproductive/developmental performance.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on group mean body weight on Day 1 of age or group mean body weight gain to Day 13 of age.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no treatment related effects on the number of implantations, litter size. The offspring survival indices were similar in all groups with no adverse effects.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no treatment related effects on sex ratio.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment related effects on the number of implantations, litter size or sex ratio. The offspring survival indices were similar in all groups with no adverse effects.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no test item related effects related to parental treatment on the ano-genital distance on Day 1 of age, corrected for the body weight.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There were no treatment related effects on the number of implantations, litter size or sex ratio. The offspring survival indices were similar in all groups with no adverse effects.

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Offspring Nipple Count
There were no nipples/areolae noted in any male offspring on Day 13.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive/developmental toxicity

Effect level:

26 900 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on developmental performance.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

 Atmosphere Analysis

Summary data are presented below:

Group	Chamber Concentration (ppm)
	Target	Achieved
2	3000	3080
3	9000	9490
4	27000	26900

 

The achieved chamber concentrations were 103, 105 and 100% of the target concentrations for Groups 2, 3 and 4 respectively.

Conclusions:

It was concluded that inhalation administration of TKN1 to parental Sprague Dawley rats at concentrations of 3080, 9490 or 26900 ppm for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment was associated with signs of parental toxicity at 26900 ppm, manifest as lower body weight gain of males and females.
In the context of this study, TKN1 showed no effects on reproductive/developmental performance.
In the context of this study, TKN1 showed no evidence of being an endocrine disrupter. There were no effects on thyroxine levels in adults or offspring, organ weights, external examination of offspring, and nipple counts in male offspring.
Within the constraints of this screening study, the no-observed-adverse-effect level (NOAEL) of TKN1 for local/systemic toxicity and reproductive/developmental toxicity was considered to be 26900 ppm.

Executive summary:

The purpose of this study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, with administration of the test item, TKN1 (an industrial chemical - refrigerant for air conditioning), by inhalation (snout only) administration for at least 4 weeks.

 

Three groups of 10 male and 10 female rats received TKN1 at target exposure concentrations of 3000, 9000 or 27000 ppm by inhalation administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the air control.

 

During the study, clinical condition, body weight, food consumption, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

 

Results

F0 responses

Summary data for chamber concentrations is presented below:

Group	Chamber Concentration (ppm)
	Target	Achieved
2	3000	3080
3	9000	9490
4	27000	26900

 

The achieved chamber concentrations were 103, 105 and 100% of the target concentrations for Groups 2, 3 and 4 respectively.

Body weight gain in animals exposed to 26900 ppm was lower in males over the four weeks of exposure and females over the two weeks of exposure prior to pairing.

There were no test item-related effects on clinical observations, food consumption, organ weights or macroscopic and microscopic pathology. Estrous cyclicity, pre-coital interval, gestation length, mating performance and fertility were unaffected by the test item.

There was no effect on circulating levels of thyroxine (T4) in adult males at termination.

 

F1 responses

The clinical condition, litter size, sex ratio, post birth survival indices and growth of offspring were unaffected by parental exposure.

The ano-genital distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

No macroscopic findings considered to be related to paternal treatment were recorded.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

 

Conclusion

It was concluded that inhalation administration of TKN1 to parental Sprague Dawley rats at concentrations of 3080, 9490 or 26900 ppm for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment was associated with signs of parental toxicity at 26900 ppm, manifest as lower body weight gain of males and females.  

In the context of this study, TKN1 showed no effects on reproductive/developmental performance.

In the context of this study, TKN1 showed no evidence of being an endocrine disrupter.  There were no effects on thyroxine levels in adults or offspring, organ weights, external examination of offspring, and nipple counts in male offspring.

Within the constraints of this screening study, the no-observed-adverse-effect level (NOAEL) of TKN1 for local/systemic toxicity and reproductive/developmental toxicity was considered to be 26900 ppm.

 

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

26 900 ppm

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available OECD 421 study included a screen for reproductive/developmental effects. Estrous cycles, pre-coital interval, mating performance, fertility and gestation length were unaffected by treatment. The mean number of implantations and the clinical condition of the offspring, their post birth growth and survival, sex ratio, ano-genital distance on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment. No test-item related microscopic pathological changes were observed in the male or female reproductive organs. There were also no macroscopic changes in the decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment. As such it was considered there were no adverse reproductive/developmental effects.

As such, the substance is not classified for reproductive toxicity.