Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Sep 2019 to 24 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP Guideline study

Justification for type of information:

For the Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate a new chemical substance notification in China has been prepared under the Regulation ‘Measures on the Environmental Management of New Chemical Substances' (Decree No. 7 of the Ministry of Environmental Protection of the P.R. China, also known as ‘China REACH’). Under this regulation an prenatal developmental toxicity study according to OECD 414 is part of the data requirements for substances that are produced or imported in volumes > 100 t/y. For this reason an OECD 414 study was performed in 2020 at Shenyang Research Institute of Chemical Industry. The results of the study are included in the dossier and serve as the valid test related to this endpoint.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2019-02-11

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

EC Number:

911-467-3

Molecular formula:

C2O2F3K and CO2F3KS

IUPAC Name:

Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Details on test material:

- Appearance: Yellow to slightly brown liquid
- Analytical purity: 30%
- Impurities (identity and concentration): Sulfates 0.02%; Sulfites 0.08%; Dimethylformamide 0.06%
- Composition of test material, percentage of components: Potassium triflinate (TFSK) 14.2%, Potassium trifluoroacetate (TFAK) 14.8%, Water: to complete the composition
- Purity test date: 2019-02-13
- Lot/batch No.: SSK1904360
- Expiration date of the lot/batch: March 27, 2022
- Storage conditions: Room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF (Beijing) Biotechnology Co., Ltd
- Age at study initiation: 11 weeks of age at arrival at the test facility
- Weight at study initiation: 267.07 - 404.02 g
- Fasting period before study: No
- Housing: animals were housed in plastic cages (L46.0xW31.5xH20.0 cm). During the acclimatization period, the animals were housed in groups of max. 2. Mated females were housed individually in cages.
- Diet (e.g. ad libitum): SPF rodent growth and breeding feed supplied by Shenyang Mao Hua biological science and Technology Co., Ltd, ad libitum
- Water (e.g. ad libitum): Purified drinking water, ad libitum
- Acclimation period: 33 days. Clinical observations were performed daily during acclimatization period, and were continued until the start of the test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled at 20 - 25°C (actual range: 22.6 - 25.6°C)
- Humidity (%): controlled 40 - 70% (actual range 34-82%)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Nov 2019 To: 5 Dec 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item in the vehicle (drinking water) were prepared daily before the administration. The test item concentrations were based on the active ingredient of the reaction mass (Potassium triflinate, TFSK: 14.2%, Potassium trifluoroacetate, TFAK: 14.8%). A dosing volume of 10 mL/kg was applied for all animals, which was adjusted based on the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CONCENTRATION VERIFICATION: The concentration of the test item was analyzed in the first and last week of the test item preparation with a validation analytical method (G1999B0040).

- Separation method: Ionic Chromatography (IC) (ICS-5000+ EG)
- Detection method: Conductivity detector.
- Conditions:
Analytical column: Dionex IonPac AS11-HC RFIC, 4 mm x 250 mm, 13 µm
Pre-column: Dionex IonPac AS11-HC RFIC, 4 mm x 50 mm, 13 µm
Column temperature: 30°C
    Flow rate: 1.0 mL/min
    Injection volume: 50 μL
    Mobile phase: 10mM potassium hydroxide aqueous solution
Retention time: About 11.2 and 13.7 min.
Run time: 17.0 minutes
- Data processing: the analytical data obtained were processed using the software of Chromeleion (c) Dionex. Since the test item is a reaction mass of two materials, the two main chromatography peaks in the chromatogram were considered as the target peak during sample analysis. The quantitative analysis was based on the sum of the two main peaks. The RSD of retention time was obtained from the first chromatography peak and the RSD of peak area was obtained from the sum of the two mean peaks in the system suitability test.
- External calibration and linearity range: A stock solution of 1.0 g active ingredient/L was diluted with ultra-pure water to obtain a series of standard solutions of 70.00, 60.00, 50.00, 40.00 and 30.00 mg/L. The accuracy of calibration of 5 concentrations should be within 90-110% by regression analysis and the coefficient of correlation (R) should be more than 0.99.
- System suitability/Method validation: To demonstrate the validity of the method, each analytical sequence consisted of at least:
+ A blank control (drinking water only), two repeats at a time.
+ Low dose (10 mg/ml), five repeats at a time.
+ Middle dose (30 mg/ml), five repeats at a time.
+ High dose (100 mg/ml), five repeats at a time.
Acceptance criteria: At the retention time of the target peak, the peak area of the blank matrix should be less than 10% of that of the lowest concentration standard solution. The determined concentrations of the low-, mid- and high-dose formulations should be 85-115% of the nominal concentration, and the RSD should not exceed 10%.
- Sample analysis: The dose formulations (nominal concentrations: 10, 30 and 100 mg/mL) were sampled and diluted within the linear range (30-70 mg/L), and determined by Ion Chromatography. The results of the determind concentration were in accordance with the target concentrations (accuracy 94.8 - 101.2%).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 2 (or one) females : one male
- Length of cohabitation: until a sperm positive smear was detected.
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy

Duration of treatment / exposure:

Females were dosed daily in the morning from GD5 to GD19 by oral gavage.

Frequency of treatment:

Once daily

Duration of test:

15 treatment days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

Active ingredient

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Active ingredient

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Active ingredient

No. of animals per sex per dose:

27 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route in rats is available for the Reaction mass of TFSK/TFAK. No effects were observed on reproductive organs, reproductive performances and progeny at the maximum dose level tested (1000 mg active ingredient/kg bw/day). The No Observed Adverse Effect Level (NOAEL) for parental toxicity, reproductive performance (mating and fertility) and effects on progeny was therefore considered to be 1000 mg/kg/day. For this reason the maximum dose level of 1000 mg a.i./kg bw/day was selected as the highest dose level in the OECD 414 study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed daily by cage-side observations until the termination. After dosage, dams were observed for morbidity and mortality twice daily (once daily on non-working days)

BODY WEIGHT: Yes
- Time schedule for examinations: All pregnant rats were weighed on GD0, then they were weighed once per 3 days during the dosing period (GD5-19), and on the day of scheduled necropsy (GD20).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: During the period of administration, all pregnant rats were provided with a known quantity of feed on the day before body-weight determination, and the remaining feed were weighed on the next day (24h ± 1.5h). The daily food consumption of each animal was calculated.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All rats were killed on GD20, and the uterus was removed for examination immediately.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included: the whole gravid uteri and total placentas were weighed. The number of corpora lutea, absorbed fetuses (early and late separately), dead fetuses (early and late separately) and viable fetuses were counted.

Fetal examinations:

- External examinations: Yes, the sex and body weight of each viable fetus were determined. Each fetus was examined for external alterations, including head, trunk, limbs and tail.
- Soft tissue examinations: Yes, one-half live fetuses of each litter were immersed in modified Davidson's fixative (the recipe for 100 ml of fixative: 14 ml ethanol, 6.25 ml glacial acetic acid, 37.5 ml saturated 37% formaldehyde and 37 ml distilled water) for one week, rinsed twice with tap water and stored in 70% isopropyl alcohol for soft tissue examination. Limbs and tail of the fetus were cut down first, then four chips were cut to examine the structural alterations of the head. Finally, thorax and abdomen of the fetus were opened to examine the size, shape and position of the organs.
- Skeletal examinations: Yes, the other half of each litter was prepared and examined for skeletal alterations. After removal of the skin and soft tissue, the remainder of the fetus was stained by using Alizarin red staining method, and stored in 70% glycerol for examination. This procedure include the skull, vertebra, sternum, ribs, limb bones and pelvis. At the same time the number of sternal ossification points, namely the number of ossified sternal segments, was recorded.

Statistics:

Average and standard deviation of the data for each group were calculated including body weight, food consumption, body weight change corrected for gravid uterine weight, number of corpora lutea and implantations, number of viable fetuses and body weight of fetuses, number of absorptive and dead fetuses.
Data statistics: If variance homogeneity test was P >0.05, Anova was used. If variance homogeneity tests P ≤0.05, Kruskal Wallis test was used. The data about ratio of viable fetuses, dead fetus and absorptive fetus, and incidence of anomalies were evaluated by the chi-square test. Anova and non-parametric analysis was done with Provantis 9.4.3.0 software, the chi-square test was done with SPSS 17.0 software.

Indices:

Pre-implantation loss (%): (No. of corpora lutea - No. of implantations) / No. of corpora lutea ×100 %
Post-implantation loss (%): (No. of implantations - No. of live fetuses) / No. of implantations ×100 %
Sex ratio distribution (%): Number of male fetuses / Number of fetuses x 100%
External abnormalities/litter (%): Number of fetuses with abnormality / Number of fetuses x 100%
Visceral abnormalities/litter (%): Number of fetuses with abnormality / Number of fetuses x 100%
Skeletal abnormalities/litter (%): Number of fetuses with abnormality / Number of fetuses x 100%

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed in the course of the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No deaths were observed in the course of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

During the administration period, the body weight and body weight change of pregnant rats in dose groups had no significant difference compared with the control group (P>0.05). The mean corrected body weight gain on GD20 (body weight minus gravid uterine weight) and net body weight change (body weight on GD20 minus body weight on GD5 minus gravid uterine weight) during the gestation period in the pregnant rats in all dose groups had no significant difference compared with the control group (P>0.05). Detailed results are provided in Table 1 see 'Any other information on results'.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During the administration period, the food consumption of the pregnant rats in all dose groups had no statistically significant difference compared with the control group (P>0.05). Detailed results are provided in Table 2 see 'Any other information on results'.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The percentage of absorbed fetuses and the pre-implantation loss of high-dose group had a significant decrease compared with the control group (P≤0.05 or P≤0.01), but without toxicological significance and that had no statistically difference in the low- and mid- dose group (P>0.05). Detailed results are provided in Table 3 see 'Any other information on results'.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

not specified

Details on maternal toxic effects:

In all dose groups, no statistically significant difference was observed in the mean numbers of corpora lutea, implantation sites and total placental weights, and the gravid-uterine weights compared with the control group (P>0.05). Detailed results are provided in Table 3 see 'Any other information on results'.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: No effects observed at the highest dose level tested.

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference in the body weight of live fetuses was observed in all dose groups compared with the control group (P>0.05).
Detailed results are provided in Table 4 see 'Any other information on results'.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

In the high-dose group there was a statistically significant increase in the percent of live fetuses (P≤ 0.05).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No statistically significant difference in the sex distribution of live fetuses was observed in all dose groups compared with the control group (P>0.05).

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

One fetus in the mid-dose group showed sign of narrow tail and 1 fetus in the high-dose group showed sign of tail absent. Two fetuses in the control group showed sign of small brain, and 1, 0, 1 and 3 fetuses in the control group, low-, mid- and high-dose groups showed signs of uronephrosis or small kidney in unilateral or bilateral kidneys. Five, 0, 5 and 4 fetuses in the control group, low-, mid- and high-dose groups showed signs of parietal imcomplete ossification, and one fetus showed signs of wavy rib, but the frequency of these abnormalities in the dose groups had no significant difference compared with the control group (P>0.05), which were considered as incidental findings. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations; some examined fetuses had less than six sternal ossification points,but the mean number of sternal ossification points in all dose groups had no statistically difference compared with the control group (P>0.05).

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Deviations from protocol: During the test the relative humidity and the realitve temperature in the animal room were beyond the controlled range of respectively 40 -70% (actual 34 -82%) and 20 -25°C (actual 22.6 -25.6°C). As the deviated relative humidity and temperature lasted for a short term, and no related adverse effects were observed in the animals, the deviaions are not considered to affect the quality or integrety of the study.

Table 1: Body weight and body weight gain of pregnant rats (g)

Day(s) relative to mating			0 mg/kg d	100 mg/kg d	300 mg/kg d	1000 mg/kg d
Body weight gain (g)	5-20 [a]	Mean SD N	121.5 20.1 27	130.1 24.4 27	124.0 23.5 27	122.6 22.7 27
Corrected body weight on GD20 (g)	[a]	Mean SD N	378.25 27.98 27	381.26 33.47 27	375.95 25.77 27	375.19 32.46 27
Corrected body weight gain (g)	[a]	Mean SD N	54.327 17.186 27	56.334 21.815 27	52.323 16.921 27	49.599 17.193 27
Net body weight change (g)	[a]	Mean SD N	25.02 12.77 27	28.44 13.52 27	24.71 20.83 27	22.37 12.81 27

[a] – Anova&Dunnett

Table 2: Food consumption of animals (g/day)

		Day(s) Relative to mating
		7 → 8	10 → 11	13 → 14	16→ 17
0 mg/kg d	Mean SD N	28.15 4.60 27	27.77 4.76 27	29.77 4.98 27	32.31 4.67 27
100 mg/kg d	Mean SD N	26.71 3.96 27	28.65 4.82 27	28.70 4.12 27	32.38 5.32 27
300 mg/kg d	Mean SD N	25.99 4.22 27	27.77 4.26 27	29.61 4.75 27	32.12 5.38 27
1000 mg/kg d	Mean SD N	25.39 4.44 27	27.92 7.17 27	27.27 4.29 27	31.71 4.76 27

Table 3: Developmental endpoint results

		0 mg/kg d	100 mg/kg d	300 mg/kg d	1000 mg/kg d
Mated female	(n)	28	28	28	28
Pregnant female	(n)	27	27	27	27
Corpora Lutea [a]	Mean SD Sum	16.9 3.7 456	17.2 2.8 465	16.7 2.9 451	16.6 3.2 449
Implantation[a]	Mean SD Sum	16.1 3.5 436	16.4 2.8 442	16.1 2.7 434	16.1 3.1 436
Gravid Uterus [a] (g)	Mean SD	96.4771 22.7950	101.6460 19.9209	99.3060 16.2687	100.2396 17.2214
Total placental weight[a] (g)	Mean SD	14.1308 3.2056	14.7799 2.9829	14.6335 2.5385	14.7466 2.7125
Live fetus[a]	Mean SD Sum	15.1 3.7 409	15.5 3.0 419	15.4 2.7 415	15.7 3.0 424
Percent[c] (%)		93.8	94.8	95.6	97.2*
Absorbed fetus[a]	Mean SD Sum	0.9 1.2 25	0.8 1.1 23	0.7 1.0 19	0.4 0.5 10
Percent[c] (%)		5.7	5.2	4.4	2.3**
Dead fetus[k]	Mean SD Sum	0.1 0.3 2	0.0 0.0 0	0.0 0.0 0	0.1 0.4 2
Percent[c] (%)		0.5	0.0	0.0	0.5
Pre-implantation loss[c] (%)		4.4	4.9	3.8	2.9
Post-implantation loss[c] (%)		6.2	5.2	4.4	2.8*

[a] – Anova&Dunnett

[k] – Kruskal-Wallis&Wilcoxon

[c] – Chi-square test: *p ≤ 0.05, **p ≤ 0.01

Table 4: Fetal examination results

		0 mg/kg d	100 mg/kg d	300 mg/kg d	1000 mg/kg d
Body weight of fetus[a](g)	Mean SD	3.974 0.308	4.068 0.273	4.019 0.264	3.965 0.322
Number of male fetuses		203	210	231	222
Number of female fetuses		206	209	184	202
Sex distribution (males/total) [c] (%)		49.63	50.12	55.66	52.35
[c] – Chi-square test

 

Applicant's summary and conclusion

Conclusions:

The No-Observed-Adverse-Effect-Level (NOAEL) of the Reaction mass of TFSK/TFAK for maternal and embryo-fetal development toxicity in rats was considered to be 1000 mg/kg/day.

Executive summary:

A GLP compliant prenatal developmental toxicity study with the Reaction mass of TFSK/TFAK was conducted in Sprague Dawley rats according to OECD Guideline 414. Mated females (27/group) were treated once daily with the Reaction mass of TFSK/TFAK by oral gavage at dose levels of 100, 300, and 1000 mg active ingredient/kg bw/day during gestation (from Gestation Day 5 to 19). A control group of 27 females receiving the vehicle (water) was included in the study.

The study animals were observed daily for mortality and clinical signs. Body weight and food consumption of the dams were also recorded. On GD 20, the study animals were necropsied and the uterine was removed for determination of gravid uteri weight and placental weight. The number of corpora lutea, absorbed fetuses, dead fetuses and viable fetuses was determined. The fetuses were removed, weighed, sexed and examined externally for defects. Approximately half of the fetuses were examined for soft tissue abnormalities. The other half was examined for skeletal abnormalities and ossification state.

No deaths or treatment-related clinical signs were observed during the study. No effects on mean body weights, body weight change and food consumptions were observed in the treated groups when compared to the control group. No adverse effect was observed in the prenatal reproductive parameters. The fetal examination showed no adverse effect in body weight and sex distribution of live fetuses in all dose groups. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations.

Based on these results, the No-Observed-Adverse-Effect-Level (NOAEL) of the Reaction mass of TFSK/TFAK for maternal and embryo-fetal development toxicity in rats was considered to be 1000 mg/kg/day.