1-Propanaminium, 2-hydroxy-N,N,N-trimethyl-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)thio]-, chloride (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 nov 2017-20 dec 2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

{2-hydroxy-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulfanyl]propyl}trimethylazanium chloride

EC Number:

811-523-6

Cas Number:

88992-45-4

Molecular formula:

C14 H19 F13 N O S . Cl

IUPAC Name:

{2-hydroxy-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulfanyl]propyl}trimethylazanium chloride

Test material form:

solid

Specific details on test material used for the study:

Concentration > 99%

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

rats (Rattus norvegicus), Wistar Hannover strain, and albinos. The rat is selected because it is the main rodent specie for reproductive and developmental toxicity studies, and Guideline OECD 421 (2016) recommends it. The rat Wistar Hannover was selected because of extensive historical data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age (on the first day of pre-exposure period): between 9 to 12 weeks old.
The animals will be housed up to 2 individuals per sex per cage during acclimation, pre-exposure period, and pre-mating period. After the pre-mating period, one male will be placed with one female for pairing. After pairing, females that present vaginal smears with the presence of sperm will be considered mated and housed individually.
During the experimental period, the rats will be housed in polypropylene cages with wire mesh tops and bedding material (autoclaved wood shavings). Cage changing will be done twice a week for all animals. The cages will be arranged on the racks in such a way that uniform ventilation and lighting is ensured during the course of the study.

The experimental room temperature will be maintained at 22 ± 3°C, with a relative humidity of 30-70%. Room ventilation will be set for 10-20 air changes per hour, and a photoperiod of 12 hours light and 12 hours dark will be maintained.
The animals will be provided with conventional laboratory diet for laboratory animals (irradiated) and filtered drinking water will be supplied by SABESP (Companhia de Saneamento Básico do Estado de São Paulo) ad libitum throughout the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on mating procedure:

After the females have been screened for normal oestrous cylces (in a 2 weeks pre-treatment period) and after a premating period of 2 weeks, they will be paired with an assigned male (1 female : 1 male) from the same dose level until evidence of mating is observed, or either 3 estrous periods or 2 weeks have elapsed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

After preparation of test solutions, an aliquot of each dose evaluated will be analyzed by chromatography to determine the effective concentration of the active ingredient (a.i.) in the solution. The acceptance limit should be less than ± 20%.

Duration of treatment / exposure:

The Test Suspensions or vehicle will be administered daily, by gavage, approximately at the same period of day (in the morning) on a 7-day-a-week basis according to the following schedule:
- males: 2 weeks prior to mating, during the mating period and until 80% of the females have delivered.
- females: 2 weeks prior to mating, during the mating period, during gestation and up to, and including, lactation day 13.

Frequency of treatment:

daily

Details on study schedule:

After the females have been screened for normal oestrous cycles (in a 2 weeks pre-treatment period) and after a premating period of 2 weeks, they will be paired with an assigned male (1 female : 1 male) from the same dose level until evidence of mating is observed, or either 3 estrous periods or 2 weeks have elapsed. Exceptions can arise in the case of occasional deaths of males. Care will be taken to avoid sibling mating. Vaginal smears will be collected daily during mating period and examined for the presence of sperm. Day 0 of gestation is defined as the day the vaginal smear is found positive for sperm. Females that show no evidence of copulation will be assumed pregnant at the end of the mating period, and housed accordingly. The day of parturition will be designated day 0 of lactation (day 0 post-partum).

All animals will be euthanized as follows:
- Adult males: after a minimum total dosing period of 28 days (alternatively, the males may be retained and continued to be dosed for the possible conduction of a second mating if considered appropriate;
- Adult females: from day 13 post-partum, after the examination of all pups;
- Adult females which have not delivered on day 25 post-coitum: from day 25 post-coitum;
- Adult females which with no evidence of mating: 24-26 days after the last day of the mating period;
- Surviving pups: from day 13 post-partum, after examination.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day

Remarks:

highest dose

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day

Remarks:

Intermediate dose

Doses / concentrations 3

Dose / conc.:

12.5 mg/kg bw/day

Remarks:

lower dose

No. of animals per sex per dose:

12 animals/sex/group and 6 satellite animals/sex on control group and on higher dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Selection of the doses
Initially, the dose levels (in mg/kg body weight/day) were selected based on the LD50 (letal dose of 50% of organisms) value, 500 mg.Kg-1 (ECHA,2015), in agreement with the Sponsor. The suggested highest dose were 400 mg.Kg-1.
In order to confirm a higher dose, the evaluations of 3 doses (400, 250 and 200 mg.Kg-1) were carried out in a pilot study with administration for 7 days. In addition, in the dose of 200 mg.Kg-1 only liver and kidney presented lightness alterations. Due to this result the study started with the doses below:
- Dose 1: 50 mg.Kg-1 (dose not expected to observe signs of toxicity);
- Dose 2: 100 mg.Kg-1 (intermediate dose);
- Dose 3: 200 mg.Kg-1 (dose at which signs of toxicity are expected, except for mortality and severe suffering)
In the first week of administration, the test systems of doses of 200 and 100 mg.Kg-1 died and the rest of them were apathetic with severe diarrhea. Thus, conducting the study with these doses was considered a risk to ethical values and the other animals in the groups were euthanized for humanitarian reasons.
Based on the facts previously reported, the study was re-conducted with the doses below:
- Dose 1: 12.5 mg.Kg-1 (dose not expected to observe signs of toxicity);
- Dose 2: 25 mg.Kg-1 (intermediate dose);
- Dose 3: 50 mg.Kg-1 (dose at which signs of toxicity are expected, except for mortality and severe suffering)

Experimental design
Group Number Nº of Animals Dose Level (mg/kg/day) Concentration (mg.mL-1)
Male Female
1 12 14 Vehicle (water) 0a
2 12 14 12,5 25b
3 12 14 25 50b
4 12 14 50 100b
a: vehicle / b: test solution

Examinations

Parental animals: Observations and examinations:

During the treatment period, all animals were observed daily, two times a day (in the beginning and at the end of each day), for mortality and morbidity.

Clinical exam
All animals were submitted a detailed clinical examination, out of the box, prior to initiation of treatment and weekly during the treatment period. Signs to be observed included changes in the skin, hair, eyes, mucous membranes, occurrences of secretions and excretions, and autonomic activity (lacrimation, piloerection and altered respiratory pattern). Changes in locomotion, posture and reaction to manipulation, as well as presence of clonic or tonic movements, stereotyped (excessive self-cleaning, repetitive circular movements) or bizarre behavior (self-mutilation, walking backwards) were recorded.

General clinical observations will be made at least once a day during the entire study period. Throughout the treatment period, cage side observations of each animal will be done at least twice a day for mortality, morbidity, pertinent behavioral changes, signs of difficult or prolonged parturition, and any signs of overt toxicity. These records will include time of onset, degree and duration of toxicity signs.
The day of parturition will be recorded and the duration of gestation will be calculated.

Males will be weighed on the first day of dosing, weekly thereafter, and at termination.
Females will be weighed on the first day of dosing and once a week during premating period, on days 0, 7, 14 and 20 during gestation, and during lactation (on days 0 and 13 postpartum).
These observations will be reported individually for each adult animal. Animals will not be weighed during mating period.

Food consumption will be determined on the same days of body weight determination.

Litter observations:

Each litter will be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups are significantly smaller than corresponding control pups) and presence of gross abnormalities.
Live pups will be counted and sexed and the litters will be weighted within 24 hours of parturition (day 0 or 1 post-partum) and at least on day 4 and 13 post-partum. The pups will be observed daily for mortality, clinical signs, or abnormal behavior, and any external malformation will be recorded.
On day 4 post-partum, the size of each litter will be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four or five pups per sex per litter. Whenever the number of males and females prevents having four or five of each sex per litter, a partial adjustment (for example, six males and four females) will be performed.
The anogenital distance (AGD) of each pup will be measured on the same postnatal day (PND) between PND 0 trough PND 4. Pup body weight will be collected on the day the AGD is measured. The number of nipples/areolae in male pups will be counted on PND 12 or 13.

Postmortem examinations (parental animals):

At termination, or if unscheduled death occurs during the study, all parents animals will be examined macroscopically for any structural abnormalities or pathological changes. Special attention will be paid to the organs of the reproductive system. Animals found dead or sacrificed moribund during the study will be subjected to gross examination as soon as possible after death. If the necropsy cannot be performed immediately, the animal will be kept in a refrigerator until the examination is performed.
In all females, the number of implantation sites and corporea lutea will be recorded (whenever possible). Vaginal smears will be examined in the morning on the day of necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of ovaries.

At scheduled necropsy, testes, epididymides, levator ani plus bulbocavernous muscle complex, Cowper’s glands and glans penis of all adult males will be weighted. The thyroid weight can be determined after fixation.

At scheduled necropsy, the following organs of all animals will be preserved:
- testes;
- epididymides;
- ovaries;
- prostate;
- seminal vesicle and coagulating gland;
- bulbourethral gland;
- uterus and cervix;
- thyroid;
- organs showing alterations.

Histopathology of ovaries, testes and epididymides (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) will be performed in high dose or test limited dose and control animals. The other preserved organs may be examined if necessary. These examinations will be extended to animals of other groups, if treatment-related changes are observed in the high dose group. All gross lesions will be examined.

Postmortem examinations (offspring):

Dead pups and pups killed at day 13 post-partum will be carefully examined externally for gross abnormalities. Particular attention will be given to the external reproductive genitals which will be examined for signs of altered development.
At day 13 post partum, the thyroid from 1 male and 1 female pup per litter will be preserved.
Histopathology of ovaries, testes and epididymides (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) will be performed in high dose or test limited dose and control animals. The other preserved organs may be examined if necessary. These examinations will be extended to animals of other groups, if treatment-related changes are observed in the high dose group. All gross lesions will be examined.

Statistics:

Quantitative variables such as body weight, food consumption, organ weights, number of fetuses and corpora lutea will be analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett’s test or Tukey if significance is detected, or by non- parametric test of Kruskal-Wallis, according to the results of tests for normality and homogeneity of variance. For qualitative or non-parametric data such as fetal variations and malformations, clinical findings, macroscopic and microscopic findings and fetal findings, comparison between means will be carried out using the Fisher Exact Test or Chi-Square Test. If necessary, additional statistical analyses will be carried out using suitable methods. The level of significance will be set at 5%.

Reproductive indices:

Indices Calculations
1. Pre-implantation loss:

Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

2. Post-implantation loss:

Number of implantation sites - Number of live concepti
_____________________________________________ x 100
Number of implantations

3. Mating index:

Number of mated animals
_____________________ x 100
Number of paired animals

4. Fertility index:

Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

5. Gestation index:

Number of females with live born pups
________________________________ x 100
Number of pregnant females

Offspring viability indices:

Indices calculations
6. Live birth index:

Number of live born pups
_____________________ x 100
Number of delivered pups

7. Viability index on day 4 post-partum:

Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups

8. Lactation index:

Number of surviving pups on day 5 post-partum
_______________________________________ x 100
Number of live born pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Daily the test system were observed in cage and weekly they were submitted a detailed clinical examination by veterinarian. During the period of study in males, all groups including control presented nasal porphyria for 1 one day. In only 4 test animals this alteration persisted for 2 days and in one for 5 days no consecutive.
In addition, 3 males and 3 females presented salivation for 1 day, after the administration of test item. Exclusively, 6 males and 7 females of dosed groups, presented wheezing.

Mortality:

no mortality observed

Description (incidence):

During the pre-exposure period there was no mortality

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight of males were evaluated during 42 days and no statistical difference was observed when comparing dosed groups with control.
As well as, no significant difference was found on body weight females. On the other hand, the evaluation of females was segmented according to the step of the study: pre-mating, gestation and lactation period. Additionally, females no pregnant were evaluated in the pre-mating period and gestation, until 26-day post-partum.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males consumption was observed statistical difference in the 3rd week (50 mg.Kg-1 versus 12.5 mg.Kg-1) and 6th week (25 mg.Kg-1 versus control group), both cases there were a decreasing of consumption related to control group.
In pre-mating period was observed statistical difference between highest dose (50 mg.Kg-1) and control group, actually depressing of means. Although, in the gestation and lactation periods no statistical difference was observed. Significant statistical difference was also observed in the feed consumption of non pregnant females, between intermediate dose (25 mg.Kg-1) and control group. However, this was not considered to be impacting to the study, because it was not indicative of being related to the exposure to the test item, since it did not occur in the highest dose group as well.
The statistical differences observed in feed consumption, both in males and females were punctual, occurred only in 1 week. Since the alteration was not persistent, it did not present a dose-response relationship, no correlated evidence was recognized in macroscopic or histological evaluation that could be suggest to be unrelated to the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Blood samples were collected from all adult animals and initially it was assessed for thyroxine levels (T4). The results were compared to the reference values to evaluate biologic relevance. Means of males and non pregnant females were within the range of reference values. As expected, pregnant females presented depressed hormonal levels.
Subsequently, the results were assessed statistically and significant differences were observed in males.
In the parental generation, the males showed a tendency to reduce the T4 values concomitant with the dose increase of the test item. However, the means were in accordance with the reference values. In addition, no macroscopic or microscopic findings were evidenced that indicated structural alterations in the thyroid. On the other hand, usually the adverse effects occur first in the functions and subsequently the structural damages are caused. Thus, the reduction of hormone expression may be a change even without structural evidence. Since the macrophage infiltrate was found with the same intensity in the control and in the treated groups.
The identical reduction in T4 expression was observed in pregnant females, and the values of intermediate and higher dose were lower than the reference value. On the other hand, there was no statistical difference when compared with control group, which presented a mean (3.89) close to the expected minimum value (3.40). Additionally, T. Takayama et. al (2016) described that the presence of macrophage infiltrate and larger colloidal lumen correspond to a two different phases of a life cycle of thyroid cells, such as development and degeneration, and it was not necessarily a toxic effect.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the histological evaluation of males no significant findings were found when compared the control with treated group. The only exception was the presence of macrophage infiltration in the great number of males, with distribution in all groups, including control group.
The uterus of non-pregnant females that presented alterations on macroscopic evaluation were investigated and the findings were described in the report (table 76).
During the microscopic investigation, it was also possible to confirm if the females did not become pregnant, and the possible reasons according to the Chart below:
Group n P NP Reasons of NP
Control 5 1 4 3 of 4 were hydrometra
12.5 mg.Kg-1 6 0 6 3 of 6 presented atrophy in uterus
25 mg.Kg-1 6 0 6 2 of 6 were hydrometra
50 mg.Kg-1 5 0 5 1-female was cervix alterations and another one with inflammation in uterus

P- Confirmed pregnant; NP- Confirmed non-pregnant

The ovaries and adrenals of pregnant females that presented alterations on macroscopic evaluation were investigated and in most of cases, the tissue were normal.
Both females pregnant and not presented macrophage infiltration in thyroids, as well as in males. The type and intensity of this sign were the same in control and treated groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Determination of the estrous cycle of the females was performed during the pre-exposure period and the evaluations were performed considering that the normal cycle is 4 to 5 days, could be extended for 6 days. Therefore, during the pre-exposure period 2 to 3 consecutive estrous cycles should be identified. Due to the occurrence of insufficient material on some days, the female was considered to have normal cycling from the identification of 2 consecutive cycles of 4 to 6 days.
After the above evaluation, 2 females of each group were excluded because of abnormal cycle.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating
The mating were performed 1:1 (one male to one female), totalizing 12 couples for group. Every day, females were examined for the presence of sperm or a vaginal plug. All females of control group and dose of 12.5 mg.Kg-1; and the majority of doses 25 and 50 mg.Kg-1 became pregnant during the first 3 days of mating period. The exception were AR 3/44 who did not become pregnant during the 14-day mating period; and AR 4/42 who become pregnant in the 4th day of mating period.
In addition, day 0 of pregnancy was defined as the day on sperm was found on vaginal smear.

Gestation
The number of females give birth were practically the same in all groups (7 females in control and dose of 50 mg.Kg-1; 6 females in the group of doses 12.5 and 25 mg.Kg-1). As well as, there was no significant difference on the duration of gestation between the groups. The great number of females give birth on 23th of gestation.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 12.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

the number of live born puppies that died up to the 4th postnatal day was significantly high at the dose of 50 mg.Kg-1.
Due to that the viability index of the highest dose was 88.5% while in the control group it was 92.9% and in the other groups approximately 100%.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The litters were evaluated statistically based on average of body weight and no significant difference was found between dosed groups and control

Haematological findings:

no effects observed

Description (incidence and severity):

Blood samples were collected in two moments: DPN 4 and DPN 13. On the DPN 4 the pup blood was a pooled by litter, however on DPN 13 blood samples were divided in males and females by litter. In both evaluations no statistical difference were observed.

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

As expected, the ADG in males was higher than females and there was no significant differences in males between tested groups and control.
On the other hand, the ADG of females of doses of 25 and 50 mg.Kg-1 was statistically different of control

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

no retention was observed in any of the animals evaluated in the control and doses of 12.5, 25 and 50 mg.Kg-1.

Gross pathological findings:

no effects observed

Description (incidence and severity):

In the macroscopic evaluation of pups no significant findings were found when compared to the control and treated groups. There were 2 rents (pups smaller than another), one in the lower dose and another in the higher dose.

Histopathological findings:

no effects observed

Description (incidence and severity):

In the microscopic evaluation of pups no significant findings were found when compared to the control and treated group.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 12.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

haematology

gross pathology

histopathology: non-neoplastic

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The results obtained in the present study indicated that oral administration by gavage of test item 2-hydroxy-N,N,N-trimethyl-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)thio]-1-propanimium chloride at doses of 12.5, 25 and 50 mg/Kg/day in Wistar rats (Rattus novergicus) during the pre-mating period, mating, gestation and lactation produced the following findings related to the test substance:
Group of 12.5 mg/Kg/day
- No adverse effects related to the test item;
Group of 25 mg/Kg/day
- Increased AGD of females puppies;
- Reduction of T4 values in parental pregnant females;
- Reduction of relative weight of uterus of pregnant females.
Group of 50 mg/Kg/day
- Survival index significantly lower;
- Increased AGD of females puppies;
- Reduction of T4 values in parental pregnant females;
- Reduction of relative weight of uterus of pregnant females.
Based on these results and the endpoints described in the literature, there was evidence that the test item affect or cause influence on the reproduction and development of Wistar rats and the reproductive NOAEL (No-Oberved-Adverse-Effect-Level) is ≥ 12.5 mg/Kg/day.

Executive summary:

Based on the available data, the the substance 2-hydroxy-N,N,N-trimethyl-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)thio]-1-propanimium should be classified in Category 1B for fertility and development in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.