1-Propanesulfonic acid, 2-methyl-2-[[1-oxo-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)thio]propyl]amino]-, sodium salt (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 - 23 Jun 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Clinical biochemistry analyses have been performed by a subcontractor laboratory

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

62880-93-7

Test material form:

solid

Specific details on test material used for the study:

Concentration > 99%

Test animals

Species:

rat

Strain:

Wistar

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 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. 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

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Limit test

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

Examinations

Parental animals: Observations and examinations:

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:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no statistical difference when comparing the control with treated group in both genders.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The males rats were observed during 49 days and during this period no statistical difference was found between control and treated group.
Therefore, the feed consumption for females rats was evaluated in periods: pre-mating, gestation and lactation. There was no statistical difference in any of the three periods.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The hormonal results of T4 were compared to the reference values, subsequently evaluated statistically between the groups.
The values obtained from the male rats in the control group are slightly above the normal value. While the treated group presents values above the control group and higher than reference value. Therefore, comparing the groups there was a statistical difference between control and treated group.
On the other hand, in females rats the values obtained is almost according to reference value, only one or 2 animals that presented results below the standard of normality.
Due to the difference found in the T4 values, for males the TSH values were also measured and no statistical differences was found.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In the histological evaluation no significant findings were found when compared the control with treated group.

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

During the pre-exposure period there was no mortality and none of the animals in any of the groups evaluated presented clinical signs.
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 3 to 4 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 3 consecutive cycles of 4 to 6 days.
In the control group, 10 females presented normal cycle and 2 females presented abnormal cycle. As AP 1/02 presented estrus prolongation with a cycle of 7 days, whereas AP 1/04 due to insufficient material in 2 days it was not possible to conclude the cycle period.
In the group at dose of 1000 mg.Kg-1, 10 females presented normal cycle and 2 females presented abnormal cycle. Since AP 2/06 and AP 2/20 had estrus prolongation with cycle of 7-10 days.
The females with abnormal cycle was excluded of the study.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Females and males rats did not present abnormalities during the mating period.
In the mating period 10 females was paired with 10 males, and all females showing evidence of copulation in control and treated group. In both groups 7 of 10 females achieving pregnancy and major of females conceiving between the days 01 to 05.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 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

clinical biochemistry

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

Description (incidence and severity):

it is possible to observe that the total number of live-born pups that remained alive on the post-natal days 4 and 5 is practically the same between both groups. During the daily observations, it was verified visually the difference in size among the pups of the same litter, and in the control group 1 male of the litters AP1/12 and AP1/14; and 1 female of litter AP1/18 were smaller than the others. Therefore, in the treated group only 1 male of the litter AP2/02 was smaller than the others. This observation was made only by visually comparing the pups' sizes, not considering the individual weights.

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Each litter was weighted and when comparing the average for control group with treated group, no statistical difference was found.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The T4 values was evaluated on two moments, day of post-natal 4 and 13.
Moreover, the literature values was only for adults rats, due to the values of pups was evaluated only statistically.
The quantity of pups did no representative on DPN 4, due to the statistical evaluation was not performed. Therefore, comparing the values of DPN 13 there was no statistical difference between control and treated groups in both genders.

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distance (DAG) of all pups was evaluated on day 4 postnatal. As recommended by OECD GD No. 43, the DAG of males is naturally larger than that of females. In the control group the mean of females pups was 2.59 cm and 5.40 cm for males pups. Additionally, the means of treated group was closely, 2.32 cm for females pups and 5.57 cm for males pups. In the both groups the proportion of DAG females/males it was more than double.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

On the 13th day post-partum the male pups were evaluated for nipple retention, and no retention was observed in any of the animals evaluated in the control and at dose of 1000 mg.Kg-1.

Organ weight findings including organ / body weight ratios:

not specified

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

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

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

the sex ratio was evaluated on the birth and the DPN 4 and the control group presented litters more homogeneous, the number of males and females per litter was very close. Although on the treated group was observed more females than males on the same litter.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

histopathology: non-neoplastic

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

During the period of study no abnormalities or differences was found in body weight, feed
consumption and clinical observations when comparing the treated with control group. Moreover,
evaluating the reproductive data obtained it is possible to observe that there were no differences
between the reproductive indices of the control and treated groups. In addition, during the gestation period according to GAD (2007) the time considered normal is 21 to 23 days, therefore
both groups presented within the expected period.
There were no significant differences between the litters of the groups evaluated, even though
there were more rumps than the control group. However, it is expected that in litters with many
puppies, that one or the other will ingest less milk, getting smaller than the others pups.
As for the difference found between the sex ratio of the litters, the OECD GD 43 (2008) reports
that changes in the sex ratio index can result from a number of factors including selective
changes in the production of X and Y sperm resulting in different proportions of male or female
conceptuses. In addition, a change in the sex ratio can indicate that the chemical has endocrine
activity and may have nothing to do with XY alterations.
Therefore, the influence in endocrine activity could be measured through T4 values. The
EOGRTS specifies the measurement of thyroxine (T4) and/or thyroid stimulating hormone (TSH)
in parental and F1 offspring at various life-stages to assess direct effects on thyroid function or
indirect effects via the hypothalamic-pituitary-thyroid axis (OECD GD 151, 2008). Since some
thyroid toxicants have been reported to induce changes in total T4 without concomitant changes
in TSH. Statistically or biologically significant changes in either one or both hormones between
treated and control groups can be evaluated in conjunction with any changes in thyroid gland
weight and histopathology.
Evaluating the other factors mentioned in the previous paragraph, such as relative thyroid
weight, no significant differences were found to corroborate the hormonal changes found. As well
as, in the histological evaluation no abnormalities was found on the thyroids. The T4 values
should be influenced by circadian cycle and the time of blood sampling (WAKABAYASHI, 2004).
In conclusion, no adverse effects were found.


The results obtained in the present study indicated that oral administration by gavage of test substance 2-methyl-2-2[(1-oxo-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8- tridecafluorooctyl)thio]propyl)amino]-1-propanesulfonic acid, sodium salt at dose of 1000 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 1000 mg/Kg/day
- No adverse effects related to the test substance;
Based on these results and the endpoints described in the literature, no supporting evidence was found that the test substance no affects or causes influence on the reproduction and development of Wistar rats and the reproductive NOAEL (No-Oberved-Adverse-Effect-Level) is ≥
1000 mg/Kg/day.

Executive summary:

No adverse effects related to the test substance were observed on reproductive and developmental functions following the oral administration of a dose of 1000 mg/kg bw/day.
The NOAEL is = or > than 1000 mg/kg bw/day.