Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 February 2016 to 21 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(5 March 2015)
Limit test:
no
Justification for study design:
This study provided initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.
The oral route was selected since it is a route of administration which is recommended by the Regulatory Authorities for this type of study.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name of the test susbtance: Trifluoromethanesulfonic acid
Synonyms: Triflic acid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
RjHan:SD (Rats CD®)
Details on species / strain selection:
The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study and the Sprague-Dawley strain was selected since background data from previous studies are available at CiToxLAB France.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: The males were approximately 10 weeks old and the females approximately 9 weeks old.
- Weight at study initiation: The males had a mean body weight of 440 g (range: 409 to 475 g) and the females had a mean body weight of 256 g (range: 230 to 279 g).
- Fasting period before study: No
- Housing: The animals were individually housed, except during mating and lactation, in cages (Tecniplast 2154, polycarbonate with stainless steel lids, 940 cm2) containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen in order to not jeopardize gestations and to avoid aggressive behavior between males around mating.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
Each cage contained cocoon and nylabone for the environmental enrichment of the animals.
The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): Yes. All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 9705482 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): Yes. The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: The animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 21 June 2016 To: 18 August 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: pH 7.2 sodium phosphate 0.5M buffer
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test was administered as a solution in the vehicle.
* Preparation procedure: According to CiToxLAB France/Study No. 43498 VAS describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study: Add the required amount of test item in some amount of vehicle and mix. Then add the vehicle until the final volume and mix. Check the pH (pH paper) of each test item dose formulation at the first preparation in the study.
Frequency of preparation: The formulations were prepared for use every 2 to 12 days [based on test item dose formulation stability (CiToxLAB France/Study No. 43498 VAS) and vehicle expiry]. The formulations were stored and delivered at room temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg bw/day
- Amount of vehicle (if gavage): 10 mL/kg/day
- Lot/batch no. (if required):
* sodium phosphate monobasic monohydrate: BCBK7914V and BCBR2016V
* NaOH 10N: 1494258
- Preparation: The vehicle was prepared using:
* sodium phosphate monobasic monohydrate (Sigma-Aldrich ref. 71504)
* NaOH 10N
* drinking water treated by reverse osmosis using ELIX 5 (Millipore SA)
- Storage conditions: After preparation, the required amount of vehicle was stored in the refrigerator set at +5°C. In the afternoon of the day before control and test item dose formulations preparation, it was placed at room temperature under magnetic stirring.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Each female was placed with the same male until mating occurred.
- Proof of pregnancy: Vaginal plug or for sperm in vaginal lavage referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Alone
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Ion Chromatography (IC) with conductivity detection was used.
Determination of test item concentrations in dose formulations: Once in Weeks 1 and 4. A sample was taken from control and test item dose formulations and analyzed using the validated method.
Acceptance criterion: measured concentration = nominal concentration ± 10%.

The validation of the analytical method was conducted in CiToxLAB France (CiToxLAB France/Study No. 43498 VAS) prior to dose formulation analysis and precise details concerning the checked parameters, acceptance criteria and obtained results were documented in the corresponding validation report.
Duration of treatment / exposure:
The dosage forms were administered daily according to the following schedule:
# males:
- 2 weeks before pairing,
- during the pairing period,
- until euthanasia on Day 32.
# females:
- 2 weeks before mating,
- during the mating period,
- during gestation,
- during lactation until day 4 post-partum inclusive,
- until euthanasia for females with no delivery.
Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
Once a day, at approximately the same time
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Concentration: 10 mg/mL
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Concentration: 30 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Concentration: 100 mg/mL
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, on the basis of the results of:
* a Japanese 4-week toxicity study (Hita Research Laboratories/Test Code No.: B11-0254; 1994) where six male and six female Sprague-Dawley rats per group received the test item in formulation in water by gavage at 0, 8, 40, 200 or 1000 mg/kg/day (dose-volume = 10 mL/kg).

During the treatment period, no premature deaths occurred. There were no effects on general health or food intake. A trend towards absence of body weight gain was observed at 1000 mg/kg/day in the last week of treatment in males. Reductions in several red blood cell parameters (males) and increase in platelet count (females) were seen at 1000 mg/kg/day. A reduction in total protein was observed in females at 1000 mg/kg group, as well as an increase in urea nitrogen and creatinine in one female. An increase in ketone bodies and glucose was observed in urines of one female at 1000 mg/kg. There were no effects on organ weight. At necropsy, boundary ridge protrusion in the forestomach (plus reddening of the mucosa in the glandular stomach in one case) was observed in males treated at 1000 mg/kg, and boundary ridge protrusion in the forestomach, roughening and blackening of the glandular stomach mucosa (one to two females), and fading of the kidney (one female) were observed in the females. At microscopy, squamous epithelium hyperplasia in the forestomach was observed in one male at 200 mg/kg/day and all males at 1000 mg/kg/day. A reduction in glandular stomach parietal cells, surface epithelial cell hyperplasia, submucosal bleeding, and loss of villi in the duodenum were also observed in the 1000 mg/kg/day males. In females of this group, squamous epithelium hyperplasia in the forestomach, a reduction in glandular stomach parietal cells, surface epithelial cell hyperplasia, loss of villi in the duodenum, renal tubular necrosis, and/or urinary casts were noted in one to three females each time.
After 2 weeks of recovery, changes due to the test item were only observed in the males at 1000 mg/kg/day (reduction in mean corpuscular haemoglobin, squamous epithelium hyperplasia in the forestomach - lower occurrence and extent compared to the end of treatment, reduction in relative liver weight, peripheral hepatocyte enlargement of the liver), * a 7-day toxicity study (2016; CiToxLAB France/Study No. 43737 TSR) where five Sprague-Dawley males received the test item in formulation in pH 7.2 sodium phosphate 0.5M buffer by gavage at 1000 mg/kg/day (dose-volume = 10 mL/kg). There were no premature deaths or relevant clinical signs. All males gained weight and the mean food consumption was correct. There were no necropsy findings.

As the study duration of the present study was also 4 weeks in males (likely 5-6 weeks for most females), and as the vehicle slightly buffered the formulations compared to the first study, 1000 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).

- Administration: As the test item is corrosive, care was taken to correctly wipe the gavage tube before administering the animals in order to remove all the dose formulation at the exterior surface of the tube.
The control and test item dose formulations were stirred just before administration. The formulations were maintained under continuous magnetic stirring and at room temperature throughout the dosing procedure.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day

BODY WEIGHT: Yes
- Time schedule for examinations:
* Males: On the first day of treatment (Day 1), then once a week until euthanasia
* Females: On the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c. (post-coitum) and on Days 1 and 5 p.p.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
* Males: Once a week, over a 7-day period, from the first day of treatment until the start of the mating period.
* Females: once a week, over a 7-day period, from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-5 p.p.

During the mating period, food consumption was not measured for males or females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period until the females were mated.
Sperm parameters (parental animals):
No data
Litter observations:
Each live pup was identified individually on Day 1 p.p., by subcutaneous injection of Indian ink.

Litter size
The total litter size and sex of each pup were recorded as soon as possible after birth. Any gross external malformations in pups were noted.
The litters were observed daily in order to note the number of live, dead and cannibalized pups.

Clinical signs
The pups were observed daily for clinical signs, abnormal behavior and external abnormalities.

Body weight
The body weight of each pup was recorded on days 1 and 5 p.p..
Postmortem examinations (parental animals):
SACRIFICE
On completion of the treatment period, all F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination:
* males: after the end of the mating period (on Day 32),
* females: on Day 5 p.p.,
* females which did not deliver: on Day 25 p.c. (after a body weight recording to check for a possible un-noticed delivery).

GROSS NECROPSY
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all F0 animals. Special attention was paid to the reproductive organs and to the stomach/forestomach and esophagus.
The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 5 p.p.
For apparently non-pregnant females, the absence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 below (in Any other information on materials and methods incl. tables) were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
Surviving pups were euthanized on Day 5 p.p. by an intraperitoneal injection of sodium pentobarbital.
Moribund pups were euthanized by an intraperitoneal injection of sodium pentobarbital. Found dead and
prematurely euthanized pups were discarded without any examination.

GROSS NECROPSY
Pups were discarded without any examination.
Statistics:
- Body weight, food consumption and reproductive data:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

- Organ weight:
See attached document: statistic_1493-13-6.doc
Reproductive indices:
#pre-implantation loss:
(Number of corpora lutea - Number of implantation sites / Number of corpora lutea) x 100

#post-implantation loss (calculated manually):
(Number of implantation sites - Number of live pups / Number of implantations sites) x 100

#mating index:
(Number of mated animals / Number of paired animals) x 100

#fertility index:
(Number of pregnant female partners / Number of mated pairs) x 100

#gestation index:
(Number of females with live born pups / Number of pregnant females) x 100
Offspring viability indices:
#live birth index:
(Number of live born pups / Number of delivered pups) x 100

#viability index on day 4 post-partum:
(Number of surviving pups on day 4 post-partum / Number of live born pups) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was noted in all males (for 4 to 17 days) and all females (for 1 to 28 days; mainly during gestation) at 1000 mg/kg/day. This clinical sign was considered to be test item treatment-related.
Chromodaccryorrhea was recorded in males with a dose-related higher incidence at 300 and 1000 mg/kg/day. This clinical sign can be spontaneously observed in laboratory rats, but a relationship with the test item treatment cannot be excluded at the high-dose at least, where half of the males were affected and two of them during 11 or 14 days, vs. 2 days for the control male.
Both clinical signs were considered to be of minor toxicological significance (non-adverse clinical signs).
Other clinical signs recorded in test item-treated animals (chromorhinorrhea, scab, hair loss, cutaneous lesion, reddish vaginal discharge, nodosities, reflux at dosing) were not attributed to the test item treatment (no dose-relationship, of common background and/or at isolated occurrence).
In controls, there was one male (F24622) with round back and emaciated appearance on Day 6. This was the consequence of absence of access to water of the animal noticed on Day 6. Free access to water was given again on the same day and the clinical signs then disappeared.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects on mean body weights and mean body weight changes.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects on mean food consumption.
The higher food consumption during Week 1 (53 g/day vs. up to 26 g/day in the same group) and in the first week of gestation at least (46 g/day vs. up to 29 g/day in the same group) of female F24730 treated at 300 mg/kg/day was likely due to food spillage (which was indeed observed for this female in Week 2 of the study).
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Testes, epididymides, ovaries and all tissues with gross findings were examined.
Administration of the test item did not produce microscopic changes in the examined tissues from males and females. The few findings recorded were of those commonly observed in this species.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no effects on mean pairing, mating and fertility data.
The higher mean pre-coital time obtained at 100 mg/kg/day compared with controls was due to two females which were blocked in diestrus for several days during the mating period. This was considered as incidental (of common background and not dose-related).
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
In absence of dose-relationship and of statistical significance, there were no toxicologically significant effects on mean delivery data.
All females gave birth on Day 21 or 22 p.c., except one female (F24738) at 1000 mg/kg/day which gave birth on Day 23 p.c. In view of the limited incidence and as there were no consequences on post-implantation loss or on pup clinical condition, this event was considered incidental.

Details on results (P0)

There were:
. no premature deaths,
. minor toxicologically significant clinical signs at 1000 mg/kg/day as ptyalism in all animals and chromodacryorrhea in half males (a relationship with the test item cannot be excluded for this latter finding),
. no effects on mean body weights, mean body weight changes and mean food consumption,
. no effects on mean pairing, mating, fertility data,
. no toxicologically significant effects on mean delivery data,
. no effects on mean organ weights and at macroscopic and microscopic examinations.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
(parental toxicity)
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the absence of adverse effects up to this dose
Key result
Dose descriptor:
NOAEL
Remarks:
(reproductive performance)
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the absence of effects on mean reproductive data up to this dose

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related pup clinical signs.
At 300 mg/kg/day, there was one litter (F24726) with two pups in poor clinical condition, one of the two pups was prematurely euthanized for ethical reasons. The dam was the one with the lowest number of corpora lutea and the percentage of highest post-implantation loss in the study. These pup clinical conditions were not found in other litters of the same group or at the high-dose; they were therefore not considered to be test item-related.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were no effects on viability and lactation indexes.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects on mean pup body weight and mean pup body weight changes.
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio: There were no effects on the sex ratio at birth.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

There were:
. no effects on pup viability,
. no test item-related clinical signs,
. no toxicologically significant effects on mean body weight and mean body weight changes,
. no indication of a test item treatment effect on the percentage of male pups at birth.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
(toxic effects on progeny)
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the absence of adverse effects in pups up to this dose

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 2: Summary of the most relevant clinical signs observed

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Pre-mating (female) or whole study (males)

 

 

 

 

 

 

Ptyalism

 

 

 

10

 

 

 

3

Chromodacryorrhea

1

1

3

5

 

 

 

 

Emaciated appearance

1

 

 

 

 

 

 

 

Round back

1

 

 

 

 

 

 

 

Gestation

 

 

 

 

 

 

 

 

Ptyalism

/

/

/

/

 

 

 

10

Lactation

 

 

 

 

 

 

 

 

Ptyalism

/

/

/

/

 

 

 

3

/: not applicable

 

Table 3: Summary of pairing, mating and fertility data

Dose level (mg/kg/day)

0

100

300

1000

Number of animals paired (M + F)

10 + 10

10 + 10

10 + 10

10 + 10

Number of males mated

10

10

10

10

Number of females mated

10

10

10

10

Mean number of days taken to mate (pre-coital time)

2.5

4.6

2.0

2.3

Number of pregnant females

9

9

10

10

Number of females with live born pup(s)

9

9

10

10

M: males; F: females

 

Table 4: Summary of delivery data

Dose level (mg/kg/day)

0

100

300

1000

Number of females which delivered

9

9

10

10

Mean duration of gestation (days)

21.8

21.8

21.8

22.0

Mean number ofcorpora lutea

16.9

16.0

15.3

15.6

Mean number of implantations

15.9

15.4

12.9

15.4

Mean pre-implantation loss (%)

5.8

3.2

18.8

1.3

Mean number of pups delivered

14.0

12.4

11.2

13.5

Mean post-implantation loss (%)a

11.7

19.0

13.2

12.9

a: calculated with Excel, no statistics performed.

Table 5: Viability and lactation indexes

Dose level (mg/kg/day)

0

100

300

1000

Viability index on Day 4p.p.(%)

96.0

94.6

96.4

100.0

Lactation index on Day 5p.p.(%)

99.2

100.0

100.0

100.0

 

Table 6: Clinical signs observed during lactation (pup incidence, in brackets: litter incidence)

Dose level (mg/kg/day)

0

100

300

1000

Twisted tail

1a(1)

 

 

 

Hematoma (neck, head, thorax, back, hindlimbs)

1a(1)

 

2b,c(1)

1d(1)

Increase in size of back

 

 

1b(1)

 

Dehydration

 

 

2b, c(1)

 

Emaciated appearance

 

 

1b(1)

 

Absence of milk in the stomach

 

 

1b(1)

 

Generalized pallor

 

 

1c(1)

 

Absence of distal segment of one forelimb and its extremity

 

 

1c(1)

 

Scab (proximal segment of forelimb)

 

 

1c(1)

 

Hypoactivity

 

 

1c(1)

 

Cold to the touch

 

 

1c(1)

 

Deformation of hindlimb

 

 

1 (1)

 

a: same pup (No. 4 from dam F24710);b: same pup (No. 1 from dam F24726);c: same pup (No. 2 from dam F24726);d: same pup (No. 10 from dam F24734).

Table 7: Sex ratio at birth

Dose level (mg/kg/day)

0

100

300

1000

% of males on Day 0p.p.

45.2

55.4

45.5

51.1

Applicant's summary and conclusion

Conclusions:
The test item, Trifluoromethanesulfonic acid, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice for males, or through gestation and until Day 4 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day.

Under the experimental conditions and results of this study:
. the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day for males and females based on the absence of adverse effects up to this dose,
. the NOAEL for parental reproductive performance was considered to be 1000 mg/kg/day, based on the absence of effects on mean reproductive data up to this dose,
. the NOAEL for toxic effects on progeny was considered to be 1000 mg/kg/day, based on the absence of adverse effects in pups up to this dose.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, Trifluoromethanesulfonic acid, following daily oral administration (gavage), 2 weeks before mating, during mating and, for the males, until euthanasia, for the females, throughout gestation until Day 4 post-partum (p.p.) inclusive.

This study provided initial information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of theconceptusand parturition.

 

Methods

Three groups of 10 male and 10 female (groups 2 to 4) Sprague-Dawley rats received the test item, Trifluoromethanesulfonic acid (batch No. STR1525030), daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 4 p.p. The test item was administered as a solution in the vehicle, pH 7.2 sodium phosphate 0.5M buffer, at dose-levels of 100, 300 and 1000 mg/kg/day.

Another group of 10 males and 10 females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dose-volume of 10 mL/kg/day was used.

The concentrations of the dose formulations were checked in study Weeks 1 and 4.

The animals were checked at least twice daily for mortality and morbidity and once daily for clinical signs during the treatment period. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 post-coitum (p.c.) and lactation on Days 1 and 5 p.p.

The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p.The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on Days 1 and 5 p.p.

Males were sacrificed after at least 4 weeks of treatment and females on Day 5 p.p.Final body weights and selected organs weights (epididymides, testes) were recorded and a macroscopicpost-mortemexamination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on ovaries (with oviducts), epididymides and/or testes from all males sacrificed at the end of the treatment period and all females sacrificed on Day 5 p.p.in the control and high-dose groups, and on all macroscopic lesions.

Pups were sacrificed on Day 5 p.p.and no macroscopicpost-mortemexamination was performed.

 

Results

Chemical analyses

No test item was observed in the control dose formulations and the test item concentrations in the analyzed dose formulations were within an acceptable range of variations.

 

F0 animals

There were:

. no premature deaths,

. minor toxicologically significant clinical signs at 1000 mg/kg/day as ptyalism in all animals and chromodacryorrhea in half males (a relationship with the test item cannot be excluded for this latter finding),

. no effects on mean body weights, mean body weight changes and mean food consumption,

. no effects on mean pairing, mating, fertility data,

. no toxicologically significant effects on mean delivery data,

. no effects on mean organ weights and at macroscopic and microscopic examinations.

 

Pups

There were:

. no effects on pup viability,

. no test item-related clinical signs,

. no toxicologically significant effects on mean body weight and mean body weight changes,

. no indication of a test item treatment effect on the percentage of male pups at birth.

 

Conclusion

The test item, Trifluoromethanesulfonic acid, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice for males, or through gestation and until Day 4p.p.for females, at dose-levels of 100, 300 and 1000 mg/kg/day.

Under the experimental conditions and results of this study:

* the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day for males and females based on the absence of adverse effects up to this dose,

* the NOAEL for parental reproductive performance was considered to be 1000 mg/kg/day, based on the absence of effects on mean reproductive data up to this dose,

* the NOAEL for toxic effects on progeny was considered to be 1000 mg/kg/day, based on the bsence of adverse effects in pups up to this dose.