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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-07 to 2017-11-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Deviations did not impact the scientific integrity of the study
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test, July 2000)
Deviations:
yes
Remarks:
Deviations did not impact the scientific integrity of the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane
EC Number:
206-581-9
EC Name:
Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane
Cas Number:
355-37-3
Molecular formula:
C6HF13
IUPAC Name:
Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AGC Chemicals Europe Ltd (UK); Batch no. 41251
- Expiration date of the lot/batch: 01 March 2017 (nominal expiry date) (taken from label)
- Purity test date: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: until 01 March 2017 (nominal expiry date) (taken from label)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Colourless liquid

OTHER SPECIFICS:
Purity: 100.00%
Molecular weight: 320
Vapour pressure: 121 mmHg at 25°C
pH (1% in water, indicative range): 7.22 – 7.78 (determined by Charles River Den Bosch)
Specific gravity/density: 1.67 at 25°C
Stability in vehicle: Stability for at least 6 hours at room temperature under normal laboratory light
conditions is confirmed at 200 mg/mL, Test Facility Study No. 515115.
(1% Aqueous carboxymethyl cellulose with 3% Tween)

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Pretest Females: 11 wks; F0 Treatment - Males: 11 wks; Females: 13 wks
- Weight at study initiation: Not specified
- Fasting period before study: Not specified
- Housing:
A] Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
B] Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
C] Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
D) Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
E] Lactation: Females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam.

Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, males were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements (males only), animals did not have access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Free access to tap-water. During motor activity measurements (males only), animals did not have access to water for a maximum of 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 2016-09-07 To: 2016-11-25

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
other: 1% Aqueous carboxymethyl cellulose with 3% Tween
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Formulations were prepared at one concentration only for all dose groups; dose volume was adjusted to reach the correct dose levels. No adjustment was made for specific gravity/density of the vehicle. Adjustment was made for specific gravity/density of the test item (1.67 at 25°C). No correction was made for the purity/composition of the test. Actual dose volumes were calculated according to the latest body weight.

- Amount of vehicle (if gavage):
Group 1: 5 mL/kg body weight.
Group 2: 0.5 mL/kg body weight.
Group 3: 1.5 mL/kg body weight.
Group 4: 5 mL/kg body weight.

- Lot/batch no. (if required): 1% Aqueous carboxymethyl cellulose (carboxymethyl cellulose: BUFA, IJsselstein, The Netherlands; water: Elix, Millipore S.A.S., Molsheim, France or Milli-Q. Millipore, Bedford, MA, USA) with 3% Tween 80 (Merck-Schuchardt, Hohenbrunn, Germany).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted during the treatment phase according to a validated method (Test Facility Study No. 515115). Due to analytical problems during analysis of formulation samples taken on 27 September 2016, new samples were taken on 11 October 2016. Results from the first measurement were not reported, but kept in the raw data. Samples of formulations were analyzed for homogeneity (formulation containing test item, used for Groups 2 - 4) and accuracy of preparation (formulation used for Group 1 (vehicle only), and formulation used for Groups 2-4). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85 - 115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males were dosed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.

Females that delivered were dosed for 50-64 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. The two females which failed to deliver healthy offspring were treated for 43 and 53 days, respectively.

Female nos. 47 (Group 1), 57, 58, 60 (Group 2) and 76 (Group 4) were not dosed at the time of littering. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 (Control)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previous 28-day repeated dose oral toxicity study in the rat, in which dose levels of 40, 200 and 1000 mg/kg were tested. In rats dosed with either 40 or 200 mg/kg, no treatment-related effects were observed on clinical signs, body weight, food consumption, hematological and blood chemical examinations, urinalysis, necropsy and histopathological examinations. In male rats dosed with 1000 mg/kg, increased liver weights were noted at the end of treatment, which returned to control group level after a 14-day treatment free recovery period.

- Rationale for animal assignment (if not random): Before initiation of pretest, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (mortality and viability)
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals directly after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes (Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected)

OTHER:
1) Functional Observations: The following functional observations tests were performed on the first 5 males per group:
A] Hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent)
B] Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
C] Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
The males were tested during Week 4 of treatment; tests were performed after observation for clinical signs.
Sacrifice and pathology:
SACRIFICE: All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.

- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals. Females which delivered were terminated PND 14-16. Females which failed to deliver (nos. 68, 73) were terminated post-coitum Day 25 (female no. 73 with evidence of mating) or 26 days after the last day of the mating period (female no. 68 without evidence of mating).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique (Ref. 1) in order to detect any former implantation sites (Salewski staining prepared at Charles River
Den Bosch using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Samples of the tissues mentioned in Table 2. and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights: The following organ weights and terminal body weight were recorded from all animals on the scheduled day of necropsy:

Epididymides
Prostate
Seminal vesicles including coagulating glands
Testes
Thyroid, including parathyroid if detectable

Absolute organ weights and organ to body weight ratios are reported.

Histopathology: All organ and tissue samples, as defined under Histopathology, were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

The following slides were examined by a pathologist:
1) The ovaries, testes, epididymides and thyroids of the animals of Groups 1 and 4.
2) Additional slides of the testes of the males of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis.
3) All gross lesions of all animals (all dose groups).
4) The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of all males that failed to sire and all females that failed to deliver healthy pups:

Group Female/Male nos. Reason
3 68/28 Not mated
4 73/33 Not pregnant

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings. A peer review on the histopathology data was performed by a second pathologist.

Blood Sampling for Thyroid Hormone Analysis:
F0-generation, males and females: End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of treatment (including all males that failed to sire). Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retro-orbital sinus and collected into serum tubes (Greiner BioOne GmbH, Kremsmünster, Austria).

After clotting and centrifugation, serum was used as listed below.
Males: 1 aliquot of 150 µL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroid-stimulating hormone (TSH).

Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroidstimulating hormone (TSH). Serum samples were stored at ≤ -75°C. Under these storage conditions, the samples are stable for 6months. Any samples remaining after 6 months will be discarded.

In general: Total T4 was measured in serum using the Immulite 1000® (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
Parameter (Abbreviation) Unit
Thyroxine (T4) µg/dL
Generation Time point of terminal blood sampling Parameter T4 TSH
F0-males After at least 28 days of treatment. Yes Not required
F0-females PND 14-16 Not required Not required
Statistics:
The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one ttest) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during the observation period.

One female treated at 1000 mg/kg (no. 72) showed piloerection on a few days prior to scheduled sacrifice. From Day 7 of the lactation period, she also had considerable weight loss and reduced food consumption. Macroscopic and microscopic findings in this female were suggestive of misgavage,and hence were unrelated to treatment with the test item. Other clinical findings noted incidentally occurred within the range of background findings to be expected for rats of this strain and age which are housed and treated under the conditions in this study. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were not affected by treatment.

The apparently lower mean body weight and body weight gain noted in females treated at 1000 mg/kg on Day 13 of the lactation period was due to the weight loss of a single female (no. 72) and considered to be unrelated to treatment with the test material.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before and after allowance for body weight were not affected by treatment.

The apparently lower mean food consumption (absolute and relative to body weight) noted in females treated at 1000 mg/kg between Days 7-13 of the lactation period was due to the reduced food consumption of a single female (no. 72) and considered to be unrelated to treatment with the test material.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum levels of T4 in F0 males were not affected by treatment up to 1000 mg/kg.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Seminal vesicle weights (absolute and relative to body weights) in males treated at 1000 mg/kg were lower than those of the controls. The effect was considered to be treatment-related. However, as the mean value, about 20% lower compared to concurrent controls, remained within the normal range, and reproductive performance was not affected, the lower seminal vesicle weight at 1000 mg/kg was considered not adverse.

There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related gross observations.

Findings of note were recorded for female no. 72 treated at 1000 mg/kg consisted of:
• A gray-white hard nodule, size 3x2 mm, at the right ventricle of the heart, microscopically correlated with marked endocarditis.
• Many gray-white hard nodules of the lung, microscopically correlated with extensive suppurative bronchopneumonia.
• Several gray-white hard nodules of the kidneys (bilateral), microscopically correlated with acute bilateral pyelonephritis.
• Thoracic cavity, pleura grown together with lungs, microscopically correlated with an extensive perivascular inflammation and adhesions of the serosa and the presence of a marked thrombus.

These findings were observed in a single female and were regarded to be suggestive for a misgavage. Therefore these findings were considered to be unrelated to treatment with the test item.

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this strain and age, and did not show a dose-related incidence trend. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic observations. Findings of note were observed in female no. 72 treated at 1000 mg/kg (described earlier under Gross pathological findings). The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this strain and age. There was no treatment related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on a lack of adverse treatment-related effects observed, the systemic toxicity No Observed Adverse Effect Level (NOAEL) was determined to be at least 1000 mg/
Kg bw/day.
Executive summary:

In a key guideline (OECD 421) oral reproductive/developmental toxicity screening test (Charles River Laboratories Den Bosch BV., 2017e), the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane) formulated in 1% aqueous carboxymethyl cellulose with 3% Tween 80 was administered by daily oral gavage to male and female Wistar Han rats (10/sex/dose) at dose levels of 0, 100, 300 and 1000 mg/Kg. Males were treated for 2 weeks prior to mating, during mating, and up to the day prior to termination (for 29 days). Females that delivered were treated for 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation (i.e. up to the day prior to scheduled necropsy; for 50-64 days). Two females that failed to deliver healthy offspring were treated for 43 or 53 days.

 

There were no treatment-related changes in the in-life parameters examined in this study up to 1000 mg/Kg (i.e. mortality, clinical appearance, body weight and body weight gain, food consumption). Males treated at 1000 mg/Kg had statistically significantly lower weights (absolute and relative to body weight) of the seminal vesicles than controls. It could not be excluded that this was related to treatment. However, as the mean value, about 20% lower compared to concurrent controls, remained within the normal range, and reproductive performance was not affected, the lower seminal vesicle weight at 1000 mg/kg was considered not adverse.

 

No treatment-related changes were noted in serum thyroid hormone T4 in males, remaining organ weights (testes, epididymides, prostate, thyroid), or findings at macroscopic and microscopic examination (testes, epididymides, ovaries, thyroid) up to 1000 mg/Kg.

   

Based on the results observed, the systemic toxicity No Observed Adverse Effect Level (NOAEL) was determined to be at least 1000 mg/Kg.