Trideca-1,1,1,2,2,3,3,4,4,5,5,6,6-fluorohexane

Endpoint summary

Description of key information

Two key guideline (OECD 407, 421) studies that investigated the repeated dose toxicity potential of 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane following oral exposure (gavage) in rats are available. The results are summarized below:

 

OECD 407

The oral repeated-dose toxicity NOEL for 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was considered to be 200 mg/Kg/day, based on increased liver weights observed in male rats exposed to the test material at 1000 mg/Kg/day for 28 days.

 

OECD 421

The oral repeated-dose toxicity NOAEL for 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was determined to be at least 1000 mg/Kg, based on lack of adverse

treatment-related effects observed at the highest dose tested, and after a maximum of 64 days exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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)

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

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.

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

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.

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.

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

Key result

Critical effects observed:

no

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-12-24 to 1994-06-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Asahi Glass Co., Ltd. (Japan): Batch no. 930817 (preliminary study), 31001 (main study)
- Expiration date of the lot/batch: Not specified
- Purity test date: 1994-02-24

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in a cold and dark place
- Stability under test conditions: stable during the dosing period

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

OTHER SPECIFICS:

Purity: 99.9%
Molecular weight: 320.05
Boiling Point: 71°C
Solubility: Oil soluble
Degree of solubility Water: <10 mg/L

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crj:CD (SPF)

Details on species / strain selection:

Not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc. (Hino Breeding Center, 735, Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, Japan)
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: 5 weeks old
- Weight at study initiation: Males: 121.5 - 135.6 grams; Females: 123.6 - 138.1 grams
- Fasting period before study: Not specified
- Housing: Individually in a hanging stainless steel cage with wire-mesh floor (165 mm W x 300 mm D x 150 mm H; Tokiwa Kgaku Kikai Co., Ltd.)
- Diet (e.g. ad libitum): MF pelleted diet (Oriental Yeast Co., Ltd.) autoclaved at 121°C for 30 minutes prior to use was supplied ad libitum
- Water (e.g. ad libitum): Water (chlorinated) from the Hita city supply was provided ad libitum via automatic waterer with sipper tubes.
- Acclimation period: 7-8 days from receipt

DETAILS OF FOOD AND WATER QUALITY: Analysis of contaminants in both diet and water confirmed that they would not affect the test system

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light (07:00 - 19:00)

IN-LIFE DATES: From: 1994-02-05 To: 1994-04-06

Route of administration:

oral: gavage

Details on route of administration:

Not specified

Vehicle:

other: 3% Polyoxyethylene (20) sorbitan mono-oleate including 1% carboxymethyl cellulose dissolved in purified water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was not soluble in olive oil
- Concentration in vehicle: 0.4, 2, & 10%
- Amount of vehicle (if gavage): 10 mL/Kg
- Lot/batch no. (if required): Not specified
- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test substance were confirmed in the labortaory using infra-red spectrophotometer and gas chromatography. Details on the same can be accessed in the Appendix in the attached study report.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily (in the morning)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle Control

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle Control (Recovery)

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Low Dose

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Intermediate Dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High Dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High Dose (Recovery)

No. of animals per sex per dose:

6/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 14-day preliminary repeated-dose oral toxicity study was carried out at three doses of 50, 250, or 1000 mg/Kg. There were no abnormaltiies in clinical signs, body weights, hematological blood chemical examinations, organ weights and pathological examinations. Based on these results, high dose level at 1000 mg/Kg and two lower doses at 200 40 mg/Kg were applied to the main study. Receovery groups were separately provided for the 1000 mg/Kg and vehicle control groups.
- Rationale for animal assignment (if not random): Body-weight stratified randomization

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed as follows: Before dosing day -2 (at grouping); During dosing period: day 1,3,5,8,10, 12,15,17,19,22,24,26, and 28; During recovery period: day 1,3,5,8,10,12, and 14.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measure as follows: Before dosing: Once; During dosing and recovery periods: Twice weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of dosing and recovery periods
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (16-20 hours)
- How many animals: All animals
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of dosing and recovery periods
- Animals fasted: Yes (16-20 hours)
- How many animals: All animals
- Parameters checked in table [No.3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During dosing period: Once (Day 28); During recovery period: Once (Day 14)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters examined: volume, colour, additional items of pH, protein, ketone bodies, bilirubin, occult blood, glucose, and urobilinogen using a test paper (N-Multistix®, Miles-Sankyo)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

Animals were subjected to a detailed gross necropsy abnormalities recorded. Following organs were recorded in wet weight: Brain, Liver, Spleen, Kidneys, Adrenal glands, Testes, Ovaries

HISTOPATHOLOGY: Yes

1) The organs and tissues mentioned in Table No. 4. were preserved in 10% formalin:

2) Light microscopic examinations were performed for the following organs and tissues after embedding in paraffin, sectioning and staining with hematoxylin and eosin.

a) Terminal necropsy of dosing period: Vehicle Control and 1000 mg/Kg groups: Liver, Spleen, Kidneys, Heart, Stomach, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Adrenal glands.

b) Gross lesions: Terminal necropsy of dosing period: 200 mg/Kg group (Male No. 24): Glandular stomach; 40 mg/Kg group (Male No. 16): Glandular stomach; 40 mg/Kg group (Female No. 49): Hindlimb

c) Terminal necropsy of recovery period: Vehicle control group (Male Nos. 10 and 11): Glandular stomach; 1000 mg/Kg group (Female No. 71): Glandular stomach; 1000 mg/Kg group (Female No. 68): Skin

Statistics:

Data regarding body weights, food consumption, hematological examinations, blood chemical examinations, urine volume and organ weights were anlayzed using the Bartlett's test for homogeneity of variance. If the variances were homogenous at a significane level of 5%, one way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analyzed by the Dunnett's test (equal number of data) or Scheffe's test (unequal number of data).

If the variances were not homogenous the Kruskal Wallis test was used. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analyzed by the non parametric Dunnett's test (equal number of data) or non parametric Scheffe's test (unequal number of data).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During Dosing Period:
Males: Reddish tear (1/12) observed in the vehicle control group. Loss of hair (2/12) and scab formation (2/12) at the left cervical region observed in the 1000 mg/Kg group.
Females: Swelling of the left hind limb (1/6) was observed in the 40 mg/Kg group.

During Recovery Period:
Males: Scab formation (1/6) at the left cervical region observed in the 1000 mg/Kg group.
Females: Scab formation (1/6) at the left cervical region observed in the 1000 mg/Kg group.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed through the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No abnormalities observed in animals of either sex in all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No abnormalities observed in animals of either sex in all dose groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period: No abnormalities observed in animals of either sex in all dose groups.

At Termination of Recovery Period:
Males: No abnormalities observed in animals in all dose groups.
Females: An increase in white blood cell count was observed in the 1000 mg/Kg dose group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period:
Males: An increase in Total Cholesterol level was observed in 200 mg/Kg dose group
Females: No abnormalities observed in animals in all dose groups.

At Termination of Recovery Period:
Males: Increases in GOT and GPT activity was observed in the 1000 mg/Kg dose group.
Females: Decreases in Total Protein, Albumin, and Creatinine levels was observed in the 1000 mg/Kg dose group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

At Termination of Dosing Period: No abnormalities observed in animals of either sex in all dose groups.

At Termination of Recovery Period: No abnormalities observed in animals of either sex in all dose groups.

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):

At Termination of Dosing Period:
Males: An increase in relative liver weight was observed in the 1000 mg/Kg dose group
Females: No abnormalities observed in animals in all dose groups.

At Termination of Recovery Period:
Males: An increase in relative testes weight was observed in the 1000 mg/Kg dose group
Females: No abnormalities observed in animals in all dose groups

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period:
Males: Blackish region of the mucosa in the glandular stomach were observed in the vehicle control group (1/6); 40 mg/Kg group (1/6); 200 mg/Kg group (1/6).
Females Swelling of the paw (1/6) in the hind limb was observed in the 40 mg/Kg group. Blackish region of the mucosa in the glandular stomach was observed in the 1000 mg/Kg group (1/6).

At Termination of Recovery Period:
Males: Blackish region of the mucosa and blackish spot of the mucosa in the glandular stomach were observed in the vehicle control group (1/6).
Females: Scab formation (1/6) at the left cervical region and blackish region of the mucosa (1/6) in the glandular stomach were observed in the 1000 mg/Kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period:
Males: Necrosis of the mucosa in the glandular stomach was observed in the vehicle control group (+, 1/6); 40 mg/Kg group (+, 1/1); and 200 mg/Kg group (+, 1/1).
Females: Proliferation of connective tissue with synovitis (1/1) in the hind limb was observed in the 40 mg/Kg group. Necrosis of the mucosa (+, 1/6) in the glandular stomach was observed in the 1000 mg/Kg group.

At Termination of Recovery Period:
Males: Necrosis of the mucosa (+, 2/2) in the glandular stomach was observed in the vehicle control group.
Females: Necrosis of the mucosa (+, 1/1) in the glandular stomach and granulation tissue with scab formation (1/1) in the skin was observed in the 1000 mg/Kg group.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 5. Relative Organ Weights
Sex	Exp Group (mg/Kg/day)	Number of Animals	Liver (g/100 g)	Testis (g/100 g)
Male	Vehicle Control	6	2.87 ± 0.11	0.90 ± 0.11
	40	6	2.81 ± 0.08	0.96 ± 0.07
	200	6	3.02 ± 0.20	0.90 ± 0.07
	1000	6	3.14** ± 0.11	0.93 ± 0.05
	Recovery
	Vehicle Control	6	2.71 ± 0.08	0.78 ± 0.05
	1000	6	2.67 ± 0.05	0.86* ± 0.05

Mean ± S.D.

* Significantly different from vehice control at P <0.05

** Significantly different from vehicle control at P <0.01

Table 6. Clinical Signs
Sex	Signs	Number of Animals (6/group)
		Administration Period (mg/Kg/day)						Recovery Period
		Vehicle Control	Vehicle Control (Recovery)	40	200	1000	1000 (Recovery)	Vehicle Control	1000
Male	No. of abnormalities detected	5	6	6	6	5	5	6	5
	Loss of hair (cervical region)					1	1
	Scab formation (left cervical region)					1	1		1
	Reddish tear	1
Female	No. of abnormalities detected	6	6	5	6	6	6	6	5
	Swelling of the left hind limb			1
	Scab formation (left cervical region)								1

Conclusions:

The oral repeated-dose toxicity NOEL for 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was considered to be 200 mg/Kg/day, based on increased liver weights observed in male rats exposed to the test material at 1000 mg/Kg/day.

Executive summary:

In a key guideline (OECD 407) short-term repeated-dose toxicity study, the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane) was orally administered via gavage once daily to Crj:CD (SD) rats (6/sex/dose) at doses of 0, 40, 200, or 1000 mg/Kg/day for a period of 28 days followed by a recovery period of 14 days. Recovery groups were separately provided for the 1000 mg/Kg/day and vehicle control groups.

 

No mortality was observed through the study period and body weight and food consumption during the dosing period remained unaffected by exposure to the test material. Treatment with the test material did not have any adverse effect on hematological and blood chemistry parameters, or on urinanalysis examinations conducted at the termination of the dosing period.

 

During the dosing period, loss of hair and scab formation at the left cervical region were observed in two males in the 1000 mg/Kg/day dose group while swelling of the left hind limb was observed in one female in the 40 mg/Kg/day dose group. Loss of hair and scab formation were considered to have been caused by the grasping of animals for dosing while the cause for swelling of the hind limb was not identified. However, the swelling of the hind limb occurred in only one female in the low dose group and was therefore not considered to be treatment-related.

 

At the termination of the dosing period, increased liver weight was observed in the 1000 mg/Kg/day males. While this appeared to be related to treatment with the test material, no histopathological changes were observed in the liver.

 

Necrospy conducted at termination of the dosing period revealed in females of the 1000 mg/Kg/day dose group, blackish region of the mucosa in the glandular stomach which corresponded to necrosis of the mucosa histopathologically. Similar lesions were also noticed with no dose-relationship in other groups including the vehicle control suggesting that these were incidental and not treatment-related.

 

In the recovery test, increases in GOT and GPT activities and testis weights were observed in the 1000 mg/Kg/day males. An increase in the white blood cell count and decreases in total protein, albumin, and creatinine levels were observed in the 1000 mg/Kg/day females. These findings were not considered to be treatment-related as there were no similar findings at the termination of the dosing period and no other changes associated with them.

 

The oral repeated-dose toxicity NOEL for 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was considered to be 200 mg/Kg/day, based on increased liver weights observed in male rats exposed to the test material at 1000 mg/Kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeat Dose Toxicity - Oral

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.

In a key guideline (OECD 407) short-term repeated-dose toxicity study (Hita Research Laboratories, 1994a), the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane) was orally administered via gavage once daily to Crj:CD (SD) rats (6/sex/dose) at doses of 0, 40, 200, or 1000 mg/Kg/day for a period of 28 days followed by a recovery period of 14 days. Recovery groups were separately provided for the 1000 mg/Kg/day and vehicle control groups.

 

No mortality was observed through the study period and body weight and food consumption during the dosing period remained unaffected by exposure to the test material. Treatment with the test material did not have any adverse effect on hematological and blood chemistry parameters, or on urinanalysis examinations conducted at the termination of the dosing period.

 

During the dosing period, loss of hair and scab formation at the left cervical region were observed in two males in the 1000 mg/Kg/day dose group while

swelling of the left hind limb was observed in one female in the 40 mg/Kg/day dose group. Loss of hair and scab formation were considered to have been caused by the grasping of animals for dosing while the cause for swelling of the hind limb was not identified. However, the swelling of the hind limb occurred in only one female in the low dose group and was therefore not considered to be treatment-related.

 

At the termination of the dosing period, increased liver weight was observed in the 1000 mg/Kg/day males. While this appeared to be related to treatment with the test material, no histopathological changes were observed in the liver.

 

Necrospy conducted at termination of the dosing period revealed in females of the 1000 mg/Kg/day dose group, blackish region of the mucosa in the

glandular stomach which corresponded to necrosis of the mucosa histopathologically. Similar lesions were also noticed with no dose-relationship in other groups including the vehicle control suggesting that these were incidental and not treatment-related.

 

In the recovery test, increases in GOT and GPT activities and testis weights were observed in the 1000 mg/Kg/day males. An increase in the white blood cell

count and decreases in total protein, albumin, and creatinine levels were observed in the 1000 mg/Kg/day females. These findings were not considered to be treatment-related as there were no similar findings at the termination of the dosing period and no other changes associated with them.

 

The oral repeated-dose toxicity NOEL for 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane was considered to be 200 mg/Kg/day, based on increased liver weights observed in male rats exposed to the test material at 1000 mg/Kg/day.

Justification for classification or non-classification

Not classified for oral repeated dose toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).