Registration Dossier

Administrative data

Endpoint:
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 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
Vehicle:
other: 1% Aqueous carboxymethyl cellulose with 3% Tween
Details on 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%.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days was allowed for mating
- Proof of pregnancy: sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing females who had not shown evidence of mating were separated from their males.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
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.
Duration of test:
Males: 29 days
Females: 50-64 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1 (Control)
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day
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 repeateddose 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

Maternal examinations:
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.

Additionally, Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

POST-MORTEM EXAMINATIONS: Yes
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 6 months. 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
Ovaries and uterine content:
Estrous cyclicity (parental animals)
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals)
Parameters examined in male parental generations: staging of spermatogenesis

The ovaries and uterine content was examined after termination: Yes
Fetal examinations:
- F1 parental animals: Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.

STANDARDISATION OF LITTERS
- Performed on day 1 postpartum: Yes
- all pups/litter ; excess pups were killed and discarded. To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in [F1 offspring]: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-15), except those used for blood sampling on PND 13-15, were sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater, The Netherlands) by intraperitoneal (ip) injection. On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter were collected into one serum tube.

On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination.

GROSS NECROPSY
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated. Necropsy was conducted on the following days:
Condition: Day of necropsy
Culling: PND 4.
Terminal sacrifice: PND 13-15.

Blood Sampling for Thyroid Hormone Analysis:
F1-generation, PND 4 pups: From 2 surplus pups per litter at culling (if possible). Blood samples from the 2 pups per litter were collected into one serum tube. If only 1 surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. Blood samples were collected by decapitation, between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter (0.4 mL in total) were collected into one serum tube (Greiner Bio-One GmbH, Kremsmünster, Austria) for possible future measurement of thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months were discarded. F1-generation, PND 13-15 pups: Blood samples were collected from 2 pups per litter (if possible from one male and one female) at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m.. Blood was collected into serum tubes. After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150 µL serum for measurement of thyroxine (T4) and the remaining volume of serum for possible future measurement of thyroid-stimulating hormone (TSH). Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.

Generation Time point of terminal blood sampling Parameter*
T4 TSH
2 pups/litter PND 4 Not required ---
2 pups/litter PND 13-15 Yes Not required
* Assessment of TSH for PND 13-15 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 13-15. Assessment of T4 and TSH for F0-females was considered not relevant because F0-males showed no treatment-related changes in T4, and since both sexes showed a similar degree of thyroid follicular hypertrophy and showed no treatmentrelated changes in thyroid weight or morphology. Assessment of TSH for F0-males was considered not relevant because F0-males showed no treatment-related changes in T4 and thyroid weight or morphology.
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.
Indices:
Reproductive indices
For each group, the following calculations were performed:
1) Mating index (%): (Number of females mated / Number of females paired) x 100
2) Fertility index (%): (Number of pregnant females / Number of females paired) x 100
3) Conception index (%): (Number of pregnant females / Number of females mated) x 100
4) Gestation index (%): (Number of females with living pups on PND 1 / Number of pregnant females) x 100
5) Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices
Survival indices:
1) Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
2) Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
3) Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
4) Percentage live females at First Litter Check (%) = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
5) Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
6) Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no differences in post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) in the treated groups compared to the controls. The survival indices across the groups were 89-94%. For females nos. 72 and 77 (both treated at 1000 mg/kg) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during (the 14-16 days of) lactation. No toxicological relevance was attached to this finding in this study.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation index and duration: There were no differences in gestation index and duration of gestation in the treated groups compared to the controls.

Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Other effects:
no effects observed
Description (incidence and severity):
Reproductive function: estrous cycle - effects observed, non-treatment-related
There were no differences in the length and regularity of the estrous cycle in the treated groups compared to the controls. All females had regular cycles of 4 days. Extended di-estrus during pairing occurred in one female of each treated group (nos. 58, 68 and 77). These females had normal litters except for no. 68 which had not mated. The incidence of females with extended di-estrus was within normal limits and did not indicate a relation with treatment.

Reproductive function: sperm measures - no effects observed
Spermatogenic staging profiles were normal for all males examined

Reproductive performance: no effects observed
There was 1 out of 10 couples (male no. 28 and female no. 68) treated at 300 mg/kg without successful mating, and 1 out of 10 couples (male no. 33 and female no. 73) treated at 1000 mg/kg without pregnancy despite proof of mating. No cause for the absences of offspring could be established from the sections examined. Based on the low incidences (within background incidence of rats of this strain and age) and absence of related histopathology changes in the reproductive organs, these failures to produce offspring were considered to be unrelated to treatment with the test material. There were no morphological findings in the reproductive organs of either sex, which could be attributed to the test material.

Mating Index: There were no differences in the mating index in the treated groups compared to the controls. One female treated at 300 mg/kg (no. 68) showed no evidence of mating. This incidental unsuccessful mating, which occurred in absence of related histopathology changes in reproductive organs or a dose-related trend, was considered to be unrelated to treatment.

Precoital time: There were no differences in precoital time in the treated groups compared to the controls. All females with evidence of mating were mated within 4 days, except for one female each in the low dose and high dose group for which it took 14 days before mating could be confirmed. At the isolated incidence and in the absence of a dose-related trend, it was considered to be unrelated to treatment.

Number of implantation sites: There were no differences in the number of implantation sites in the treated groups compared to the controls.

Fertility and Conception index: There were no differences in fertility and conception indices in the treated groups compared to the controls. Except for one female treated at 1000 mg/kg (no. 73), all mated females were pregnant. This incidental non-pregnancy without related histopathology changes in reproductive organs was considered to be unrelated to treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no differences in body weights of pups in the treated groups compared to the controls.

The pups of one female treated at 1000 mg/kg (no. 72) showed growth retardation between lactation Days 7-13. This was due to the poor health condition of the dam and considered to be unrelated to treatment with the test item. Additionally, pup body weights of female no. 75 (treated at 1000 mg/kg) were reduced. As this was limited to one litter only, this effect was considered not to be treatment related.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
At first litter check, one pup in the 100 mg/kg group (litter no. 55) and one pup in the 1000 mg/kg group (litter no. 75) were found dead. This incidental pup mortality was unrelated to treatment with the test material.

There were no differences in live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) in the treated groups compared to the controls. The live birth indices across the groups were 99 or 100%.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio: There were no differences in sex ratio in the treated groups compared to the controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no differences in litter size in the treated groups compared to the controls.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
Viability index: There were no differences in viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) in the treated groups compared to the controls. The viability indices across the groups were 98 or 100%.

Post-natal loss up to PND 4 was limited to two pups of the control group (litter nos. 45 and 46) which went missing on PND 2 or 3, and three pups at 1000 mg/kg (two pups of litter no. 75 went missing on PND 2, one pup of litter no. 77 was found dead on PND 4). Pups found missing were most likely cannibalized. This incidental post-natal loss was unrelated to treatment with the test material.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.

The nature and incidence of the few findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
No clinical signs that were considered to be related to treatment occurred among pups. The clinical signs observed remained within the range considered normal for pups of this age and showed no dose-related trend. They were therefore considered to be unrelated to treatment.

Clinical Biochemistry: There were no differences in serum T4 levels in male and female PND 13-15 pups in the treated groups compared to the controls.

Sexual maturation:
Anogenital distance: There were no differences in anogenital distance (absolute and normalized for body weight) in male and female pups in the treated groups compared to the controls.
Areola/nipple retention: There were no differences in the areola/nipple retention in the treated groups compared to the controls. No nipples were observed in any of the male pups examined on PND 13

Lactation index : No pups were found dead or went missing between lactation Days 5 and 13. For each group, the number of live offspring on Day 13 after littering was the same as the number of live offspring on Day 4 (after culling), resulting in a lactation index of 100% for all groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

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

In a key Guideline (OECD 421) oral reproductive/developmental toxicity screening test, 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.

 

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/Kg).No treatment related changes were noted in any of the reproductive  parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs)

 

No developmental toxicity was observed up to the highest dose level tested (1000 mg/Kg).No treatment related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, post-implantation survival index, viability and lactation indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum thyroid hormone T4 (PND 13-15) and macroscopy).

 

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