Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 October 2016 to 02 January 2017
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from moisture
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Formulations of the test item in paraffin oil were found to be homogenous at both concentrations tested (12.5 mg/mL and 250 mg/ mL). Formulations at 12.5 mg/mL and 250 mg/mL were also found to be stable under all conditions i nvestigated (6 hours at room temperature, 10 days at room temperature, at 2-8 °C and at -15 to -35 °C).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item and further vortexing it for 2-3 minutes. To protect from hydrolysis by humidity, the test item and formulation containers were purged with argon after the preparation before closing.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable- Final preparation of a solid: not applicable

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: males: 261 - 301 g; females: 174 - 203 g
- Fasting period before study: none
- Housing: - Animals were housed in groups of 2 animals / sex / cage in IVC cages during the premating period for both males and females and during postmating period for males depending on the mating status.
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water (e.g. ad libitum): Tap water, sulphur acidified to a pH of approximately 2.8, ad libitum- Acclimation period: 6 days
DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): : 55 +/- 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
paraffin oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item before further vortexing for 2-3 minutes. To protect from hydrolysis by humidity, the test item and formulation containers were purged with argon after the preparation and before closing the containers. The test item or vehicle were administered at a single dose daily to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured. The test item formulation was prepared at least once every ten days based on available stability data. The prepared formulation was stored at room temperature under argon.
VEHICLE
- Justification for use and choice of vehicle: The vehicle, light liquid paraffin, was selected based on the test item’s characteristics, dose range finding study and testing guideline after consultation with the sponsor.
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): 0000720349 / 0000855509 - Purity: not specified
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until confirmation of mating.
- Proof of pregnancy: vaginal plug/sperm in vaginal smear referred to as day 0 of pregnancy
- After unsuccessful pairing replacement of first male by another male with proven fertility was considered.
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): After the confirmation of mating, females were kept individually during gestation/lactation period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected for the investigation of homogeneity and substance concentration. Samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (40 samples).
Duration of treatment / exposure:
females: up to 54 days
males: 28 days
Frequency of treatment:
7 days a week
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters: not applicable
- Selection of parents from F1 generation when pups were [...] days of age: not applicable
- Age at mating of the mated animals in the study: 13-14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
low dose (LD)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
mid dose (MD)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
high dose (HD)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
high dose (HD): At the end of the 2nd week of the premating period, the animals of the HD group were not administered with the test item for 2 to 3 days due to observed morbidity and mortality. From the respective premating day 14 / mating day 1 onwards surviving HD animals were treated with a reduced dose of 125 mg/kg bw/day.
No. of animals per sex per dose:
test animals: 10 males and 10 females per sex per group
recovery animals: 12 males and 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected according to the results of a previous dose range finding study and in consultation with the sponsor.
- Rationale for animal assignment (if not random): random
Positive control:
Not included

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day, preferably at the same time each day, twice daily for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- Cage side observations included: general clinical observations, health condition of the animals, morbidity and mortality.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter. Clinical observations included check for spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded if present.
BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as on day 4 post-partum along with the pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, runs and presence of gross abnormalities. Within 24 hours of parturition, live pups were weighed.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not specified

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not specified
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after completion of the mating period on treatment day 29.
- Maternal animals: Females along with their pups were sacrificed on post-natal day 4. Non-pregnant females of the main groups were sacrificed on study day 26 using the sperm-positive vaginal smear.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera (See Table #2).

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table #2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to examinations as follows: gross external abnormalities

GROSS NECROPSY
- Gross necropsy consisted of external abnormalities examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Histopathology was not performed on tisses of offsprings.
Statistics:
A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
Reproductive indices:
Copulation index, fertility index, delivery index
Offspring viability indices:
Viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Remarkable clinical signs related to the neuromuscular system were observed dose-dependently in males and females of the MD and the HD group. Predominant signs were ataxia, paresis and hypotonia. Mainly the hind limbs appeared to be affected. Neuromuscular clinical signs were progressing in most animals in the course of the treatment period. Additionally, abnormal breathing was observed in several animals of the HD group mostly shortly before euthanasia for animal welfare reasons. Onset of neuromuscular clinical symptoms was from treatment day 10 onwards. Overall there was a tendency for males being affected earlier and more severely when compared to females, which mostly led to an earlier interim sacrifice of males than females. General signs of reduced health condition such as reduced spontaneous activity, wasp waist (sunken flanks) and piloerection were also observed dose-dependently in the MD and the HD group. The neuromuscular clinical signs and, ultimately, the moribund condition of several animals in the MD and the HD group leading to euthanasia for animal welfare were related to adverse effects of the test item. There were no clinical signs of toxicological relevance in the LD group during the treatment period of this study.
Most surviving animals of the HD recovery group which were affected with neuromuscular clinical signs partly remained those signs during the treatment-free recovery period of this study. In one female signs diminished over the course of the recovery period.
Mortality:
mortality observed, treatment-related
Description (incidence):
Four males of the MD group, five animals per sex of the HD group and four males and three females of the HD recovery group were sacrificed during the study due to reduced health condition for animal welfare reasons. One female of the LD group and one male of the MD group were found dead during the course of the study.
The cause of morbidity/death was a peripheral nervous system polyneuropathy alone in twelve animals, and a combination of polyneuropathy and lung disease in five animals. Lung disease only was considered the cause of death in one animal. In two animals, accidental aspiration of the test item was considered the cause of death. In one animal, the cause of death could not be established.
The single mortality without preceding signs of morbidity in the LD group was related to misgavage.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test item-related, adverse effects were noted for body weight and body weight development in the MD and the HD group. From the second week of the premating period onwards, males of the HD group showed a marked, statistically significant loss of body weight. When considering the whole treatment period, mean change of body weight was -5.80 g in the male HD group when compared to a gain of +66.60 g in the respective controls leading to a 28 % lower mean body weight at the end of the treatment period. Slight but statistically significant loss of mean body weight was also observed in males of the MD group in the last two weeks of treatment resulting in an 11 % lower mean body weight at the end of the treatment period when compared to controls. In females, similar effects on body weight development were noted in the MD and HD group as in males but with slightly less severity. Females of the HD group showed statistically significant loss of mean body weight in the second week of the premating period (- 9.00 g compared to a gain of +11.50 g in controls). Furthermore, mean body weight was statistically significantly lower in the second half of gestation period and during lactation period. The greatest difference to controls was seen at the end of the treatment period on lactation day 4 (14 % below controls). Similarly in females of the MD group, slightly and statistically significantly lower mean body weight was noted from the second week of gestation onwards with the greatest effect on gestation day 20 and lactation day 4 (10 % below controls, respectively). Lower mean body weight was assumed to be partly related to microscopically observed muscle atrophia.
Similar effects on body weight and body weight development were also noted during the treatment period of males and females in the HD recovery group. During the recovery period, body weight development was noted to slightly recover which correlated to the observed slight improvement of the general health condition and neuromuscular symptoms after ceasing of treatment with the test item. However, animals did not fully recover in this 14 to 16 day recovery period. No effects of toxicological relevance on body weight and body weight development were noted in the male and female LD group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males and females, there was a slight tendency towards lower food consumption of the HD group in the second week of the premating period of this study but without statistically significant difference to controls. Mean food consumption was comparable between the male and female MD and LD group and controls. Marginally to moderately and statistically significantly lower food consumption in the first and second week of gestation in the female HD group when compared to controls. Marginally but statistically significantly lower food consumption in all dose groups during lactation period when compared to controls did not follow a clear dose dependency. In the male HD recovery group mean food consumption was moderately lower from the second week of the treatment period onwards with achieving statistical significance in the last week of treatment. In the female HD recovery group mean food consumption was slightly lower in the fourth and the last week of the treatment period with achieving statistical significance in the last week. Mean food consumption in recovery groups was comparable to controls.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmoscopic findings in any of the animals of this study.
Haematological findings:
no effects observed
Description (incidence and severity):
At the end of the treatment and the recovery period, no toxicologically relevant effects on parameters of haematology and blood coagulation were observed.
At the end of the recovery period, results of haematology and blood coagulation could only be evaluated from two males and two to three females of the HD recovery groups. Thus, results have only limited conclusiveness. Most mean values for haematology and coagulation in the HD recovery groups were comparable to controls and slight and non-consistent differences to the control group and deviating individual values were considered as incidental. Slight to moderate mean differences to controls for some parameters of clinical biochemistry were seen without consistency in only individual animals and/or only one gender, not at the end of the treatment period and without any microscopic organ correlate. Thus, no adversity was assumed.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At the end of the treatment and the recovery period, no toxicologically relevant effects on parameters of clinical biochemistry were observed.
At the end of the recovery period, results of clinical biochemistry could only be evaluated from two males and two to three females of the HD recovery groups. Thus, results have only limited conclusiveness. Most mean values for haematology and coagulation in the HD recovery groups were comparable to controls and slight and non-consistent differences to the control group and deviating individual values were considered as incidental. Slight to moderate mean differences to controls for some parameters of clinical biochemistry were seen without consistency in only individual animals and/or only one gender, not at the end of the treatment period and without any microscopic organ correlate. Thus, no adversity was assumed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on parameters of urinalysis in any group at the end of the treatment and the recovery period.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Neuromuscular symptoms in animals of the MD and the HD group were also noted during weekly detailed clinical observations as dose-dependently affected gait (ataxic and hypotonic gait). Females were slightly less affected. In the same dose groups a slight tendency towards reduced spontaneous activity was noted when compared to controls. Furthermore, there was a tendency towards slightly reduced reactivity concerning response to handling, fear, arousal, finger approach and head touch in males but not females of the MD and HD groups.
In the functional observation battery at the end of the treatment period, impairments for several of the parameters tested such as reduced equilibrium reflex, positional passivity, visual placing, grip strength, sensitivity to pinching the tail, limb tone, hind limb reflex, toe pinch reflex, righting reflex on the ground and/or air righting reflex were noted in animals of the MD and the HD group. Females were slightly less affected. At the end of the recovery period, some of the observed impairments were still observed in some of the surviving animals. Slightly lower number of rearings in the HD group at the end of the treatment and the recovery period were assumed to be related to the observed neuromuscular symptoms.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At histopathological examination, the polyneuropathy was noted in peripheral nerves, including the brachial, femoral and sciatic nerves and distal extensions (tibial, peroneus, and soleus nerves). It was characterized by multifocal nerve fiber affection showing focal nerve fiber swellings, swellings of Schwann cells, myelin break down and multifocal axonal fragmentation. The myelin breakdown appeared to be the initial lesion. However, in more advanced lesions, Bügner’s bands were present (collapsed myelin sheets devoid of axons). This is deemed to be indicative for a progressing injury and the Bügner’s bands may be considered as an indicator for a primary axonal damage that already chronified. Subsequently, the nerve fibers rarefied. The progressing state is supported by the clinical symptoms noted (ataxia, paresis and hypotonia).
Brachial nerves (cranial peripheral nerves) were less affected than distal nerves. Femoral and sciatic nerves, including tibial, peroneus, and soleus nerves were affected similar in incidence and sever ity. In decedents, the severity of the lesion was in most cases higher than in survivors. In decedent animals from the MD group, there was not an obvious difference to the affection of animals from the HD group. However, in survivors, the lesion was less pronounced in animals of the MD group when compared to animals of the HD group.
In animals with high severity affection, also the nerve roots of the spinal cord were affected, but less in severity. Peripheral ganglia did not show any damage. In rats, especially the L4 level is very sensitive, i.e., the L4 level is the major contributor to the sciatic nerve [19]. In the affected animals, motoneurons in the spinal cord were, however, not affected. In general, no injury was noted in the optic nerves, spinal cord and brain. The findings did not recover in surviving animals that underwent a treatment-free period. However, all findings noted in the LD group were within the range of back ground lesions.
Minor severity degrees of myofiber atrophy (reduced fiber diameter with tinctoral changes) and/or degeneration (necrosis, disruption of fibers) were noted in muscles from a few animals (mainly decedents) of the MD and the HD group. There was no obvious difference between the quadriceps and gastrocnemius muscles. The quadriceps is innervated by the femoral nerve, and the gastrocnemius is innervated by the peroneal nerve (as an extension of the sciatic nerve). Therefore, it is not unusual to find lesions in both muscles. The incidence and severity was, however, low for both muscles. It might be considered that this finding is also supporting a progressing state, i.e., there was in the most duration of the study a single fiber degeneration compensated by unaffected nerve fibers during the major part of the study. The findings were not recorded in surviving recovery animals.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
The copulation index (no. of animals copulated/no. of pairs x 100) was markedly reduced in the HD group (43 %) when compared to 100 % in controls, 100 % in the LD group and 90 % in the MD group. One single female no. 82 of the MD group did not copulate in the course of the mating period. In the HD group, 4 of 7 live females did not copulate.
The fertility index (number of pregnant females/number of copulated females x 100) was 100 % in the LD group, 78 % in the MD group and 100 % in the HD group when compared to 100 % in the control group. Histologically there were no findings in reproductive organs that could be related to non-mating of females with their male pairing partners. Sperm staging did not reveal abnormalities in maturation. The stages were complete.
The delivery index (no. of dams with live pups born / no. of pregnant dams x 100) was comparable between the groups with 90 % in the LD group, 100 % in the MD group and 100 % in the HD group when compared to 100 % in the control group. One female of the LD group could not deliver because of its prescheduled death.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were noted on male and female fertility at any dose tested. The reduced copulation index was considered secondary to the more generalized physical debilitation (polyneuropathy) and was not considered as an adverse reproductive effect.
Remarks on result:
other: reproduction

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
All pups survived from PND 0 to PND 4 except for one pup missing of a control dam on PND 4 (attributed to cannibalism by the dam) which was considered as incidental.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no biologically or statistically significant effect on pup mean weight on post-natal day (PND) 0 and 4 in the dose groups when compared to the control group. A tendency towards marginally and not statistically significantly lower pup mean weight on PND 0 was noted in the MD and the HD group (6.14 g and 6.13 g) when compared to 6.34 g in controls. However, values were in the normal range of biological variation.
Slightly lower mean total litter weight was observed in the MD and the HD group on PND 0 and when compared to controls. This reached statistical significance for the MD group on PND 0 (21 % below controls) and for the MD and the HD group on PND 4 (21 % and 20 % below controls, respectively) without clear dose dependency. This was related to a tendency towards marginally (not statistically significantly) lower number of pups and pup weight in the MD and the HD group.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no remarkable, toxicologically relevant external abnormalities.
Single findings in few pups (one LD pup discoloured dark, one MD pup small in size) were considered as incidental.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in F1.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Concentration and homogeneity of the test item in the vehicle were analyzed in formulations of all dose groups in study weeks 1, 3, 5 and the last week. Nominal concentrations were confirmed for all dose groups, as measured mean concentrations were within acceptance criterion of 10 %. All samples were homogenous, as COV was below or equal 10 %.

See attachment for results tables.

Applicant's summary and conclusion

Conclusions:
In the Combined Repeated Dose Oral Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, conducted according to an appropriate OECD test guideline and in compliance with GLP, the reported NOAEL value for reproductive toxicity for triethoxy(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane was ≥ 125 mg/kg bw/day.