4-[fluoro(dimethyl)silyl]butanenitrile

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 16, 2016 - June 1 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

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) adopted on 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-[fluoro(dimethyl)silyl]butanenitrile

EC Number:

812-925-4

Cas Number:

1639345-42-8

Molecular formula:

C6H12FNSi

IUPAC Name:

4-[fluoro(dimethyl)silyl]butanenitrile

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han) (Full Barrier)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation:males: 330 - 392 g (mean: 365.32 g, ± 20% = 292.26 – 438.38 g) females: 219 - 247 g (mean: 232.12 g, ± 20% = 185.70 – 278.54 g)
- Housing: Animals were housed in groups of 5 animals/sex/cage in IVC cages (type IV, polysulphone cages) during the premating period for both males and females and during postmating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. Animals of the recovery groups were housed in groups of 5 animals / sex / cage throughout the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Adequate acclimatisation period (at least 5 days) under laboratory conditions


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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

TEST ITEM FORMULATION
- Rate of preparation of diet (frequency): The test item formulation was prepared once every ten days based on available stability data.
- Mixing appropriate amounts with (Type of food): The test item formulation was prepared by weighing the test item into a tared plastic vial on a suitable precision balance, adding the vehicle to give the appropriate final concentration of the test item formulation and further vortexing it for 2-3 minutes. The amount of test item required to achieve the targeted dose concentration of active ingredient within the formulation sample was calculated based on the concentration of active ingredient within the test item (99%).
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the physical properties of the test item (hydrolysis in water) and testing guideline, corn oil had been selected as vehicle.
- application volume (if 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study.
Study pre start stability analysis was included samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day -15 to -35 °C (10 samples).
Prestart homogeneity investigation included the samples collected from various levels (top, middle and bottom) of high dose and low dose groups (6 samples).
The test item was shown to be homogenous (after 30 minutes without stirring) and stable under all tested conditions.
During the study, samples were collected for the investigation of nominal substance concentration. As prestart homogeneity was demonstrated it was not further investigated additionally during study sampling.
Samples for the nominal concentration verification were taken in study week 1 (first day of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples).
All formulation samples collected during the study were stored at room temperature and analysed within 10 days using a GC/FID method.

Details on mating procedure:

M/F ratio per cage: 1:1 (male to female)
- Proof of pregnancy: The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day ‘0’ of gestation

Duration of treatment / exposure:

The animals of the main groups were treated with the test item formulation or vehicle for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
The males of the recovery groups were treated for 28 days and were subjected to necropsy 15 days after the last administration (end of recovery period). The females of the recovery groups were treated up to the first scheduled necropsy of dam and were subjected to necropsy 15 days thereafter (end of recovery period).

Frequency of treatment:

7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

LD = low dose

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

MD = medium dose

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

HD = high dose

No. of animals per sex per dose:

main groups: 10
recovery groups: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses were selected based on the results of a 14 day dose range finding study (non-GLP).
The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, at the same time each day after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals of the main groups and the recovery groups outside the home cage in a standard arena except in male recovery animals in the first week of recovery period when detailed clinical osbervations were inadvertently not made.

BODY WEIGHT: Yes
- Time schedule for examinations: he 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), on PND 4 and PND 13 along with pups. All animals were weighed at termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: the week before the first treatment from all animals and during the last week of the treatment. In case of animals of the recovery groups on all males and females once before the first exposure, during the last week of treatment as well as in the last week of the recovery period.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment and in all males and females at the end of the recovery period prior to or as part of the sacrifice of the animals.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females
- Parameters checked: haematocrit, haemoglobin content, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular concentration, reticulocytes, platelet count, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, coagulation parameters (prothrombin time, activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each main group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. In case of recovery groups, clinical biochemistry were measured for all males and females at the end of the recovery period.
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females
- Parameters checked: alanine aminotransferase, aspartate-aminotransferase, alkaline phosphatase, creatinine, total protein, albumin, urea, total bile acids, total cholesterol, glucose, potassium, sodium

URINALYSIS: Yes
- Time schedule for collection of urine: An urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance was recorded. In case of recovery groups, urinalysis was performed for all males and females at the end of the recovery period.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked: specific gravity, nitrite, pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, blood, leukocytes.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Multiple detailed behavioural observations were made in the week before the first treatment from all animals and during the last week of the treatment in 5 randomly selected males and during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each main group outside the home cage using a functional observational battery of tests. In case of animals of the recovery groups FOB were made on all males and females once before the first exposure, during the last week of treatment as well as in the last week of the recovery period.
- Dose groups that were examined: all
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

IMMUNOLOGY: No

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Not applicable - pups delivered

Statistics:

A statistical assessment of the results of body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry 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. Results of absolute and relative organ weights were statistically analysed 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. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.6.01 software Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Indices:

Mean litter size
Postnatal survival between birth and PND 0 or PND 4
Postnatal survival for all other intervals

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In males of main study and recovery groups, predominant clinical sign observed during the treatment period (premating day 1 to mating day 14 in main study males and day 24 to 28 in recovery males) was slightly to moderately increased salivation in few animals of MD, HD and HDR group. Isolated incidences of eschar/crust/alopecia at left cheek in 1 male (no.3) of control group were considered to be incidental. No clinical signs were observed in recovery males during recovery period.

In females of main study and recovery groups, predominant clinical signs observed during the treatment period (PMD 2 to PND 12 in main study females and day 28 to 48 in recovery females) were moving the bedding, slightly to severely increased salivation in few animals of LD, MD, HD and HDR group. Isolated incidences of alopecia on various body parts and red nasal discharge were observed on few occasions in very few female animals in all group including control group and considered to be incidental in nature. No clinical signs were observed in recovery females during recovery period.

None of the females showed signs of abortion or premature delivery. However, female no. 60 (C) was euthanised in moribund condition due to prolapse of uterus on PND 0.

During the weekly detailed clinical observation, no relevant differences between the groups were found.

Mortality:

mortality observed, treatment-related

Description (incidence):

During the treatment period of this study, few mortalities/moribund sacrifice observed were male no. 47 (HDR) was euthanised in moribund condition on day 28, female no. 51 (C) was found dead on post natal day (PND) 4, female no. 60 (C) was euthanised in moribund condition due to prolapse of uterus on PND 0, female no. 77 (MD) was found dead after dose application on pre mating day (PMD) 4, Female no. 81 (HD) was euthanised in moribund condition on gestation day (GD) 23 due to animal welfare reasons, female no. 82 (HD) was euthanised in moribund condition on PND 9 due to animal welfare reasons and female no. 87 (HD) was found dead on gestation day (GD) 22.Histopathologically, the cause of death/morbidity in animal no. 51 (C) and 60 (LD) was considered to be due to gavaging error and uterine prolapse, respectively. The cause of morbidity of animal no. 47 was considered to be induced nephropathy. The cause of death/morbidity could not be established for the other animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, there was no statistically significant difference observed on body weight between the LD, MD dose groups and the control group during the entire study period. However, lower group mean body weight was observed in HD group throughout the study period when compared with the control although statistical significance was achieved only on mating and postmating day 14 and on day of terminal sacrifice. In recovery males, statistically significantly lower group mean body weight was observed on day 8 and day 28 in HDR group when compared with recovery control group. However, no such effect was observed on group mean body weight in HDR group male animals during the recovery period. Group mean body weight gain in HD group was statistically significantly lower throughout the study period except during premating day 7-14 and premating day 14 to mating and post mating day 7 when compared with the control. In recovery males, statistically significantly lower group mean body weight gain was observed in HDR group males during week 4 (day 22-28) when compared with the controls. However, during recovery period, higher body weight gain observed in HDR group males when compared to the control although statistical significance achieved only in week 5 (i.e. first week of recovery period).
In females, no statistically significant effect on body weight and body weight gain was observed during premating period, gestation and lactation period in treatment groups when compared with the controls except statistically significantly lower group mean body weight In HD group on lactation day 4. No effect was observed on group mean body weight and body weight gain in recovery female animals except statistically significantly higher body weight gain in first week of recovery period in HDR group when compared with the controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, In correlation to the body weight and body weight gain, the food consumption in main study males during premating period and recovery males during treatment and recovery period tended to increase with the progress of the study in the control, the LD, the MD and the HD group. However, In HD group during premating period and HDR group during treatment period, food consumption was lower compared to the control group.
In females, no statistically significant effect on food consumption was observed during premating period, gestation and lactation period in treatment groups when compared with the controls. No effect was observed on food consumption in recovery female animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males sacrificed at the end of treatment period and at the end of recovery period, there were no treatment related effects or statistically significant effect observed on clinical biochemistry parameters except marginal (within historical control data range) but statistically significantly lower albumin in HD group compared to controls. All mean and most of the individual values were comparable with controls and within the historical control data range. In females sacrificed at the end of treatment period and at the end of recovery period, there were no treatment related effects or statistically significant effect observed on clinical biochemistry parameters.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In males, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period and also at the end of recovery period. There were no biologically relevant differences in body temperature between the groups. In females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period and also at the end of recovery period except statistically significantly higher not supported rearing count was observed in last week of the recovery period in HDR group when compared with the controls.There were no biologically relevant differences in body temperature between the groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males and females sacrificed at the end of treatment period and recovery period, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group except statistically significantly lower absolute epididymides, testes and glans penis weight in HD male compared to the controls. There were also statistically significantly higher relative liver weights in HDR males and statistically significantly higher relative heart weights in HDR females when compared to the respective recovery controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance. However, enlarged kidneys noted in four males in HD group (33, 37, 39 and 40) correlated histologically to an induced nephropathy

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 60 mg/kg group, an induced nephropathy was recorded. In general, males were more affected than females. There were enlarged kidneys in four males at 60 mg/kg. The nephropathy consisted of tubular dilation. Increased incidences of tubular basophilia (males only), tubular hyaline casts (males only), tubular cell necrosis and related granular casts (the latter in males only), interstitial fibrosis (mainly in the corticomedullary unction) (males only, and inflammatory (single case of pyelitis and pyelonephritis) and reactive changes (urothelial hyperplasia). After the recovery period, the findings did not fully recover. The residuals indicated repair (tubular dilation, tubular basophilia, interstitial fibrosis, interstitial inflammation). In one decedent, there were findings as in the main test animals, i.e., granular casts, tubular cell necrosis and pyelonephritis.
In the adrenal gland zona fasciculata, vacuolation (fatty change) increased in incidence and/or severity in both sexes at 60 mg/kg. Furthermore, there were a few cases of diffuse, cortical hypertrophy in females at all doses (no dose-relationship). This finding may be related to stress and hence is not considered a primary effect induced by the test item.
There were no findings in testes at all. During sperm staging, all stages were complete. There was no indication for sperm resorption, maturation arrest or any finding indicative for an affection of the male reproductive system.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Results (fetuses)

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

On PND 13 sex ratio was statistically significantly higher in HD group when compared with the controls. Group Mean number of female pups were statistical significantly lower in HD group on PND 0, 4 and 13 compared to control. This decrease in number of female pups in HD group was attributed to low or no female pups in just few females (81,84 and 89) of the HD group. Therefore this effect on female pup number and sex ratio was not considered to be toxicologically relevant.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

A slightly higher mean mortality of pups between PND 0 and PND 4 was observed in the HD group (25 %) compared to the control group (12.04 %). No effect on pup survival was observed in treatment groups during PND 4-13. This effect on pup survival in HD group from PND 0-4 was considered as test item related and was attributed to complete litter loss from just 2 females (84 and 89) from HD group between PND 1-2.

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

In males statistically significant higher absolute and relative anogenital distance (AGD) of male pups was observed in HD group when compared with the controls. This effect on anogenital distance in HD group was considered as test item related. However, whether this finding is indicative for a possible androgenic effect or not cannot be decided based on this screening study.
In females, statistically significant lower female pup weight, cube root of female pup weight in HD group and statistically significant higher anogenital distance and relative anogenital distance of female pups was observed in all treatment groups when compared with the controls. However, parameters like body weight, litter size and sex ratio were not affected in LD and MD groups and these parameters are correlated with AGD. Furthermore, the statistically significant mean values in LD and MD group for female anogenital distance and relative anogenital distance could also be attributed to variation in individual data values for absolute and relative anogenital distance from 2 females from each group (62, 68 from LD and 74 and 79 from MD) and in control group also 2 females (55 and 58) had values comparable to LD and MD groups.
Therefore effect on AGD in LD and MD group can not be considered as test item related. Effect on anogenital distance and female pup weight in HD group was considered as test item related. However, whether this finding is indicative for a possible androgenic effect or not cannot be decided based on this screening study.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in postnatal survival

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

no

Relevant for humans:

not specified

Any other information on results incl. tables

Thyroid Hormone (T4) Analysis:

No test item related effect of toxicological relevance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups and recovery groups when compared to the controls.

Applicant's summary and conclusion

Conclusions:

Based on the slightly higher mean mortality of pups between PND 0 and PND 4 observed in the HD group (25 %) the dosage level of 20 mg/kg/day (=the medium dosage level evaluated) was considered to be the no-observed-adverse-effect level (NOAEL) for developmental toxicity of the test substance when administered orally by gavage to Wistar Crl: WI(Han) (Full Barrier) rats.