Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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 moist
- 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 investigated (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:
other: liquid paraffin
Details on oral 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 and further vortexed 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. 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
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
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.
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: random
- Rationale for selecting satellite groups: In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects, additional animals in recovery groups were observed for a period of 14 days (males) or 16 days (females).
- Post-exposure recovery period in satellite groups: 14 days (males) or 16 days (females)
- Section schedule rationale (if not random): random
Positive control:
not included

Examinations

Observations and examinations performed and frequency:
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.

FOOD CONSUMPTION: food consumption was measured weekly, except for food consumption measurements which were not taken during the mating period in females and the mating and post-mating period in males

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the lactation period in 5 randomly selected females (only lactating females were evaluated except for non-pregnant females no. 83 and 92 of the HD group) of each main group outside the home cage using a functional observational battery of tests.
- Dose groups that were examined: each dose level

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment for main groups and at the end of recovery period for recovery group
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females of each main group and all recovery males and females
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment for main groups and at the end of recovery period for recovery group
- Animals fasted: Not specified
- How many animals: 5 randomly selected males and females of each main group and all recovery males and females
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment for main groups and at the end of recovery period for recovery group
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the lactation period in 5 randomly selected females (only lactating females were evaluated except for non-pregnant females no. 83 and 92 of the HD group) of each main group outside the home cage using a functional observational battery of tests.
- Dose groups that were examined: each main group
- Battery of functions tested: sensory activity, grip strength, motor activity, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, 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.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No 2)

HISTOPATHOLOGY: Yes (see table No 2)
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.

Results and discussion

Results of examinations

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 consumprion 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
Description (incidence and severity):
At the end of the treatment period, mean absolute but not relative (to body weight) liver weight was moderately and statistically significantly lower in the male HD group (27 % below controls) and slightly and not statistically significantly lower in the male MD group (18 % below controls) when compared to the control group. A marginal tendency towards lower absolute liver weight was also observed in the female MD and HD group (10 % and 11 % below controls, respectively) but without achieving statistical significance. Changes to organ weights could be related to microscopic liver findings in few animals of the MD and the HD group (hepatocellular necrosis). No such findings occurred after the recovery period.
Further differences in organ weights in thymus, spleen and adrenals between the dose groups and controls at the end of the treatment period were assumed to be stress-related signs in accordance with microscopic findings. Moderately and statistically significantly lower mean absolute thymus weight was noted in the male HD group with values 33 % below controls. A tendency towards lower thymus weight was also seen in the male MD group (19 % below controls), the female MD group (24 % below controls) and the female HD group (31 % below controls) and but without achieving statistical significance. Mean absolute spleen weight was slightly, dose-dependently lower in the male MD group (15 % below controls) and the male HD group (21 % below controls) but did not achieve statistical significance. No dose-dependent or statistical significant chances to spleen weight were observed in the female dose groups. Marginally but statistically higher relative (to body) weight of adrenals was noted in the male HD group (34 % above controls) but not the female HD group.
Description (incidence and severity):
The lung disease consisted of a strong increase of subacute peri-/vasculitis, which was noted in animals of the HD group, especially in decedents and was related to an interstitial edema in some animals. It could be related to artificial aspiration of test item. Occasionally minor severity degrees of peri-/vasculitis were noted in control animals. However, the lesions noted in the HD animals were unusual and are deemed to be contributive to morbidity/death. The precise reason for these lesions remains unclear but is so unusual that it is deemed to be test item-related. In the heart of one decedent animal, there was peri-/arteritis in an intramural coronary artery. This is also an unusual finding and should not be noted in rats at these ages. A relationship to the peri-/vasculitis observed in lungs should be considered.
Next to misgavaging as cause of death in one female of the LD group, in two other decedents, aspiration of the test item was considered as the contributing factor to death. This is supported by a number of findings also recorded in survivors including controls, including aspirated foreign material, alveolar macrophages and alveolar/bronchiolar hyperplasia. The incidence of interstitial inflammation increased in the HD group may be related to aspiration of the test item. BALT hyperplasia was noted as a reactive lesion only in one HD decedent male. Such accidental aspiration is sufficiently published in literature and is an accidental issue even in cases of correct oral dosing whereby a small amount of the compound may be inadvertently deposited at the laryngeal orifice and would be inhaled during inspiration. In addition, the vehicle liquid paraffin is a viscous fluid, which increases the possibility of aspiration.
Findings in the liver consisted of a minor severity of hepatocellular hypertrophy in a few decedent MD and HD animals. In addition, minimal to slight hepatocellular necrosis was noted in a few decedent animals. It remains unclear if the hepatocellular necrosis is of agonal nature. Therefore, the adverse vs non-adverse nature of the hepatic lesions cannot be judged. However, no findings occurred after the recovery period.
A number of stress-related findings were noted including a diffuse hypertrophy of the zona fasciculata in the adrenal cortex of a few animals of the MD and the HD group, mainly decedents. Also, the incidence and severity of thymic atrophy increased in the HD animals, again in decedents.
Neuropathological findings:
not examined
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 severity. 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 background 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.
Details on results:
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 %.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
peripheral nervous system
Organ:
other: brachial, femoral and sciatic nerves
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Mortality Table - Main Groups and Recovery Groups - Summary

Group

Gender

Gender

Day and cause of death

Male

Female

C

0/10

0/10

-

CR

0/6

0/6

-

LD

0/10

1/10

Found dead: no. 70 (GD 14)

MD

5/10

0/10

Euthanised: no. 21 (Day 19)
Euthanised: no. 23 (Day 19)
Euthanised: no. 25 (Day 19)
Found dead: no. 27 (Day 24)
Euthanised: no. 28 (Day 10)

HD

5/10

5/10

Euthanised: no. 34 (Day 12)
Euthanised: no. 35 (Day 12)
Euthanised: no. 38 (Day 11)
Euthanised: no. 39 (Day 14)
Euthanised: no. 40 (Day 14)
Euthanised: no. 84 (GD 0)
Euthanised: no. 87 (GD 2)
Euthanised: no. 89 (PMD 14)
Euthanised: no. 90 (PMD 14)
Euthanised: no. 91 (PMD 14)

HDR

4/6

3/6

Euthanised: no. 48 (Day 15)
Euthanised: no. 49 (Day 12)
Euthanised: no. 50 (Day 15)
Euthanised: no. 52 (Day 12)
Euthanised: no. 99 (Day 22)
Euthanised: no. 100 (Day 29)
Euthanised: no. 104 (Day 30)

Animals affected / total number of animals

Given in brackets: study day of death

GD = gestation day; PMD = premating day

Table 2: Clinical observations - Males- Treatment priod - summary

Clinical Finding

C

CR

LD

MD

HD

HDR

Total number of animals examined

10

6

10

10

10

6

abnormal breathing

0

0

0

0

5

1

alopecia

0

0

0

1

0

0

ataxia

0

0

0

8

10

6

hypotonia

0

0

0

4

9

5

paresis

0

0

0

6

9

6

piloerection

0

0

0

5

8

6

spontaneous activity reduced

0

0

0

4

8

5

wasp waist

0

0

0

5

9

5

Table 3: Clinical observations - Males- Recovery period - summary

Clinical Finding

CR

HDR

Total number of animals examined

6

2 (4 of 6 died during treatment period)

ataxia

0

2

hypotonia

0

2

paresis

0

2

piloerection

0

2

wasp waist

0

2

Table 4: Clinical Observations - Females - Treatment Period - Summary

Clinical Finding

C

CR

LD

MD

HD

HDR

Total number of animals examined

10

6

10

10

10

6

abnormal breathing

0

0

0

0

5

2

alopecia

0

0

1

0

1

0

ataxia

0

0

0

1

9

4

hypotonia

0

0

0

0

8

4

paresis

0

0

0

0

9

4

piloerection

0

0

0

1

5

3

slow movements

0

0

0

0

2

2

spontaneous activity reduced

0

0

0

0

3

0

wasp waist

0

0

0

1

8

4

Table 5: Clinical Observations - Females - Recovery Period - Summary

Clinical Finding

CR

HDR

Total number of animals examined

6

3 (3 of 6 died during treatment period)

ataxia

0

1

hypotonia

0

1

paresis

0

1

wasp waist

0

1

Table 6: Mean Body Weight (g) - Males - Main Groups

Group

 

Premating

Premating

Premating

Mating and Postmating

Mating and Postmating

Terminal Sacrifice

Day 1

Day 7

Day 14

Day 7

Day 14

C

Mean

291.50

313.20

334.00

341.70

358.10

361.70

C

SD

19.05

24.00

28.88

31.90

36.98

37.34

C

N

10

10

10

10

10

10

LD

Mean

291.80

312.20

330.60

338.30

350.40

352.30

LD

SD

18.74

22.14

26.24

29.56

33.28

31.86

LD

N

10

10

10

10

10

10

LD

%

100

100

99

99

98

97

MD

Mean

292.00

311.60

326.22

325.50

318.00

321.40

MD

SD

17.59

21.10

24.69

27.59

25.76

25.28

MD

N

10

10

9

6

5

5

MD

%

100

99

98

95

89

89

HD

Mean

291.20

311.50

294.00*

298.40*

277.00***

277.80***

HD

SD

16.00

15.52

28.35

9.24

30.75

31.57

HD

N

10

10

7

5

5

5

HD

%

100

99

88

87

77

77

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 7: Mean Body Weight (g) - Males - Recovery Groups

Group

 

Study Day 1

Study Day 7

Study Day 14

Study Day 21

Study Day 28

Study Day 35

Study Day 42

CR

Mean

292.00

321.00

342.50

355.83

372.17

386.00

395.67

CR

SD

14.16

27.14

28.98

32.11

34.10

34.82

39.21

CR

N

6

6

6

6

6

6

6

HDR

Mean

290.50

320.50

296.25

332.50

297.5*

285.5*

311.50*

HDR

SD

14.52

18.03

49.04

26.16

19.09

30.41

31.82

HDR

N

6

6

4

2

2

2

2

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

Table 8: Mean Body Weight (g) - Females - Main Groups

Group

 

Premating

Premating

Premating

Gestation

Gestation

Gestation

Lactation

Lactation

Lactation

Day 1

Day 7

Day 14

Day 0

Day 7

Day 14

Day 20

Day 0

Day 4

C

Mean

195.80

203.80

215.30

214.80

238.30

269.50

341.50

259.60

278.20

C

SD

7.16

10.77

8.69

8.09

9.39

9.14

13.79

15.48

9.16

C

N

10

10

10

10

10

10

10

10

10

LD

Mean

194.60

205.30

213.30

213.80

235.50

263.78

332.89

249.89

264.56*

LD

SD

7.85

9.96

10.29

10.67

9.35

9.16

15.09

12.10

12.09

LD

N

10

10

10

10

10

9

9

9

9

LD

%

99

101

99

100

99

98

97

96

95

MD

Mean

192.90

205.20

209.90

212.80

233.20

256.60*

309.00***

241.00*

251.57***

MD

SD

6.33

7.71

9.27

8.64

7.01

9.21

8.06

14.76

13.96

MD

N

10

10

10

5

5

5

5

7

7

MD

%

99

101

97

99

98

95

90

93

90

HD

Mean

195.80

207.00

198.00**

203.50

223.00

251.00*

307.00**

232.67*

238.00***

HD

SD

7.80

10.33

11.99

0.71

2.83

1.41

1.41

12.50

8.54

HD

N

10

10

10

2

2

2

2

3

3

HD

%

100

102

92

95

94

93

90

90

86

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 7: Mean Body Weight (g) - Females - Recovery Groups

Group

 

Treatment Period1

Treatment Period 7

Treatment Period 14

Treatment Period 21

Treatment Period 28

Treatment Period35

Recovery Period1

Recovery Period 7

Recovery Period 14

Recovery Period 17

CR

Mean

194.33

210.33

213.00

218.00

224.17

226.67

233.83

238.33

245.17

243.17

CR

SD

11.00

11.15

11.14

12.90

15.25

15.74

16.12

13.56

13.26

16.06

CR

N

6

6

6

6

6

6

6

6

6

6

HDR

Mean

192.50

201.33

207.17

199.67

204.60

207.67

215.33

217.00*

225.33*

226.33

HDR

SD

9.63

11.29

13.67

23.52

27.11

13.65

13.32

8.54

4.93

8.14

HDR

N

6

6

6

6

5

3

3

3

3

3

Table 8: Mean Body Weight Gain (g) - Males - Main Groups

Group

Premating Day 1-7

Premating Day 7-14

Mating and Postmating Day 14-Day 7

Mating and Postmating Day 7-14

Premating Day 1 - Postmating Day 14

Premating Day 1 - Terminal Sacrifice

C

Mean

21.70

20.80

7.70

16.40

66.60

70.20

SD

7.94

6.05

5.48

6.60

21.91

23.10

N

10

10

10

10

10

10

LD

Mean

20.40

18.40

7.70

12.10

58.60

60.50

SD

4.77

5.42

5.19

5.07

16.99

16.53

N

10

10

10

10

10

10

MD

Mean

19.60

17.22

-3.33*

-7.40**

24.20**

27.60**

SD

5.40

7.63

9.27

15.14

20.19

21.04

N

10

9

6

5

5

5

HD

Mean

20.30

-18.43***

-6.20*

-21.40***

-5.80***

-5.00***

SD

4.37

38.53

14.91

24.74

24.55

25.11

N

10

7

5

5

5

5

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Tabl 9: Mean Body Weight Gain (g) - Males - Recovery Groups

Group

Treatment Period Day 1-7

Treatment PeriodDay 7-14

Treatment PeriodDay 14-21

Treatment PeriodDay 21-28

Recovery PeriodDay 28-35

Recovery PeriodDay 35-42

CR

Mean

29.00

21.50

13.33

16.33

13.83

9.67

CR

SD

13.67

5.79

4.46

4.46

3.54

6.74

CR

N

6

6

6

6

6

6

HDR

Mean

30.00

-22.75*

-3.00**

-35.00***

-12.00**

26.00*

HDR

SD

5.55

38.39

4.24

7.07

11.31

1.41

HDR

N

6

4

2

2

2

2

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

Table 10: Mean Body Weight Gain (g) - Females - Main Groups

Group

Premating Day 1-7

Premating Day 7-14

Gestation Day 0-7

Gestation Day 7-14

Gestation Day 14-20

Gestation Day 0-20

Lactation Day 0-4

C

Mean

8.00

11.50

23.50

31.20

72.00

126.70

18.60

C

SD

6.88

4.28

3.75

3.33

7.09

10.72

8.90

C

N

10

10

10

10

10

10

10

LD

Mean

10.70

8.00

21.70

28.22

69.11

120.44

14.67

LD

SD

4.92

4.52

6.82

4.55

11.50

17.95

3.35

LD

N

10

10

10

9

9

9

9

MD

Mean

12.30

4.70

20.40

23.40*

52.40**

96.20**

10.57

MD

SD

4.24

4.03

7.20

6.27

8.85

7.76

7.46

MD

N

10

10

5

5

5

5

7

HD

Mean

11.20

-9.00***

19.50

28.00

56.00

103.50

5.33*

HD

SD

4.34

18.87

3.54

4.24

2.83

2.12

10.02

HD

N

10

10

2

2

2

2

3

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001

Table 11: Mean Body Weight Gain (g) - Females - Recovery Groups

Group

Treatment PeriodDay 1-7

Treatment PeriodDay 7-14

Treatment PeriodDay 14-21

Treatment PeriodDay 21-28

Treatment PeriodDay 28-35

Recovery PeriodDay 1-7

Recovery PeriodDay 7-14

CR

Mean

16.00

2.67

5.00

6.17

2.50

4.50

6.83

CR

SD

4.65

3.67

2.83

3.54

5.36

5.28

4.36

CR

N

6

6

6

6

6

6

6

HDR

Mean

8.83*

5.83

-7.50

-2.00

-13.33**

1.67

8.33

HDR

SD

5.49

4.79

15.14

10.84

7.37

7.09

8.96

HDR

N

6

6

6

5

3

3

3

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

Table 11: See attachment for postmortem observations

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 general systemic toxicity for triethoxy(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane was 50 mg/kg bw/day.