Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24.01.2008 to 02.03.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: OPPTS 870.3650
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Triethoxyoctylsilane
- Substance type: Alkoxysilane
- Physical state: Colourless liquid
- Stability under test conditions: Stable, will react with moisture
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc, Portage.
- Age at study initiation: 9 weeks minimum
- Weight at study initiation: Males: 210.1 to 254.6 g ; Females: 159.6 to 201.7 g
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages (except during cohabitation when female was put into male home cage, and pregnant animals were moved into shoebox cages).
- Diet (e.g. ad libitum): Ad libitum except during FOB and motor activity assessment.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 21.3
- Humidity (%): 39 - 60 %
- Air changes (per hr): 13.5
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28.01.2008 To: 07.06.2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance and vehicle were administered to each dose group at an equal dose volume (2ml/kg bw) based on the most recent body weight. Dosing solutions were individually prepared. Based on stability data, dosing solutions were prepared twice during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the physical and chemical properties of the test substance, dried and deacidified peanut oil was considered to be the most appropriate vehicle for oral administration.
- Concentration in vehicle: Dependent on dose
- Lot/batch no. (if required): 080408 and 090408
- Purity: No data
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of mating
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually in a shoe box cage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solution analyses were performed by a GC/FID method to verify concentration, stability, and homogeneity of the test substance in the vehicle. Dosing solutions were analysed prior to study inititation to ensure stability and homogeneity. Stability was determined on study days 0, 4, 15, 21 and 32. Dosing solutions were also analysed for concentration verification following each dose solution preparation.
Duration of treatment / exposure:
Males: 28 days
Toxicity phase females: 29 days
Reproductive phase females: two weeks prior to mating, then through pregnancy, and to post-partum day 3.
Frequency of treatment:
Daily, seven days per week
Details on study schedule:
N/A screening study
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (group 1), 100 (group 2), 300 (group 3) and 1000 (group 4) mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a range finding study.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily for mortality, morbidity and moribundity. General clinical observations were made at least once per day. Clinical observations were not performed on the day of the detailed clinical observations.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals received a detailed physical examination once before the first dose and weekly thereafter. Examinations included but were not limited to changes in skin, fur, eyes, mucous membranes, occurence of secretions and excretions, and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behaviour were recorded.

From day 20 after evidence of mating, pregnant animals were checked at least three times daily for evidence of parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were determined beginning with randomisation into test groups, on the first day of dosing, at least weekly thereafter and the day of necropsy. During gestation the pregnant animals were weighed on gestation days 0, 7, 14 and 20, within 24 hours after parturition and on day 4 post-partum or upon terminal euthanasia of the dam. Litter weights were measured within 24 hours of parturition and on day 4 post-partum or upon terminal euthanasia of the dams.

FOOD CONSUMPTION:
Individual animal food consumption was recorded at least weekly on an individual animal basis for the periods listed below.
Male rats: Two week pre-mating only. Feeder weights were taken on days 1, 8 and 15.
Female toxicity phase: Day 1 of dosing to necropsy. Feeder weights were taken on days 1, 8, 15, 22 and the day prior to necropsy.
Reproductive phase females: Two week pre-mating period, gestation and post-partum. Feeder weights were taken on days 1, 8, 15 and on gestation days 0, 7, 14, 20 and on day 0 and 4 post-partum.

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: No

OPHTHALMOSCOPIC EXAMINATION: No
Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testes weight, epididymis weight.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, see table 2. All males and toxicity phase females had a complete gross necropsy that included examination of the external surface and all orifices of the body, the cranial, thoracic and abdominal cavities and their contents. Based on clinical findings during the study, all reproductive females were also subjected to a complete gross necropsy as per the males and toxicity phase females. In addition, the number of corpora lutea and the number of implantation sites were recorded. The uteri of females with positive evidence of mating that failed to produce a litter were stained to enable counting of possible reabsorbed implant sites. Pups found dead were examined for external gross abnormalities only.

At necropsy, a number of organs from males and toxicity group females weighed (see Table 2).

HISTOPATHOLOGY: Yes (see table 2). Tissues and organs required for histopathological examination from the males and toxicity group females were collected from all animals in all treatment groups. Based on various clinical signs in the reproductive phase females, the tissues and organs from all treatment groups of animals were also collected.
Postmortem examinations (offspring):
Pups found dead were examined for external gross abnormalities only.
Statistics:
Mean values of the following were evaluated appropriately: body weights and body weight changes, food consumption, haematology and serum chemistry, terminal organ weights and body weight ratios, gestation length, litter size, live litter size, litter weight, ratio of live births/litter size, litter sex ratio, implantation sites, corpora lutea, mating and fertility indices and neurotoxicological parameters.
Reproductive indices:
Reproductive-type parameters evaluated were evidence of mating, pregnancy and duration of gestation. In particular, the number of corpora lutea and the number of uterine implantation sites were determined for all females.
Offspring viability indices:
Pups were examined daily for survival, external abnormalities and clinical signs during the lactation period. Each litter was examined as soon as possible after delivery (within 24 hours) to determine the number and sex of the pups, the number of pups alive, number of pups dead, runts and the presence of any gross abnormalities. All pups were counted, weighed, sexed and the sex ratio calculated. Litter weights were taken within 24 hours of completion of parturition and on post-partum day 4 or earlier if the dam was euthanised prior to post-partum day 4. Developmental parameters evaluated included mean litter size, offspring survival, sex ratio and offspring body weight gain.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): All toxicity group males and reproductive group females in Group 1-3 survived until scheduled necropsy. One nongravid female with no evidence of mating was euthanised 25 days after the last day of mating. There was a dose-related increase in soiling around the nose and chin and generalised soiling around the muzzle in the Group 3 and 4 males and Group 4 reproductive toxicity females. These clinical findings were statistically significant (p<0.01) in groups 4 males and reproductive toxicity group females. Three females with evidence of mating and positive evidence of pregnancy failed to deliver and were euthanised on gestation day 26, 25 and 25. Due to the severity of the clinical signs four Group 4 reproductivity phase females were euthanised between study days 24 and 43.

There were statistically significant changes in the incidence of neurological clinical signs only in Group 4 reproductive group females compared to controls. HInd limb dragging (5/10) and incoordinated gait (5/10) ocurred in Group 4 reproductive group females. None of these neurological signs were noted until study day 32, with the exception of one animal that exhibited incoordinated gait on day 21 and dragging hind limbs by day 25. These effects were also associated with a generalised decrease in overall activity (p<0.01) in 5/9 affected females. Rapid respiration occurred in 3/10 animals (p<0.05) beginning on the day prior to and/or on the day these animals were euthanised in extremis.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): There were no significant differences in weekly mean body weights and body weight gains in all male groups compared to controls, but there was a 6.8% decrease in mean overall body weight gain in the Group 4 compared to controls (p<0.05). Mean body weights and body weight gains across all reproductive group female dose levels were comparable to control throughout the premating period. A 19.7% decrease in gestation week 2 mean body weight gains occurred in Group 3 reproductive group females compared to the controls (p<0.05) but were similar to control values throughout the remainder of the study. Mean body weights of Group 4 reproductive group females were statistically decreased compared to controls throughout gestation (p<0.01). Associated body weight gains in this group were also decreased by 45.9% (p<0.01) by the end of gestation week 1 and 29.1% (p<0.01) by the end of the gestation period. There was also a significant decrease in Group 4 reproductive female mean body weights throughout the post-partum period compared to controls (p<0.01). Although Group 4 reproductive group females exhibited a 73.9% decrease in mean body weight gains between post-partum day 0 to 4, this did not attain significance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): The mean duration of gestation was statistically increased (5.6%) in Group 4 reproductive females compared to controls (p<0.01). One pregnant reproductive group female from Group 4 that exhibited severe clinical signs also failed to initiate parturition by gestation day 24 and was subsequently euthanised on that day. Of the seven dams in Group 4 that initiated parturition, four of these dams had dystocia. All these dams also had clinical neurological signs of hindlimb weakness/dragging that likely contributed to the dystocia. Of these, one animal was euthanised prior to completion of parturition due to severity of clinical signs. Mean percent post-implantation loss was statistically increased in Group 4 (39.9%) compared to controls (5.7%) (p<0.01). Post-implantation loss values include loss of fetuses and/or pups that occurred between implantation and first litter check.

ORGAN WEIGHTS (PARENTAL ANIMALS): Organs were not weighed in maternal animals. A dose-related increase in mean absolute (p<0.05) and relative (p<0.01) liver weights occurred in Groups 3 and 4 males, resulting in an 11.7% and 17.3% increase in relative liver weights at these dose levels compared to controls.

GROSS PATHOLOGY (PARENTAL ANIMALS): There were no gross pathological findings in male rats. The Group 4 reproductive phase females exhibited various gross findings that included a generalised thinness of the body in 8/10 animals, decreased thymus size in 6/10 animals and spleen size in 7/10 animals. Five of these animals also had notable decreases in hind leg skeletal muscle mass. These macroscopic findings are consistent with the clinical observations of incoordinated gait and/or hindlimb dragging and microscopic neurological findings that occurred in all of these animals. In addition to the other gross findings, two animals had decreased ingesta in all or parts of the intestinal tract. No gross findings occurred at the lower dose levels.

The uteri of reproductive group females with positive evidence of mating that failed to produce a litter were stained to enable counting of possible reabsorbed implant sites. No implantation sites were found in these animals.

HISTOPATHOLOGY (PARENTAL ANIMALS): Lymphoreticular system: A statistically significant increase in the incidence of mild to moderate atrophy of the spleen (p<0.02) and thymus (p<0.02) was identified in 5/10 Group 4 reproductive group females and not in the controls. These findings are consistent with the gross findings of decreased spleen and small thymus in several of the reproductive group females.

The main finding in the central nervous system was white matter degeneration of the brain and spinal cord in Group 4 toxicity group and reproductive group females, with an increased incidence in the reproductive group females compared to the toxicity group females. The cerebellum and medulla appeared to be the most prominently affected areas of the brain. There was an increase in the incidence of minimal white matter degeneration (vacuolation) of the brain in 4/10 Group 4 toxicity group females and one control. In the Group 4 reproductive group females this effect attained statistical significance, with 8/10 Group 4 animals exhibiting mild to marked vacuolation in the brain and no effects in the controls (p<0.01). The spinal cord of the toxicity group females also exhibited diffuse white matter vacuolation in both the sensory and motor nerve roots in 5/10 Group 4 and no control toxicity group females (p<0.01). Minimal to marked demyelination of the spinal cord also occurred in 9/10 Group 4 reproductive group females and no controls (p<0.01). Although the incidence of the effects in the brain were less prominant and did not attain statistical significance in the toxicity group females, there was evidence of minimal white matter vacuolation of the brain in 4/10 Group 4, 1/10 Group 3 and 1/10 control females. there were no effects on the central nervous system in males.

In the peripheral nerves examined, the sciatic and tibial nerves, there was a statistically significant increase in the incidence of minimal to severe demyelination/degeneration in 8/10 (sciatic) (p<0.01) and 9/9 (tibial) (p<0.01) Group 4 reproductive group females and not in the controls. Affected animals tended to show the change in multiple sites unless only the spinal cord was involved. Minimal to mild nerve degeneration also occurred in 3/10 Group 4 toxicity group females and not in controls, but did not attain statistical significance. The only neurological effect in the males was minimal sciatic nerve degeneration in 2/10 Group 4 males, which also occurred in 1/10 controls.

Based on clinical signs in the Group 4 reproductive group females, two skeletal muscle sections of the hindlimb were evaluated. There was a statistically significant increase in Group 4 reproductive Group female adductor muscle degeneration (10/10) compared with controls (4/10) (p<0.01). There was a trend toward significance in the incidence of degeneration of the gastrocnemius muscle in Group 4 reproductive group females (9/10) compared to controls (5/10), and statistically significant increase in the severity of degeneration compared to controls. Animals affected with neurological findings also showed gross muscle atrophy, diffuse decreased muscle fibre size, fibre fragmentation, increased density of myofibre nuclei, and focal areas of inflammation around necrotic fibres. The animals with these skeletal muscle findings also had notable decreases in hind leg skeletal muscle mass. These findings are consistent with denervation atrophy and considered secondary to the nerve changes. These data indicate that the severity of the clinical and microscopic tissue neuromuscular effects is a likely reflection of duration of exposure rather than an effect specific to the reproductive status of the animal as the toxicity group females were only dosed for 29 days compared up to 45 days for the reproductive females.

These neurological findings did not occur at any of the lower doses or in males.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Mean percent pre-implantation and post-natal loss were unaffected by treatment. Mean percent post-implantation and post-natal loss were unaffected by treatment. Mean percent post-implantation loss, however, was statistically increased in Group 4 (39.9%) compared to controls (5.7%) (p<0.01). Post-implantation loss values include loss of fetuses and/or pups that occurred between implantation and first litter check.

There was a statistically significant increase in the mean ratio of male to female pups/litter on post natal day (PND) 0 in Group 2 and in the number of live male pups/litter on PND 4 after adjusting for the mean litter size. These effects did not occur at any other dose levels and are likely not attributable to the test substance. Total litter size was unaffected. The number of live male and female pups/dam at first litter check (PND 0) in Group 4 were statistically significantly decreased compared to controls (p<0.01), resulting in a 39.3% decrease (p<0.01) in percent viability pups/dam at first litter check compared to controls. Pup viability was unaffected at all lower doses. On PND 4, several dams in Group 4 had been euthanised due to severity of various clinical signs and/or difficulty during labour. Only four dams continued through PND 4. Of these litters, the total viable pups on PND 4 was decreased compared to controls, resulting in a 25.2% decrease in percent viability of pups/dam on PND 4 compared to controls (p<0.01). This significant decrease was due to a single dam that had a 14.3% post-natal loss of offspring. The remaining dams had no post-natal loss of pups between PND 0-4.

On PND 0, mean litter weights and average pup body weights were similar to controls. By PND 4, weights in Group 4 were decreased compared to control weights (p<0.02). Average pup weightson PND 4 of the surviving pups were decreased by 25.2% and average pup weight gains of the surviving pups were decreased by 60% compared to controls (p<0.01). The loss of pup body weights in the Group 4 surviving pups resulted in a 50.1% decrease in mean live litter weights compared to controls.

All of the dams with complications during parturition and/or changes in litter parameters exhibited various degrees of hindlimb weakness that likely contributed to the difficulty in delivering the pups and consequent decreases in average pup weights and pup body weight gains and pup viability on PND 0 through to PND 4.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 3 Results for reproductive and developmental parameters.

 Reproductive parameter
     Control  Group 1  Group 2  Group 3  Litter size
 Corpora lutea  Mean  19.0  17.6  18.4  17.3  NA
   SD  2.7  3.1  2.8  3.4  
   N  9  10  9  8  
 Total implants  Mean  17.4  15.4  15.8  15.3  NA
   SD  1.9  1.3  1.8  2.4  
  N  9  10  9  8  
 % pre-implantation loss  Mean  7.3  11  13.4  10.3  NA
   SD  10.4  11.8  12  12.1  
   N  9  10  9  8  
 % post-implantation loss  Mean  5.7***  5.3  7.6  39.9*** NA 
   SD  4.8  5.5  10.9  29.5  
   N  9  10  9  6  
 Length of gestation  Mean  21.4***  21.7  21.7  22.6***  NA
   SD  0.5  0.5  0.5  0.7  
   N  9 10  9  8  
 Total litter size PND 0  Mean  16.4  14.6  14.6  15  NA
  SD  1.7   1.6  2.4  1.7  
   N  9  10  9  6  
 Live male pups PND 0  Mean  8.1***  8.4  7.8 4.7***   ***
   SD  1.6  2.3  1.9  1.8  
   N  9  10  9  6  
 Live female pups PND 0  Mean  8.3***  6.2  6.8  4.3***  
   SD  1.4  1.8  0.7  3.4  
   N  9  10  9  6  
 Dead male pups PND 0  Mean  0***  0  0  2.5***  
   SD  0  0  0  1.9  
   N  9  10  9  6  
Dead female pups PND 0   Mean  0***  0  0  3.2***  
   SD  0  0  0  2.9  
   N  9  10  9  6  
 Undetermined sex (dead)  Mean  0**  0  0  0.3***  
   SD  0  0  0  0.5  
   9  10  9  6  
Male/female ratio PND 0  Mean  1.0*  1.5*  1.1  1.0  
   SD  0.3  0.7  0.3  0.3  
   N  9  10  9  6  
 Total viable pups PND 0  Mean  16.4***  14.6  14.6  9.0***  ***
   SD  1.7  1.6  2.4  4.4  
   N  9  10  9  6  
 Total dead pups PND 0  Mean  0***  0  0  6***  
   SD  0  0  0  4.9  
   N  9  10  9  6  
 % post-natal loss  Mean  0  0.6  2.5  3.6  
   SD  0  2  3.8  7.1  
   N  9  10  9  4  
 % viable at birth  Mean  100***  100  100  60.7***  
   SD  0  0  0  29.5  
   N  9  10  9  6  
 Litter weight (g) PND 0  Mean  103  93.8  93.7  90.1  ***
   SD  13  8.9  14.4  7.4  
   N  9  10  9  6  
 Average pup weight (g) PND 0  Mean  6.3  6.5  6.5  6  **
   SD  0.4  0.5  0.6  0.5  
   N  9  10  9  6  
 Live male pups PND 4  Mean  8.1***  8.4*  7.8  5.5  ***
   SD  1.6  2.3  1.9  1.3  
   N  9  10  9 4  
 Live female pups PND 4  Mean  8.3  6.1  6.4  5.5  
   SD  1.4  1.6  1.0  3.4  
   N  9  10  9  4  
 Dead male pups PND 4 Mean   0  0  0  0  
   SD  0  
   N  9  10  9  4  
 Dead female pups PND 4  Mean  0  0.1  0.3  0.3  
   SD  0  0.3  0.5  0.5  
   N  10  9  4  
 Male/female ratio PND 4  Mean  1.0  1.5  1.2  1.3  
   SD  0.3  0.7  0.3  0.6  
   N  9  10  9  4  
 Total viable pups PND 4  Mean  16.4***  14.5  14.2  11***  ***
   SD  1.7  1.6  2.5  3.9  
   N  9  10  9  4  
 % viable pups PND 4  Mean  100***  99.4  97.6  74.8***  
   SD  0  2  3.7  22.7  
   N  9  10  9  4  
 Litter weight PND 4  Mean  169.4***  148.5  143.7  84.6***  ***
   SD  23.6  14.2  16.4  31.2  
   N  9  10  4  
Average pup weight PND 4    Mean  10.3***  10.3  10.3  7.7***  ***
   SD  1.0  0.9  1.3  0.7  
   N  9  10  9  4  
 Average pup weight change (g) PND 4  Mean  4***  3.8  3.8  1.6***  ***
   SD  0.6  0.5  1.1  1.0  
   N  9  10  9  4  

  Note: * next to the control indicate a significant treatment-related effect in the ANCOVA, * in the litter column indicate a significant litter effect in the ANCOVA with p values *p<0.05; **p<0.02; ***p<0.01 in the control columns and significance in the litter sizes with p-values of *p<0.05, **p<0.02 or ***p<0.01 in the litter significance column.                                                                                                                                                        

Applicant's summary and conclusion

Conclusions:
In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted to OECD 422 and to GLP (reliability score 2) on triethoxyoctylsilane, reproductive effects (increased post-implantation loss, prolonged gestation duration and dystocia) only occurred at the 1000 mg/kg bw/day dose level in association with marked maternal toxicity. As such it is not possible to determine with confidence if the 1000 mg/kg bw/day dose level represents the NOAEL. Therefore, the maternal and reproductive toxicity NOAELs are both considered to be 300 mg/kg bw/day.