Amines, N-tallow alkyltrimethylenedi-, propoxylated

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 2010-01-19 to 2010-08-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

other: draft report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined repeated dose Toxicity study with the Reproduction/ developmental Toxicity screening Test

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-Propanol, 1,1'-[[3-[(3-aminopropyl)amino]propyl]imino]bis-, N-tallow alkyl derivs.

EC Number:

307-276-4

EC Name:

2-Propanol, 1,1'-[[3-[(3-aminopropyl)amino]propyl]imino]bis-, N-tallow alkyl derivs.

Cas Number:

97592-79-5

IUPAC Name:

97592-79-5

Constituent 2

Reference substance name:

2-Propanol, 1,1'-[[3-[(3-aminopropyl)amino]propyl]imino]bis-, N-tallow alkyl derivs.

IUPAC Name:

2-Propanol, 1,1'-[[3-[(3-aminopropyl)amino]propyl]imino]bis-, N-tallow alkyl derivs.

Details on test material:

- Name of test material (as cited in study report): POLYRAM SL
- Chemical Name: 2-Propanol, 1,1'-[[3-[(3aminopropyl)amino]propyl]imino] bis-, N-tallow alkyl derivatives
- Physical state: yellow viscous liquid
- Composition of test material, percentage of components: Fatty amine alkoxylated: 88.2%, Amines, tallow alkyl: 4.8 %, N-(tallow alkyl) triproplylenetetramine: 2.5%, Non amine: 2.5%, water content: 2.1%
- Purity test date: 12/01/2010
- Lot/batch No.: 96102715
- Expiration date of the lot/batch: 15/10/2011
- Storage condition of test material: at room temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 8-9 weeks.
- Weight at study initiation: Females: 162-200 g, (mean: 177.58 g, ± 20%= 35.52 g), Males: 246-303 g, (mean: 276.95 g, ± 20%= 55.39 g)
- Fasting period before study: no
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding, except for mating where 1 male and one female were cohoused
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Except for density (d= 0.910), no correction factor was used when weighing the test item. Based on available stability data (BSL 2010,study n°100107) The test item formulation was prepared on a daily basis: the test item was dispersed in sterile water and formulates were kept under magnetic stirring until the end of daily administration. Homogeneity of the aqueous dispersion was checked by visual inspection.

VEHICLE
- Justification for use and choice of vehicle: the vehicle was chosen due to solubility of the test item.
- Concentration in vehicle: 2.4, 4.8 and 9.6 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 7494 A 191, Provider: Braun

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Principle and validation of method used for chemical analysis of dosage forms:
Analysis of the test item in aqueous formulation samples was performed using LC MS/MS detection. Validation of the method was based on the ICH Q2(R1) guideline adopted in October 1994 and accordingly the following parameters were checked: specificity, precision and accuracy, repeatability, linearity, Sensitivity Evaluation Test (SET) and stability of the test item in working solutions. The validation of the analytical method was conducted in BSL/Study No. 100107 and precise details are documented in this report.

- Determination of test item concentration in dosage forms:
The dosage form samples were assayed using the above validated method. Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation)
Acceptance criterion was measured concentration = nominal concentration ± 15%

- Determination of dosage form stability:
Suitability of the proposed preparation process was confirmed by analysis of the stability, as described in BSL/Study No. 100107. Results of this study indicated satisfactory stability of the dosage forms for at least 6 hours at room temperature and 12 weeks at -20°C.
Homogeneity of the test item in the vehicle was analyzed for the low and high dose concentrations.Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparations in study week 1 and 5.

Duration of treatment / exposure:

- Males: 14 days before mating, during the mating period (up to 2 weeks) and until sacrifice (i.e. at least 4 weeks in total)
- Females: 14 days before mating, during the mating period (up to 2 weeks), during pregnancy, during lactation until day 3 post-partum inclusive.

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 12, 24, 48 mg/kg bw
Basis:
other: nominal

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the dose range finding study "BSL 2010b/K1 SS/ Repeated-dose toxicity: oral, 14-day dose range finding study" reported in section 7.5.1 Repeated dose toxicity: oral.
- Rationale for animal assignment: assigned to the dose/control groups based on their body weight
- Rationale for selecting satellite groups: no satellite groups

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day
- Cage side observations comprised: behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter
- Detailed clinical observations (outside the home cage in a standard arena) comprised: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoe, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), and piloerection and pupil size. In addition changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Males: on the first day of treatment (day 1), then once a week until sacrifice.
- Females: on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 post-coitum and days 0 and 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: On corresponding day of body weight measurement after beginning of the dose administration. Food consumption was not measured during mating period in males and females and post mating period in males.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE : No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in week before treatment and during the last week of treatment in males and on day 3 of the lactation in females (only lactating females were evaluated)
- Dose groups that were examined: five randomly selected males and females from each group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at respective terminal sacrifice (males: treatment day 29 or d 30; females: post natal day 4)
- Anaesthetic used for blood collection: Yes, Ketamin/Xylazin, 2:1
- Animals fasted: No
- How many animals: in five males and five females randomly selected from each group (same as for clinical chemistry)
- Parameters checked: See table 1 in results and discussion free-text for details.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at respective terminal sacrifice (males: treatment day 29 or d 30; females: post natal day 4)
- Animals fasted: No
- How many animals: in five males and five females randomly selected from each group (same animals as for hematology)
- Parameters checked: See table 1 in results and discussion free-text for details.

URINALYSIS: Yes
- Time schedule for collection of urine: before final sacrifice in males only(same animals as for haematology)
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked: See table 1 in results and discussion free-text for details.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in week before treatment and during the last week of treatment in males and on day 3 of the lactation in females (only lactating females were evaluated)
- Dose groups that were examined: five randomly selected males and females from each group
- Battery of functions tested:
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

Sacrifice and pathology:

GROSS PATHOLOGY / HISTOPATHOLOGY

1. A complete macroscopic post-mortem examination was performed on all animals, also those found dead. Special attention was paid to the reproductive organs. A careful examination of the stomach lining was performed due to the strong irritant / corrosive properties of the test item.

2. Microscopic examination
* Preservation of tissues
The ovaries, uterus with cervix, vagina, testes, epididymides, prostate, seminal vesicle with coagulating gland were preserved for all adult animals.
The tissues listed in table 2 (see table 2 in results and discussion free-text for details) were preserved for five randomly selected sacrificed-as-scheduled males, for five randomly selected females having delivered and being sacrificed on day 4 post-partum of each group, and for animals found dead unless otherwise specified.

* Microscopic examination was performed on:
All macroscopic lesions
All animals found dead
Ovaries, uterus with cervix, vagina, testes, epididymides, prostate, seminal vesicle with coagulating gland of all adult animals.
All tissues listed in table 2 for five randomly selected sacrificed-as-scheduled males, for five randomly selected females having delivered and being sacrificed on day 4 post-partum of the control- and high-dose groups
The duodenum, jejunum, ileum and mesenteric lymph nodes from five randomly selected animals of the low- and intermediate-dose groups.

Other examinations:

Special analysis of female reproductive organs and pups are described under chapter 7.8.1 of this dossier

Statistics:

Parameters like body weight change and food consumption was calculated for each animal as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and presented as percentage.
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. Statistical analysis performed with GraphPad Prism V.x software (p<0.05 was considered as statistical significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

Mortality:
There were no treatment-related deaths but five premature deaths were observed. One female of each dose- group died: on day 14 for the low-dose group, on day 5 for the mid-dose group and on day 8 for the high-dose group. Two males from the mid-dose group died on days 10 and 6 respectively. The macroscopic examinations of the five decedents together with the histopatological findings confirmed that the death was due to misgavaging or regurgitation/aspiration of the test item (see table 3 in remarks on results for details).

Clinical signs:
In the high-dose group, respiratory sounds were observed in 7 out of 10 males generally for 1 to 3 consecutive days although in 1 male it was observed during 7 consecutive days. The number of male rats showing loud breathing increased from 3 to 7 between weeks 1 and 4. In females, respiratory sounds were observed in 8 out of 10 animals including one found dead on day 8 due to aspiration of the test item in the lungs. Respiratory sounds were observed for 1 to 2 days during the premating period in 5 females and throughout gestation in 5 females also.
In the mid-dose group, respiratory sounds were observed in 3 out of 10 males including one found dead on day 10 due to a gavaging error. The 2 remaining animals had respiratory sounds on day 9 and from day 23 until terminal sacrifice respectively. Respiratory sounds were occasionally observed in 1 female of this dose-group on GD 5.
In the Low-dose group, respiratory sounds were occasionally observed in 3 males during week 2 or 3 and in 1 female the day before its death causing by a gavaging error. In the control group, one male had occasional loud breathing during week 3.

Salivation was observed in all females of the high-dose group throughout gestation and during the premating period for 4 of them. In males, the number of animals showing ptyalism increased from 6 to 9 between weeks 1-2 and 4. Salivation was generally observed for 1 to 2 consecutive days in both sexes although in 2 males it was observed during the 7 consecutive days of the week 4.
In the mid-dose group, salivation was noted in 5 males for 1 to 4 days during weeks 3 or 4 and as early as day 9 in 1 of them. 2 females showed salivation throughout gestation and 1 female with successful mating but not pregnant showed salivation in week 2 of treatment.

Piloerection was observed in 9 out of 10 males during week 4 of treatment at high dose and was coupled with excessive salivation in 6 of them. Piloerection was also observed in week 1 for 1 male. In females, piloerection was noted in 4 out of 10 animals including one found dead on day 8 due to aspiration of the test item in the lungs. In the surviving animals, 2 had piloerection coupled with salivation during week 2 of gestation and 1 had only piloerection on PND1. In the mid-dose group, piloerection was noted in one male during the last week of treatment (week 4). (See table 4 in remarks on results).

BODY WEIGHT AND WEIGHT GAIN

No test item-related effects on body weight and body weight change in any dose group were observed.

FOOD CONSUMPTION

In males, statistical analysis of the food consumption data revealed no significant effect in treated groups when compared with controls.
In females, no effect on food consumption was observed in treated groups during premating and gestation period as compared to controls. However, a statistically significant decrease in food consumption in the high-dose group was observed during post natal days 0-4 when compared with controls (- 17.4% compared to controls). This statistically significant and dose dependent effect on food consumption could be considered as a treatment related effect.

OPHTHALMOSCOPIC EXAMINATION

No effects were found.

HAEMATOLOGY

In male, there were no treatment-related effects in the hematological parameters measured. In females, statistically significant decreases in haemoglobin and hematocrit were seen in the high-dose group when compared with controls. While being in the normal biological range, the decrease in haemoglobin was considered to be treatment-related due to dose dependency. However, lack of consistent and dose dependent pattern of effect on hematocrit and Mean Corpuscular Haemoglobin (MCH) indicates no toxicological relevance.

CLINICAL CHEMISTRY

No treatment related effect on any of the clinical biochemistry parameter in male and females was observed when compared with controls.

URINALYSIS

There were no effects of treatment on urine parameters.

NEUROBEHAVIOUR

There were no treatment-related observations noted during the Functional Observation Battery and there were no effects on spontaneous locomotor activity at any dose-level.

ORGAN WEIGHTS

There were no treatment-related effects on the absolute and relative organ weights.

GROSS PATHOLOGY

There were no treatment-related macroscopic findings.

HISTOPATHOLOGY: NON-NEOPLASTIC

Test item-related histopathological changes were observed in the small intestine (duodenum, jejunum and ileum) and in the mesenteric lymph node:

In the duodenum, villous infiltration with histiocytes graded as minimal were observed in 3 out of 5 males of the high-dose group. No particular findings were found in the duodenum of females and mid-dose and low-dose males.
In the jejunum, villous infiltration with histiocytes generally graded as minimal or mild were observed in all males and all but one females of the high-dose group. No particular findings were found in the jejunum of the females treated with the mid-dose or the low-dose whereas 1 male in each of these dose groups showed a minimal infiltration with histiocytes.
In the ileum, villous infiltration with histiocytes graded as minimal to moderate were seen in all males and females of the high and mid-doses groups. 1 female and 2 males from the low-dose group showed a minimal or mild infiltration with histiocytes.
In the small intestine, histiocytic infiltration were observed with a dose-related trend at all dose-levels in the ileum of males and females and from the mid-dose level in the jejunum of males. In the absence of any degenerative changes, this observation was considered to be non adverse.

In the mesenteric lymph nodes, infiltration with histiocytes were observed in all males whatever the dose and in all females of the mid and high-doses groups and in 2 out 5 females of the low-dose group. The grade of the histiocytic infiltration increased in a dose-dependent manner in both sexes being minimal in 5 animals and mild in 2 animals of the low-dose level and mild in 2 animals and moderate in 8 animals of the high-dose level. In the absence of degenerative changes, this observation was considered to be non adverse.

Effect levels

Dose descriptor:

NOAEL

Effect level:

24 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Changes in haematological parameters and decrease in food consumption during the post –natal period in the next higher dose (ie 48 mg/kg bw/day)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 3: Mortality Table

Group	Treatment period
	Male	Female
C (0 mg/kg bw)	0/10	0/10
LD (12 mg/kg bw)	0/10	1/10
MD (24 mg/kg bw)	2/10	1/10
HD (48 mg/kg bw)	0/10	1/10

Animals affected / total number of animals

Table 4: Clinical Observations

Males
Major Clinical Findings (No. of animals showed particular clinical sign at least once during treatment period)	C (0 mg/kg bw)	LD (12 mg/kg bw)	MD (24 mg/kg bw)	HD (48 mg/kg bw)
Total number of animals examined	10	10	10	10
Respiratory sounds	1	3	3	7
Salivation	0	0	5	10
Piloerection	0	0	1	9
Females
Major Clinical Findings (No. of animals showed particular clinical sign at least once during treatment period)	C (0 mg/kg bw)	LD (12 mg/kg bw)	MD (24 mg/kg bw)	HD (48 mg/kg bw)
Total number of animals examined	10	10	10	10
Respiratory sounds	0	1	1	8
Salivation	0	0	3	10
Piloerection	0	0	0	4

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxic effects was considered to be 24 mg/kg bw/day because of changes in haematological parameters and decrease in food consumption during the post –natal period observed in females treated at 48 mg/kg bw/day.

Executive summary:

The potential toxicity of the structural analogue ' 2-Propanol, 1,1'-[[3-[(3-aminopropyl)amino]propyl]imino]bis-, N-tallow alkyl derivs.' was evaluated following repeated oral administration to rats according to OECD Guideline 422 and EPA guideline OPPTS 870.3650. The study was conducted in compliance with the principles of Good Laboratory Practice regulations.

Three groups of 10 males and 10 females Wistar rats received the test substance daily, by gavage, before mating and through mating and, for the females, through gestation until day 3 post-partum. The dose-levels were 12, 24 or 48 mg/kg bw/day. Another group of 10 males and 10 females received the vehicle, sterile water, alone, under the same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg bw/day.

Animals were examined daily for clinical signs and mortality and detailed clinical observations were performed once before the beginning of the treatment period and then once a week until the end of the study. Body weight was measured weekly, food consumption also except during the mating period. A Functional Observation Battery includingsensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations was performed in the week before treatment and at the end of the study. Blood and urine samples were taken for analysis of hematology, blood biochemistry and urine parameters at the end of the study from randomly selected five males and five females (except urine) from each group.

Males were sacrificed after completion of the mating period on treatment days 29 and 30 and females on day 4 post-partum. All animals were subjected to a complete macroscopic post-mortem examination with a careful examination of the stomach lining due to the strong irritant / corrosive properties of the test item. A microscopic examination was performed on selected organs of five randomly selected males and five randomely selected females having deliver from the control and high-dose groups, on the non-pregnant females and on all macroscopic lesions. 

There were no treatment related deaths but 5 premature deaths (1 female, 1 female and 2 males and 1 female from the low, mid and high-dose groups respectively) were observed.The macroscopic examinations of the five decedents together with the histopatological findings confirmed that the death was due to misgavaging orregurgitation/aspiration of the test item.

Salivation was observed in all males and females of the high-dose group and in 5 males and 3 females of the mid-dose group. In addition, most of the animals treated at the highest dose had piloerection and respiratory sounds. In the mid-dose group, only 1 male had piloerection while respiratory sounds were noted in 3 males and 1 female respectively. In the low-dose group, respiratory sounds were occasionally observed in few animals as well as in one animal of the control group. No other clinical signs were observed at 12 mg/kg/day.

No relevant differences were observed concerning functional and behavioural examinations in male and female treated groups when compared with controls.

No test item related effects on body weight and body weight change wereobserved. In males, statistical analysis of the food consumption data revealed no significant effect in treated groups when compared with controls. In females, no effect on food consumption was observed in treated groups during premating and gestation period as compared to controls. However, statistically significant decrease in food consumption in the high-dose group was observed during post natal days 0-4 when compared with controls. There were no effects in females food consumption at 24 or 12 mg/kg/day.

Laboratory investigations showed that females treated at 48 mg/kg bw/day had a statistically significant decrease in haemoglobin and hematocrit when compared with controls. While being in the normal biological range, the decrease in haemoglobin was considered to be treatment-related due to dose dependency. However, lack of consistent and dose dependent pattern of effect on hematocrit and Mean Corpuscular Haemoglobin (MCH) indicates no toxicological relevance. There were no treatment-related effects on hematological parameters in males and no effects on biochemical parameters whatever the sex.

No treatment-related effects on organ weights and no particular macroscopic findings were observed.

At microscopic examination, histiocytic infiltration was observed with a dose-related trend at all dose-levels in the ileum of males and females and from the mid-dose level in the jejunum of males. In the absence of any degenerative changes, this observation was considered to be non adverse.In the mesenteric lymph nodes, infiltration with histiocytes were observed in all males whatever the dose and in all females of the mid and high-dose groups and in 2 out 5 females treated at low dose. The grade of the histiocytic infiltration increased in a dose-dependent manner in both sexes being minimal in 5 animals and mild in 2 animals of the low-dose level and mild in 2 animals and moderate in 8 animals of the high-dose level. In the absence of degenerative changes, this observation was considered to be non adverse.

In conclusion, parameters like body weight, neurology, clinical biochemistry, gross necropsy, organ weight remained unaffected up to 48 mg/kg bw/day. At the highest dose-level, most of the animals had piloerection. Salivation and respiratory sounds were also observed but these clinical signs are frequently observed with strong irritant / corrosive substances administered by gavage and were therefore not considered as toxicologically relevant. At the highest dose-level, changes in haematological parameters and decrease in food consumption during the post –natal period were also seen in females. No such findings were noted at the lower dose-levels. Test item-related histopathological changes were observed in small intestine and the mesenteric lymph node in all treated groups. Both males and females had infiltration with histiocytes that probably contained test-item lipid complexes which are poorly degraded by lysosomal enzymes. As no degenerative changes were associated, this effect was therefore considered to be non‑adverse.

Based on the data generated from this study, A No Observed Effect Level (NOEL) could not be established for the parental toxicity due to histopathological findings in small intestine and mesenteric lymph nodes in all treated groups. As no degenerative changes were associated, these findings were therefore considered to be non‑adverse and the No Observed Adverse Effect Level (NOAEL) was considered to be 24 mg/kg/day.

 

Results on the reproductive toxicity potential of the test item are presented in section 7.8.1 "Toxicity to reproduction" of this dossier.