bis(hydrogenated tallow C16-18-alkyl)hydroxylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-08-17 to 1996-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Reference 2

Reference Type:

other: Amendment Final Report

Title:

Unnamed

Year:

2000

Report date:

2000

Reference 3

Reference Type:

other: Analytical Report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted May 12, 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)

Version / remarks:

Vol. 50, No. 188, September 27, 1985; revised May 20, 1987

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

-

EC Number:

418-370-0

EC Name:

-

Cas Number:

143925-92-2

Molecular formula:

C32-36 H67-74 N1 O1

IUPAC Name:

N-hexadecyl-N-octadecylhydroxylamine

Test material form:

solid

Details on test material:

- State of aggregation: solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production, CIBA-GEIGY Limited, 4332 Stein / Switzerland
- Age at study initiation: approximately 5 weeks
- Weight at study initiation: males 144.3 g to 201.3 g; females 136.6 g to 169.8 g
- Fasting period before study: no
- Housing: in groups of 5 in macrolon cages type 4 with wire mesh tops and standardized granulated soft wood bedding
- Diet: Pelleted, certified standard diet (Nafag No. 8900 FOR GLP) ad libitum
- Water: tap water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ±10 %
- Air changes: approximately 16 - 20 per hour
- Photoperiod: 12 hours light and 12 hours dark

IN-LIFE DATES: From: 1995-09-18 To: 1995-12-20 or 1996-01-16 (satellite groups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

and Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test item in the selected vehicle at the appropriate concentrations were freshly prepared once per experimental week and stored at 4°C until use. Aliquots were taken daily from the refigerator, adapted to room temperature, and administered within two hours.

VEHICLE
- distilled water containing 0.5% carboxymethylcellulose and 0.1% Tween 80
- Amount of vehicle (if gavage): 10 mL / kg bw / day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item dosage forms were taken during the study for analytical determination of concentration, homogeneity and stability. The analytical results indicate that the doses were accurately formulated during the toxicity study. The results also confirm that the formulations were homogeneous and stable from the time of preparation to completion of dosing.

Duration of treatment / exposure:

93 to 94 days

Frequency of treatment:

once daily (7 days / week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Vehicle and high dose group: 10 males and 10 females, plus 10 / sex as recovery group
Ultra low, low and medium dose: 10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a 14-days range finding study in rats (gavage), CIBA-GEIGY Study No. 954027, where no signs of overt toxicity were observed after oral administration of test item at 10, 50, 250 and 1000 mg / kg bw / day.
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:

MONITORING OF MAIN GROUP and RECOVERY GROUP ANIMALS
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice (a.m. and p.m.) on working days, once (a.m.) on weekends and holidays

CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (recorded at least weekly)

BODY WEIGHT: Yes
- Time schedule for examinations: individually at weekly (midweek) weighing sessions. The first weights were recorded during the acclimatization period. Daily body weights for accurate dosing were measured but not recorded.

FOOD CONSUMPTION:
- Food consumption was determined weekly cagewise. The individual food consumption values were calculated from the food consumption per cage and the number of animals present. Mean daily food consumption was calculated as g food / kg body weight / day.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time and dose group schedule for examinations: animals of the highest dose group and of the control group were examined prior to dosing (day -4), towards the end (day 88) of the treatment period, and towards the end of the recovery period (day 116).
- Parameters examined: surroundings of the eyes, of sclera, cornea, iris and adaptation of the pupil to the ophthalmoscopic light beam.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on all surviving animals of each dose group after 1 month of treatment, at the end of the treatment period; at the end of the recovery period on all surviving animals of the control and high dose group.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- Parameters examined: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, thrombocyte counts, leucocyte count, differential white cell count: neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells
- Coagulation: prothrombin time
- Methemoglobin

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see HAEMATOLOGY
- Animals fasted: Yes
- Parameters examined: glucose, urea, creatinine, total bilirubin, total proteins, albumin, globulin, albumin/globulin ratio, cholesterol, sodium, potassium, calcium, chloride, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Parameters examined: "detailed necropsy" (not further specified)

- Organ weights: body (exsanguinated), brain, liver, kidneys (paired), adrenals (paired), thymus, ovaries, testes (paired), spleen, thyroid. Missing organ weights according to actual OECD guideline (adopted 1998): heart, uterus, epididymides

- Preservation of tissues: skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland (both), liver, pancreas, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), kidney (both), urinary bladder, prostate, seminal vesicle, testis (both), epididymis (both), uterus, vagina ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain (medulla/pons, cerebellar cortex, cerebral cortex), spinal cord (cervical, midthoracic, lumbar), eye with optic nerve (both), orbital gland (both), extraorbital lacrimal gland (both), Zymbal gland (both), muzzle, tongue, any tissue with gross lesions

HISTOPATHOLOGY: Yes, surviving animals from ALL main groups
- Parameters examined: all tissues preserved, except tongue (no deficiencies regarding OECD guideline adopted 1998)
- Recovery group (experimental group II): liver and mesenteric lymph node of both sexes, and the kidneys and skeletal muscle of males.

Statistics:

For each time point and parameter:
- Univariate statistical analysis (not specified)
- Nonparametric methods (Lehmann)
- Lepage's two-sample test (combination of Wilcoxon and Ansari-Bradley statistics)
- Test for trends from control by Jonckheere's test for ordered alternatives
- Tests for normality of distribution or homogeneity of variances: not specified

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The few clinical signs observed during the study were either of accidental nature (eye, injured) or at an incidence which is not uncommon in gavage studies (skin lesion, hair loss). The treatment with the test article was, therefore, not considered to have produced any relevant clinical signs.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during this study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development during this study was not considered influenced by treatment. The changes to the mean body weight in high dose females (1000 mg/kg) during the recovery period may partially be associated with the minimally depressed food intake during the
last week of the recovery period, however they can not fully explain the considerable depression. Since a relation to treatment is unlikely this change is considered incidental and, therefore, not related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During the treatment period the overall mean food intakes in all treated groups were essentially equal to the control groups. In high dose animals, the changes to the food consumption during the recovery period were below the 5% level and, therefore, considered of no toxicological relevance. In addition, the magnitude of this change was to small to account for the depressed body weight development in high dose females during the last week of the recovery period.

Food efficiency:

no effects observed

Description (incidence and severity):

All food consumption ratios were within the expected range and were not considered influenced by treatment.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The examinations revealed no evidence of ocular toxicity.

Description (incidence and severity):

At treatment end, minimally higher numbers of neutrophilic granulocytes, monocytes and large unstained cells were recorded for females treated at 1000mg/kg, and were associated with lower values of relative lymphocyte counts. Reversibility of these minor alterations within the recovery period was demonstrated based on the absolute values of differential cell counts. Although statistically significant differences were also observed in certain other hematology parameters, they were not considered indicative of a treatment-related effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At treatment end, increased activities were recorded for alanine aminotransferase and aspartate aminotransferase in females of groups 4 and 5 (200 and 1000 mg/kg), and also in individual males of group 5 (1000 mg/kg). Furthermore, females of groups 4 and 5 had minimally lower plasma glucose levels and minimally higher cholesterol levels. In addition, plasma bilirubin levels were slightly increased in individual females of group 5. These alterations were completely reversible during the recovery period except for increased liver enzyme activities, which were only partially reversible in fenales and not reversible in males. There were other blood chemistry parameters reaching a level of statistical significance in their difference between treated and control groups , which were not considered to be treatment-related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The organ weight analysis revealed no statistically significant differences between control and treated animals. However, the mean spleen weight in high dose females was increased by 22% when compared with the control mean. In the absence of any histopathologic changes this finding was considered incidental and of no toxicological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few macroscopical findings which were recorded in this study were comparable in nature and incidence to those occurring spontaneously in our colony of rats. Thus, no experimental relevance was attributed to these findings.

Description (incidence and severity):

- Liver was affected in females of group 4 and in males and females of group 5. The lesion consisted of a minimal to moderate necrosis of single hepatocytes in females of group 4 and in males and females of group 5, and slight to marked granuloma formation in females of groups 4 and 5. After 4 weeks of recovery (Experimental group II) these lesions were not reversed.
- A slight to moderate accumulation of phagocytic cells was present in mesenteric lymph nodes in males and females of groups 4 and 5. This lesion was characterized by aggregates of sinus histiocytes in medullary sinuses which extended into paracortical and cortical areas. The lesion was not reversed within 4 weeks of recovery.
- Kidneys were affected in males of groups 4 and 5. The lesion consisted of a minimal to moderate hyaline change in cortical tubular epithelial cells in males of groups 4 and 5 and a minimal to moderate focal chronic tubularlesion in males of group 5. These lesions were fully reversed within 4 weeks of recovery.
- Necrosis with fragmentation of single muscle fibres reported as a minimal myopathy was present in males of group 5. This lesion was fully reversed after 4 weeks of recovery.
- Additionally, a variety of other changes was found in this study. They commonly occur in our colony of laboratory rat, and neither their incidence nor their distribution and morphologic appearance gave any indication of a treatment-related association.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

3

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (actual dose received)

System:

immune system

Organ:

mesenteric lymph node

Treatment related:

yes

Applicant's summary and conclusion

Executive summary:

The test article was administered by gavage for 3 months at daily doses of 0, 10, 50, 200, and 1000 rog/kg body weight to a total of 140 albino rats. In each dose group 10 animals per sex and group were sacrificed at the end of the treatment period; 10 animals per sex in the control and high dose groups, were kept for a 4-week recovery period before sacrifice. Administered quantities of the test article suspension were adjusted daily to individual body weights. Clinical signs, body weight, food consumption and mortality were monitored throughout the study for all animals. Ophthalmoscopic examinations were performed prior to dosing and towards the end of the treatment and recovery period. Clinical laboratory investigations were performed at treatment end and recovery end. At scheduled sacrifices, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation and were examined microscopically. The results of this study are summarized as follows:

There were no relevant clinical signs and no changes to the behavior observed. All animals survived to scheduled necropsies. Body weight development in all male and female groups was not considered influenced by treatment. There were no relevant differences in mean food intakes between treated and control groups of both sexes. The food consumption ratios obtained for treated and control groups were essentially similar. There was no evidence of ocular toxicity. There were no changes to absolute or relative mean organ weights which attained a level of statistical significance. At treatment end, minimally higher numbers of neutrophilic granulocytes, monocytes and large unstained cells were recorded for females treated at 1000 mg/kg, and were associated with lower values of relative lymphocyte counts. Reversibility of these minor alterations within the recovery period was demonstrated based on the absolute values of differential cell counts At treatment end, increased activities were recorded for alanine aminotransferase and aspartate aminotransferase in females of groups 4 and 5 (200 and 1000 mg/kg), and also in individual males of group 5 (1000 mg/kg). Furthermore, females of groups 4 and 5 had minimally lower plasma glucose levels and minimally higher cholesterol levels. In addition, plasma bilirubin levels were slightly increased in individual females of group 5. These alterations were completely reversible during the recovery period except for increased liver enzyme activities, which were only partially reversible in females and not reversible in males. There were no treatment-related macroscopical changes. At microscopical examination treatment-related changes were present in males and females of groups 4 and 5 (200 and 1000 mg/kg), respectively. These changes consisted of: necrosis of liver hepatocytes and granuloma formation in the liver of females of group 4 and in males and females of group 5; phagocytic cells in the mesenteric lymph node in males and females of groups 4 and 5 - these lesions of the liver and mesenteric lymph node were not reversed within 4 weeks of recovery; chronic tubular lesion and hyaline change in the kidney of males of groups 4 and 5; myopathy of skeletal muscle in males of group 5. The lesions of the kidney and skeletal muscle were fully reversed within the 4-week recovery period.

In conclusion, under the conditions of this study, treatment with the test item was well tolerated up to the limit dose of 1000 mg/kg body weight and did not result in any overt signs of toxicity. At 200 and 1000 mg/kg, changes to clinical laboratory parameters and/or histopathology revealed the liver, kidney, and mesenteric lymph node as main targets of organ toxicity. Except for increased liver enzyme activities, liver hepatocyte necrosis and granuloma formation, and phagocytic cells in mesenteric lymph nodes, all findings were completely reversible. It can be inferred from the observations made during the above study, that a "no-observable effect level" (NOEL) for the test item when administered by daily oral gavage for 3 months to animals of both sexes is equal to the "no-observable adverse effect level" (NOAEL) and is defined at 50 mg/kg body weight per day.