A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 1991 to 19 August 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented detailed study conducted according to good laboratory practices and following EEC and OECD guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

87787-81-3

EC Number:

618-070-1

Cas Number:

87787-81-3

IUPAC Name:

87787-81-3

Constituent 2

Reference substance name:

A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

EC Number:

413-800-3

EC Name:

A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

Cas Number:

87787-81-3

Molecular formula:

C44H79NO3

IUPAC Name:

2-(dioctadecylcarbamoyl)benzoic acid; 2-(ditetradecylcarbamoyl)benzoic acid; 2-[(propan-2-yloxy)carbonyl]benzoic acid; 2-[octadecyl(tetradecyl)carbamoyl]benzoic acid; 2-octadecyl-2,3-dihydro-1H-isoindole-1,3-dione; 2-tetradecyl-2,3-dihydro-1H-isoindole-1,3-dione; benzene-1,2-dicarboxylic acid; dioctadecylamine; ditetradecylamine; octadecyl(tetradecyl)amine

Constituent 3

Reference substance name:

reaction mass of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid dihydrogenated alkyl (C14-C18)amine

IUPAC Name:

reaction mass of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid dihydrogenated alkyl (C14-C18)amine

Constituent 4

Reference substance name:

Stepan TAB-2

IUPAC Name:

Stepan TAB-2

Details on test material:

The test sample was designated Stepan TAB-2, which had the appearance of an ivory solid. Batch number of test material was 198308. Purity was not noted, but treated as 100% pure. The test substance was typical commercial quality, and consists of the multicomponent composition detailed in the general information section of this dossier. Expiry date of the test material was April 30, 1992.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 30 males and 30 females were used. The number of animals per group was 5 males and 5 females. Animals were Wister rat, outbred, SPF quality, obtained from BRL Ltd., Basel, Switzerland. The animals were approximately 6 weeks old at start of treatment. Identification was with earmarks and tattoos. Animals were randomised using a computer-generated random algorithm according to body weight with all animals within ±20% of the sex mean. Animals were acclimitised at least 7 days. Veterinary examination was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
Animals were housed in an air-conditioned room with 15 air changes per hour and controlled environment with optimal conditions considered being a temperature of 21°C and a relative humidity of 55%. Occasional fluctuations from these optimal conditions were noted, but were considered not to have adversely affected study integrity. Lighting was 12 hours artificial fluorescent light and 12 hours dark per day. On 2 occasions the temperature and relative humidity could not be recorded due to technical failure. Animals were housed 5 to a cage (same sex) in stainless steel suspended cages with wire mesh floors. Free access to standard pelleted laboratory animal diet (Kliba, Klingentalmuehle AG, 4303, Kaiseraugst, Switzerland). Each batch was analysed for contaminants. Tap water was ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Specific gravity 0.92

Details on oral exposure:

Animals were dosed using oral gavage with corn oil as the vehicle. Dosing was once daiiy for 28 days, seven days per week.
Dose levels were: Group 1: 0 mg/kg bw/day (vehicle control); Group 2: 50 mg/kg bw/day; Group 3: 200 mg/kg bw/day; Group 4: 1000 mg/kg bw/day.
Dose volume was 5 ml/kg body weight. Dose volumes were calculated weekly according to the latest body weight.
Test substance formulation was carried out daily immediately prior to dosing. Homogeneity of test substance in vehicle was assured by stirring and shaking prior to dose administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared after termination of treatment (similar procedures as during the course of the study) were analysed to check stability and accuracy of preparation. The test material was determined by NOTOX to be stable in corn oil for at least 4 hours.

Duration of treatment / exposure:

28 days of dosing, (followed by a 14-day recovery period for some animals).

Frequency of treatment:

Once daily, seven days per week for 28 days, followed by a 14 day recovery period.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

five male and five female

Control animals:

yes, concurrent vehicle

Details on study design:

A total of 30 males and 30 females were used. The number of animals per group was 5 males and 5 females. Animals were Wister rat, outbred, SPF quality, obtained from BRL Ltd., Basel, Switzerland. The animals were approximately 6 weeks old at start of treatment. Identification was with earmarks and tattoos. Animals were randomised using a computer-generated random algorithm according to body weight with all animals within ±20% of the sex mean. Animals were acclimitised at least 7 days. Veterinary examination was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day) to provide a basis for selection of dose levels for the main study. No differences of biological significance were observed in clinical appearance, body weight, macroscopic appearance or liver weights between the treated groups. Slightly higher food consumption by males receiving 1000 mg/kg/day was considered to have occurred fortuitously. Based on these observations, treatment levels for a study of 28 days duration were selected to be 0, 50, 200 and 1000 mg/kg/day)

Dosing formulations were prepared by heating the test substance up to 50°C to melt the substance, and subsequently weighted into a glass flask on an analytical balance, and the vehicle (w/w) added. Adjustment was mad for specific gravity of vehicle. Test substance formulations were heated t approximately 50°C to dissolve the test material.

Clinical Laboratory Investigations:

Blood samples were collected under light ether anaesthesia from main and recovery group animals on day 29 and recovery group animals on day 43. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retroorbital sinus and collected into tubes prepared with EDTA for haematological parameters (0.6 ml), tubes prepared with citrate for blood clotting times (1.0 ml) and untreated tubes for clinical biochemistry parameters (>2.0 ml).

Positive control:

No data.

Examinations

Observations and examinations performed and frequency:

The following observations were recorded:
Clinical signs: at least once daily during treatment and weekly during recovery (final observation not recorded) Severity of observations were graded.
Mortality/viability: Twice daily
Food Consumption: Weekly
Water Consumption: Subject appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Body weights: weekly and on the day preceding termination, prior to overnight fasting.
Ophthalmoscopic examinations: Both eyes were examined following instillation of tropicamide solution (5 mg/ml) at week 4 (all groups) and at week 5 (recovery groups).

Clinical Laboratory Investigations:

Blood samples were collected under light ether anaesthesia from main and recovery group animals on day 29 and recovery group animals on day 43. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retroorbital sinus and collected into tubes prepared with EDTA for haematological parameters (0.6 ml), tubes prepared with citrate for blood clotting times (1.0 ml) and untreated tubes for clinical biochemistry parameters (>2.0 ml).

The following haematology parameters were determined: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, red cell distribution width, total leucocyte count, differential leucocyte count (Neutrophils, basophils, lymphocytes, monocytes).

The following haematology parameters were determined from blood containing citrate as an anti-coagulant: Prothrombin time, partial thromboplastin time.

Clinical Biochemistry:
The following clinical biochemistry parameters were determined from serum samples after clotting and centrifugation: Glucose, urea, creatinine, bilirubin, cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine kinase, lactate dehydrogenase, sodium, potassium, chloride, calcium, phosphorus, total protein, albumin protein, globulin protein, albumin globulin ratio.

Sacrifice and pathology:

Necropsy:

All animals surviving to the end of the observation period (day 29 for the main groups and day 43 for the recovery groups) were deeply anaesthetized by ether vapour and then exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.

Pathology:

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution: adrenal glands, aorta, brain, cecum, cervix, clitoral gland, colon, duodenum, epididymides, esophagus, eyes with optic nerve and Harderian gland, female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, larynx, lacrimal gland (exorbital), liver, lung infused with formalin, lymph nodes – mandibular and mesenteric, nasopharynx, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid (including parathyroid), tongue, trachea, urinary bladder, uterus, vagina, all gross lesions.

The following organ weights (and terminal body weight) were recorded at the scheduled dates of necropsy: Adrenal glands, brain, heart, kidneys, liver, spleen, testes, and ovaries.

Histotechnology: All organ and tissue samples, as defined under Histopathology (see below) were processed, embedded and cut at a thickness of 204 micrometers and stained with haematoxylin and eosin.

Histopathology: Slides of adrenals, heart, kidneys, liver, spleen and stomach, collected at termination from all animals of the control and high dose group and all gross lesions of all animals were examined by a pathologist. All abnormalities were described and included in the report.

Other examinations:

No further details. An extensive number of detailed tables are included in the report to record raw data from the study.

Statistics:

Statistical Analysis:
The following statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:

Univariate one-way analysis of variance was used to assess the significance of intergroup differences.

If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.

All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data, and medians were calculated for discrete data (scores) in the summary tables.

Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances, and then rounded off to two decimal places. Therefore two groups may display the same printed means for a given parameter, yet display different test statistics values.
The exact fisher-test was applied to the ophthalmoscopic examination data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Analysis of dose preparations: Test substance formulations in corn oil were noted as stable for at least 4 hours at all concentrations tested. In addition, these test substance preparations formed homogenous suspensions. Analysis of the accuracy of dose preparations revealed values within acceptable range of nominal concentrations for this type of formulation.

Observations:

Clinical Signs:

During the 4-week treatment period, alopecia and excessive salivation were noted in control and treated animals. In one control male, alopecia continued to be observed during the recovery period.

Alopecia, sometimes accompanied by scabs or wounds, was observed in 6/10 control males, 1/5 females receiving 50 mg/kg bw/day and 4/5 females receiving 200 mg/kg/day. These incidences of alopecia were in excess of that normally seen in rats of this age and strain, and the higher incidence in particular 2 cages (control males and group 3 females) was considered to be the result of increased grooming activity or scratching within the cage.

Excessive salivation was noted on 2 occasions in 1 control female and over intermittent periods in males receiving 200 or 1000 mg/kg bw/day. Excessive salivation is often observed in rats of this age and strain, following oral administration of a xenobiotic agent via a stomach tube, and may be associated with bad taste or irritant effect of the test substance. The incidence and severity of alopecia and excessive salivation were considered not to represent significant indications of toxicity.

Mortality:
No treatment related mortality occurred during the 4 weeks of treatment and subsequent 2 weeks of recovery. The spontaneous death of one female of the recovery group occurred in response to ether anaesthesia during blood sampling procedures.

Body Weight: Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week treatment period and the subsequent 2-week recovery period.

Food Consumption: There were no differences in food consumption before or after allowance for body weight between treated and control animals after 4 weeks of treatment or 2 weeks of recovery.

Othalmoscopic Examination: At opthalmoscopic examination at weeks 4 or 6 (2 weeks recovery) there were no differences noted between control and treated rats that could be attributed to treatment with Stepan TAB-2. An increased incidence of anterior lens opacity was noted to have arisen in 4/5 females during the recovery period. Since this finding occurred in females only and no corroborative changes in other toxicological parameters were noted in this study, no toxicological significance was attached to this finding.

Clinical Laboratory Investigations:

Haematology:

Haematological parameters of treated rats were considered not to have been affected by treatment.
After four weeks of treatment statistically significantly increased mean corpuscular haemoglobin in males receiving 1000 mg/kg bw/day, increased mean corpuscular haemoglobin concentration in males receiving 1000 mg/kg bw/day, increased platelets numbers in females receiving 50 mg/kg bw/day and decreased partial thromboplastin time in females receiving 1000 mg/kg bw/day were in the range of normal biological variation and considered to have occurred fortuitously. Statistically significantly decreased white blood cell numbers, arising between males receiving 1000 mg/kg bw/day and control males were the result of control values at the higher limit of that normally seen in rats of this age and strain, and therefore considered to be of no toxicological significance.

After 6 weeks (4 weeks of treatment and 2 weeks of recovery) the only change noted between control rats and rats receiving 1000 mg/kg bw/day was a statistically significantly decreased white blood cell number in females. Since no changes were noted in white blood cell numbers of females after 4 weeks treatment and no corroborative findings were noted in other toxicological parameters examined, this change was considered to have occurred by chance.

Clinical Biochemistry: There were no differences noted between control and treated rats that were attributed to treatment with Stepan TAB-2. At week 4 of treatment and week 2 of recovery, all values in treated animals achieving a level of statistical significance when compared to controls, were considered to be within the range of normal background variation and not to be toxicologically significant.

Pathology:

Macroscopic Examination:

Macroscopic observations at necropsy did not reveal any findings after 4 weeks of treatment or 2 weeks of recovery.
Organ Weights and relative organ weights of treated animals were similar to those of control animals. Statistically significant differences were noted to have arisen after 4 weeks of treatment among animals receiving 200 mg/kg bw/day (liver, adrenal, heart and brain weight) and in 1 occasion (heart weight) among females receiving 1000 mg/kg/day when compared to control animals. However, these changes bore no relationship with treatment or were considered to be the result of high control values (heart weight) and therefore not toxicologically significant.
Microscopic examination: There were no microscopic findings noted that were considered to be treatment related. The small number of changes recorded in treated animals were within the range commonly seen in rats of this age and strain.

Discussion:
Formulations in corn oil appeared to be stable for at least 4 hours and homogeneous. Actual concentrations remained within acceptable range of nominal concentrations for this type of formulation.

There were no changes in clinical appearance, body weights, food consumption, opthalmoscopic examination, clinical laboratory investigations, macroscopic examination, organ weights and microscopic examination that were considered to be treatment related.

Effect levels

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects or mortality was observed at the highest dose level of 1000 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this 28-day repeat dose study of Stepan TAB-2, conducted in Wistar rats by the oral gavage route, the results determined a definitive No Observed Effect Level (NOEL) of 1000 mg/kg/day.

Executive summary:

A 28 day repeat dose study was conducted on Stepan TAB-2 in the rat using the oral gavage route. The study was conducted following good laboratory practices, and according to OECD Guideline 407, "Repeated Dose Oral Toxicity - Rodent: 28 or 14 -day study,." and according to EEC Directive 84/449/EEC, Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.7: "Sub-acute Toxicity - Oral." Wistar rats were dosed at 0 (vehicle only), 50, 200, 1000 mg/kg bw/day, using corn oil as vehicle. Standard clinical, body weight, organ weight, food consumption, Ophthalmoscopic examinations, mortality, and pathology examinations were made and recorded. No treatment-related changes or mortalities were detected at any dose level. A definitive No Observed Effect Level (NOEL) of 1000 mg/kg/day was determined for this study..