1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-(C8-18(even numbered) and C18 unsaturated acyl) derivs., hydroxides, inner salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-07-24 to 1990-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 12, 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-18(even numbered) acyl derivs., hydroxides, inner salts

EC Number:

931-296-8

Molecular formula:

not applicable

IUPAC Name:

1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-18(even numbered) acyl derivs., hydroxides, inner salts

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

dihydrogen oxide

Test material form:

solid - liquid: aqueous solution

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CF (SD)BR Sprague Dawley
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: males: 115 - 174 g, females: 97 - 150 g
- Fasting period before study: no
- Housing: individually housing in solid floor macrolone cages of type III with stainless steel lids
- Diet: ad libitum, commercial powdered diet for laboratory animals (Ssniff R 10, Ssniff Spezialdiäten GmbH, 4770 Soest, West Germany); one exception of an overnight fast prior to blood sampling
- Water: ad libitum, tap water
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25°C (except on 13 occasions when temperature was once below 19°C and 6 x above 25 °C)
- Humidity: 30 . 70 %
- Photoperiod: artificial (fluorescent) light, 12 hrs dark / 12 hrs light)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The aqueous test item was further diluted with aqua destillata to achieve the scheduled doses.

VEHICLE
- Justification for use and choice of vehicle (if other than water): used vehicle was water
- Concentration in vehicle:
-- nominal: at dose levels of 0, 250, 500, and 1000 mg/kg bw/day: week 1 and 2: 0 g/500 ml, 12.5 g/500 ml, 25 g/500 ml , 50 g/500 ml
week 13: 0 g/800 ml, 20g/800 ml, 40g/800 ml, 80g/800 ml
-- analytical measured (median values of four/two measurements each): at dose levels of 0, 250, 500, and 1000 mg/kg bw/day: week 1 and 2: 0g/500 ml, 14.7 g/500 ml, 24 g/500 ml, 59.95 g/ml; week 13. 0 g/800 ml, 23 g/800 ml, 39.25 g/800 ml, 87.2 g/800 ml
- Total volume applied: 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (10 ml each for testing laboratory as reference sample and 10 ml for the study sponsor) of each test formulation were taken freshly and under conditions of use (first day and after seven days) in weeks 1, 2, and 13 and were sent to the study sponsor for analysis. The test article concentrations in the test article mixtures as well as the stability over seven days resulted to be good.

Duration of treatment / exposure:

Duration of test: 92 days
Exposure period: 90 days

Frequency of treatment:

5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

based on product, corresponding to 75 mg a.i./kg bw/d

Doses / concentrations 2

Dose / conc.:

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

Remarks:

based on product, corresponding to 150 mg a.i./kg bw/d

Doses / concentrations 3

Dose / conc.:

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

Remarks:

based on product, corresponding to 300 mg a.i./kg bw/d

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The selection of the dose levels was based on the results of a preliminary toxicity study in the rat (HLD Project No. 348-156), see study 61789-40-0_8.6.1_Goldschmidt_1991_OECD 408_14-d dose finding also cited in this IUCLID

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes; All animals were examined twice daily at the beginning and end of the working day for morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
- Observation of ill health or overt signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly during the treatment period, and on the day of necropsy or the day before

FOOD CONSUMPTION: yes
- Food consumption of the males and females was recorded twice weekly during the treatment period
- Food consumption was calculated as mean daily food consumption per measuring period

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals of group 1 (0 mg/kg/bw) and 4 (1000 mg/kg/bw) were examined once predose and during final week of treatment. Group 2 (250 mg/kg/bw) and 3 (500 mg/kg/bw) were additionally examined during the final week of treatment
- Dose groups that were examined: all dose groups
- Parameters examined: ocular fundus with macula lutea, papilla, and ocular vessels; before the examination, mydriatic agent (Mydriaticum "Roche"@,Hoffmann-La Roche Aktiengesellschaft, 7889 Grenzach-Wyhlen 1, Germany) was instilled into the eyes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during the final week of treatment (week 13)
- Anaesthetic used for blood collection: Yes; Blood samples were collected by orbital sinus puncture under light ether anesthesia
(Diethyl ether, Asid Bonz & Sohn GmbH, 7030 Böblingen, Hoechst Aktiengesellschaft, Germany)
- Animals fasted: Yes, overnight fast (about sixteen hours)
- How many animals: all animals
- Parameters examined: hemoglobin concentration (Hb), mean cell volume (MCV), red blood cell count (RBC) and derived indices: mean cell hemoglobin (MCH),
packed cell volume (PCV), and mean cell hemoglobin concentration (MCHC), prothrombin time, total and differential white blood cell count (WBC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during the final week of treatment (week 13)
- Animals fasted: Yes, overnight fast (about sixteen hours)
- How many animals: all animals
- Parameters examined: glutamate oxalacetate transaminase (GOT, AST), glutamate pyruvate transaminase (GPT, ALT), alkaline phosphatase (AP), sodium (Na+),potassium (k+), chloride (Cl-), total protein (TP), blood urea (BU), albumin, albumin/globulin ratio (A/G ratio), glucose, cholesterol, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: during the final week of treatment (week 13)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, Urine samples were collected by placing the animals in metabolism cages at 14.00 hours or 15.00 hours without access to food or water. All urine passed between 14.00 hours and 06.00 hours or between 15.00 hours and 07.00 hours the following morning, respectively, were then collected.
- Parameters examined: measured: ph, volume, specific gravity; semiquantitative estimations of protein, blood, glucose, ketones, bilirubin, urobilinogen, reducing substances, colour, microscopy of centrifuged deposits

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, A full external and internal examination was made and all lesions were recorded. The necropsies were conducted over three days. As far as possible, equal numbers of male and female animals were killed on each day.
- Parameter examined: Individual organ weights; organ /body weight ratio (%), individual animal pathology
- The following organs were weighed before fixation: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroids (with para- thyroids), paired organs were weighed separately.

HISTOPATHOLOGY: Yes
- Samples of the following tissues (with exception of the eyes which were fixed in Davidson's fluid) were preserved in 10 per cent neutral buffered formalin:: adrenals, aorta (arch and anterior abdominal), bone marrow, brain (cerebral, cortex, thalamus, midbrain, medulla, cerebellum), cecum, colon, duodenum, epididymides, esophagus, eyes (and optic nerve), heart, ileum, jejunum, kidneys, liver, lungs (with mainstem bronchi), lymph nodes (mandibular and mesenteric), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submaxillary), sciatic nerve, seminal vesicle, skeletal muscle, skin and mammary gland, spinal cord, spleen, sternum, stomach, testes, thymus, thyroids (with parathyroids), tongue, trachea, urinary bladder, uterus, all unusual lesions (according to OECD guideline 408)
- Samples examined: The above tissues from all animals in groups 1 and 4 were embedded in paraffin wax, sectioned at a nominal thickness of 5 µm, stained with hematoxylin and eosin, and were examined by the study pathologist. Due to treatment-related histopathological changes seen in high dose animals, stomach tissue of group 2 and 3 animals had been additionally examined microscopically.

Statistics:

For body weight, body weight gain, and food consumption, the Levene's test for homogeneity of variance was performed followed by one-way Analysis of Variance.
In case of significant results for the ANOVA (p < 0.01 and p < 0.05), the Dunnett's test for multiple group comparisons (p < 0.05) was performed.
For organ weights, the Analysis of Variance was performed with one factor TREATMENT followed by the Student-Newman-Keuls test.
For clinical chemistry data, hematology data, and organ/body weight ratio, the Analysis of Variance was performed with one factor TREATMENT - based on taking the ranks of the variables - and followed by the Student-Newman-Keuls test for multiple group comparisons.
The statistical evaluation was performed with the standard software package SAS (Statistical Analysis System) release 6.03 excluding the analysis for body weight, body weight gain, and food consumption which was performed with the statistical package of the on-line data collection system TERASYS.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical changes observed in any animal throughout the experimental period which would be related to treatment with the test article. On a few occasions, wounds, cuts, and scratches, nose bleed, crusted nose, and nasal discharge were observed in single animals. The findings are considered to be not treatment-related.

Mortality:

no mortality observed

Description (incidence):

- males: One male control animal and one group 3 (intermediate dose group) male died accidentally on days 29 and 19 respectively. These unscheduled mortalities were considered to be incidental and due to experimental procedure.
- female: One female group 3 animal (intermediate dose group) died accidentally on day 26 and three high dose females died accidentally on days 26, 41, and 90 of study. These unscheduled mortalities were considered to be incidental and due to experimental procedures.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Body weight gain (day 1 to 91) was considered normal for male and female animals of all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- There was no treatment-related adverse effect on overall food consumption (weeks 1 to 13) for test item treated males and females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- There were no treatment-related ocular findings.

Haematological findings:

no effects observed

Description (incidence and severity):

-There were no treatment-related hematological changes in animals of either sex.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- Although statistical analysis revealed a few significantly different values between the tested groups, there were no treatment-related changes in clinical chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

- Although there was an increased incidence of positive reactions for hemoglobin concentration in the urine of high dose males exclusively, these findings are considered to be of minor biological significance since these results could not be confirmed by microscopic urine analysis.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- Examination of absolute and relative organ weights including weights of ovaries and testes did not reveal any treatment-related changes in male and female animals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Macroscopic necropsy findings revealed some stomach ulcer at fundus and cardia region in one high dose male and female each.
There were no other treatment-related macroscopic necropsy findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Forestomach: Forestomach gastritis was seen in high and mid dose males and females. From the samples examined the foresomach gastritis was chronic and diffuse in affected animals and was characterised by squamous hyperplasia, with submucosal oedema and inflammatory cell infiltration in some animals. The severity of the forestomach gastritis was judged by the pathologist as minimal to moderate.
-- high dose group: Forestomach gastritis was present in six high dose group males, and three high dose females.
-- mid dose group: Forestomach gastritis was present in two intermediate dose group males and two intermediate dose group females.
-- low dose group: Forestomach gastritis was not present in the stomachs of low dose group animals.
- All other examined organs including epididymides, testes, prostate, seminal vesicle, ovaries, mammary gland and uterus: There was no evidence of any systemic toxicity due to test article administration in any of the other organs examined.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOEL

Remarks:

systemic effects

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no systemic effects

2

Dose descriptor:

LOEL

Remarks:

local effects

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: local irritative effects at the side of application (forestomach gastritis), judged as not relevant to humans due to significant different anatomic situation and exposure probability in humans

3

Dose descriptor:

NOEL

Remarks:

local effects

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: local irritative effects at the side of application (forestomach gastritis), judged as not relevant to humans due to significant different anatomic situation and exposure probability in humans

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Up to and including the highest tested dose of 300 mg a.i./kg bw/d ( = 1000 mg product (a.i. ca. 30 %)/kg bw/d, C8-18 AAPB was tolerated with no evidence of any systemic toxicity in this 90 day rat gavage study.
The only intolerance seen at the mid and high dose level was a local irritative effects at the side of application (forestomach gastritis). This substance related finding is judged as not relevant in view of a potential serious health risk for humans due to significant different anatomic situation and exposure probability in humans. Therefore the NOEL relevant to human DNEL calculation is the is the NOEL for systemic effects which is the highest tested dose of 300 mg a.i./kg bw/d ( = 1000 mg product (a.i. ca. 30 %)/kg bw/d.

Executive summary:

In a subchronic toxicity study according OECD guideline 408 (1981), C8 -18 AAPB (30.3 % a.i. ) was administered to 10 male and 10 female Sprague-Dawley rats per dose by gavage at dose levels of 0, 250, 500, 1000 mg/kg bw/day (corresponding to ca. 75, 150, and 300 mg active ingredient/kg bw) for 90 days. The aqueous test item was further diluted with aqua destillata to achieve the scheduled doses. Concentrations in test formulations were analytically verified. The substance was tolerated without any systemic effects. Up to and including the highest dose tested of 1000 mg/kg bw, there were no compound related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights including weights of ovaries and testes, systemic organ pathology and histopathology including inspection of epididymides, testes, prostate, seminal vesicle, ovaries, mammary gland and uterus. The only treatment related effect seen in this study was a local inflammatory response at the site of application (forestomach gastritis) most probably caused by an irritant effect of the test item. These appeared in gross pathology findings in form of some stomach ulcer at fundus and cardia region in one male and one female rat at 1000 mg/kg bw/day, and in microscopic findings in form of squamous hyperplasia, submucosal edema, inflammatory cell filtration at a dose level of >= 500 mg/kg bw/day (2/10 male and 2/10 female rats at a dose level of 500 mg/kg bw, and at 1000 mg/kg bw in 6/10 males and 3/10 females). The severity of the forestomach gastritis was judged by the pathologist as minimal to moderate. Forestomach gastritis is a common finding in rat gavage studies on irritative test items. This treatment related finding is generally forced by the gavage exposure regime with constantly repeated bolus ingestion and missing when the test items are applicated via food or the drinking water. The reversibility of AAPB induced rat forestomach gastritis and its missing when the test item is applicated via food have been proven in a subacute gavage study with recovery group (see study 61789-40-0_8.6.1_Henkel_1991_OECD 407) and in a subchronic food study (see study 61789-40-0_8.6.2_90days_Unilever_A03_FT890785, both studies also reported in this IUCLID), respectively. A forestomach or a functional correlate to the rat forestomach is missing in humans.The irritative rat forestomach gastritis is judged as not relevant in view of a potential serious health risk for humans due to significant different anatomic situation and exposure probability in humans. Therefore, the NOEL derived from this study relevant to human DNEL calculation is the NOEL for systemic effects which is the highest tested dose of 300 mg a.i./kg bw/d ( = 1000 mg product (a.i. ca. 30 %)/kg bw/d. The LOEL local effects (500 mg/kg bw/day, corresponding to ca. 150 mg active ingredient/kg bw), based on local irritative effects at the site of application (forestomach gastritis), is judged as not relevant to humans due to significant different anatomic situation and exposure probability in humans.