1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-(C12-14(even numbered) acyl) derivs., hydroxides, inner salts

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

assessment of reproduction organs during Repeated Dose 90-Day Oral Toxicity in Rodents

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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) and C18 unsaturated acyl) derivs., hydroxides, inner salts

EC Number:

931-333-8

Cas Number:

147170-44-3

Molecular formula:

not applicable

IUPAC Name:

1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-(C8-18(even numbered) and C18 unsaturated 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:

water

Test material form:

solid - liquid: aqueous solution

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Colworth Wistar
- Age at study initiation: 4-6 weeks
- Weight at study initiation (g): males: 132.9 - 138.3, females 101.7 to 104.5 (group mean values)
- Fasting period before study:
- Housing: singly housed in stainless steel or galvanised cages with stainless steel mesh floors awl doors, such that there was one animal per cage; 48 (four rows x six columns) cages were accommodated on each holding battery.
- Diet: ad libitum, MODAIN purified diet
- Water: e.g. ad libitum, potable tap water from the local water mains
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-2
- Humidity (%): 55 +/-10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: MODAIN purified diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): most probably every other week (7 batches in the course of the 13 week study were prepared)
- Mixing appropriate amounts with (Type of food):
-- The formulation of the MODAIN purified diet (%) was Maize starch 65.0 %, Casein (spray dried) 20.0%, Solkafloc 5.0 %, Maize oil 5.0%, Modified AIN-76 minerals 3.5 %, AIN-76A vitamins 1.0 %, DL methionine 0.3 %, Choline bitartrate 0.2 %.
-- The test item was added to the diet at the expense of maize starch for the 0.40% and 1.00% dietary levels. The 1.00% diet was used to prepare the 0.10% dietary level by a 1:10 dilution with control MODAIN purified diet. Similarly the 0.40% diet was used to prepare the 0.04% dietary level by a 1:10 dilution with control MODAIN purified diet
- Storage temperature of food: cold

Details on mating procedure:

animals were not mated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diets prepared for feeding at the beginning (Batch 1), middle (Batch 4) and end of the study (Batch 7) were sampled and analysed for test item to confirm dietary concentration, and for moisture, protein, fat, fibre and ash to confirm composition. Control MODAIN purified diet is routinely analysed for nutrients and contaminants. For confirmation of dietary concentration, the test item was extracted from the diet samples by sochlet extraction with a methanol: ammonia (100:1) mixture. The extracts were concentrated and analysed by HPLC on a 3µ Nucleosil C18 column using a mobile phase of methanol: water: ammonia (90:10:1) at a flow rate of 0.8 ml/min with detection by a light scattering detector. Quantitation was achieved by comparison of sample peak heights with those of external standards in the range 0.3 - 1.5 mg/mL.
Homogeneity and stability of the test substance within the diets were determined on a 5 kg batch of each diet containing (A) 1.00%, (B) 0.40% and (C) 0.10% test item (the 0.04% dietary level would be covered by the fact that it was prepared by a 1:10 dilution of the 0.40% dietary level with control MODAIN purified diet). Each batch was sampled at 5 positions in the mixing bowl as follows: A top centre, B middle centre, C bottom centre, D left centre and E right centre. Duplicate analyses were completed on each sample after it was prepared. The remainder of each batch of diet was divided into 2 parts, which were stored in an animal room (21 ± 2°C) or a cold room (4°C). Samples were taken for analysis after 7 and 14 days storage. Recovery tests were also carried out on diets spiked with the test item at levels covering the range of incorporation of the test item in the diet.

Duration of treatment / exposure:

mains study: 13 weeks
recovery period: further 32 days

Frequency of treatment:

daily

Details on study schedule:

animals were not mated

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.00%, 0.04%, 0.10%, 0.40% and 1.00%
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
9.5, 24, 97 and 247 mg a.i./kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

main study groups: 12 males and 12 females per group
additional recovery groups: 5 males and 5 females (control and high dose group)

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked at least twice each day (once on Saturdays and Sundays) for signs of ill-health and reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals vere given a detailed examination twice a week.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day that treatment commenced and then at weekly intervals for the duration of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
-- Food intake was recorded twice weekly for each animal throughout the study and weekly intakes were calculated.

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was recorded twice weekly for each animal throughout the study and weekly intakes were calculated.

OTHER:
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at start and prior to the completion of the 13 week treatment
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the completion of the 13 weeks treatment feeding period and at the end of the recovery period
- Anaesthetic used for blood collection: Yes, Halothane anaesthesia
- Animals fasted: No
- How many animals: All surviving rats at the completion of the 13 weeks treatment feeding period and at the end of the recovery period.
- Parameters examined: Haemoglobin (Hb), Red cell count (RBC), Platelets (PLT), Reticulocytes (RET), White cell count (WBC), Lymphocytes (LYM), Neutrophils (NEU), Monocytes (MON), Eosinophils (EOS), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Several indices are derived from these measurements: MCV - mean cell volume (mean of red blood cell volume histogram - femtolitres), HCT - haematocrit (red blood cell count times the MCV - litre/litre), MCH - mean cell haemoglobIn (haemoglobin divided by the red blood cell count picograms), MCHC - mean cell haemoglobin concentration (haemoglobin divided by the HCT grams/100 ml red cells), the following blood coagulation factors were measured: Prothrombin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the completion of the 13 weeks treatment feeding period and at the end of the recovery period
- Animals fasted: No
- How many animals: All surviving rats at the completion of the 13 weeks treatment feeding period and at the end of the recovery period.
- Parameters examined: in plasma: Sodium, Potassium, Chloride, Calcium, Inorganic phosphate, Magnesium, Aspartate transaminase, Alanine transaminase, Lactate dehydrogenase, 1-Hydroxybutyrate dehydrogenase, Creatine kinase, Alkaline phosphatase (pH 9.8), Pseudocholinesterase, 5-Nucleotidase, Triglyceride, Total cholesterol, Urea, Glucose, Creatinine; in serum: Total protein, Albumin; Serum electrophoresis separation yas performed on cellulose acetate plates followed by staining with Ponceau S and quantification of the fractions using the Profil Ecran scanning densitometer were performed for: Albumin Alpha-1-globulin, Alpha-2-globulin, Beta globulin, Gamma globulin, Albumin/globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected from a selection of the 13 week rats (eight male and eight female rats per group) and the recovery rats prior to treatment, after 32 and 88 days of treatment, and after 13 weeks treatment plus 32 days recovery time for recovery rats only.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined analytically: Urine volume, Refractive index, Sodium, Potassium, Chloride, Calcium, Inorganic phosphate, Magnesium, Urea, Creatinine N-acetyl-beta-D-glucosaminidase, Gamma glutamyl transferase, Alanine aminopeptidase
- Parameters examined semi-quantitative using dip-stick strips: leukocytes, nitrite, glucose, protein, erythrocytes and haemoglobin

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

not applicable

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
- All the animals which survived to the completion of the 13 weeks treatment feeding period and at the end of the recovery period received a detailed
necropsy. All macroscopic abnormalities were recorded as well as an assessment of the level of intra-abdominal fat deposition.
- Brain, heart, liver, kidneys, spleen, testes, caecum, empty caecum and adrenal glands were weighed prior to fixation. The ratio of organ weight to 100 g of body weight at necropsy was calculated in each case and recorded as relative organ weight.
-- The following tissues were taken from each rat and preserved in 10% buffered formalin: Adrenal glands, Brain, Colon, Heart, Kidneys, Lungs, Mammary glands, Ovaries and fallopian tubes, Pituitary, Sciatic nerve, Sternum, Thyroid and parathyroid, Uterus, Aorta, Caecum, Duodenum, Ileum, Larynx Lymph nodes (cervical and mesenteric), Muscle, Prostate, Spinal cord, Stomach, Tongue, Bladder, Cervix, Head, Jejunum, Liver, Oesophagus, Pancreas, Rectum, Spleen, Thymus, Trachea
-- The following tissues were taken from each rat and preserved in Bouin's fixative: Epididymides, Seminal vesicles, Vagina, Femur and stifle joint, Skin, Sali vary glands, Testes
-- The eyes and harderian glands were taken from each rat and fixed in Davidson's fluid.

HISTOPATHOLOGY: Yes
- Tissues were processed by conventional histological methods into paraffin wax and sections 4µm (nominal) in thickness were prepared and stained with haematoxylin and eosin for histological examination. Additional portions of liver were fixed in 10% formol saline for the preparation of cryostat sections that were subsequently stained with Oil Red 0 to demonstrate neutral fat.
- Full histological examination was confined to rats from the high dose group (1.00% test item) and control group.

Postmortem examinations (offspring):

not applicable

Statistics:

- For body weights, food intakes, food efficiencies and water intakes, haematology, clinical chemistry and organ weights (including the ratio of organ weight to body weight at terminal kill), a statistical analysis was conducted using Student's t-test, F-test and Duncan's Multiple range test.
- A t-test versus control was used to show any significant differences between control group and any of the other treatment levels at the 5%, 1% and 0.1% probability levels. In conjunction with the F-ratio test in the analysis of variance, a Duncan's multiple range test at the 5% protection level was included to allow all pair-wise comparisons amongst the treatment levels to be carried out.

Reproductive indices:

not applicable

Offspring viability indices:

not applicable

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

no toxic symptoms, signs of distress or decrease in activity

Mortality:

no mortality observed

Description (incidence):

All rats survived to the end of the 13 week feeding period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Statistically significant lower body weights and body weight gains were observed over the 13 weeks of treatment for only the female rats fed 0.10%
test item when compared with controls. These were both considered to be marginal effects and were not dose related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- No statistically significant differences in food intakes were observed over the 13 weeks of treatment for either male or female rats fed the test item when compared with controls.
- the dietary dose levels compared to the following mean daily ingestion levels based on product and based on the a.i. of 33.8 %
-- 0.04 %: 28 mg test item/kg body weight/day; 9.5 mg a.i./kg bw/day
-- 0.10 %: 71 mg test item/kg body weight/day; 24 mg a.i./kg bw/day
-- 0.40 %: 288 mg test item/kg body weight/day; 97 mg a.i./kg bw/day
-- 1.00 %: 731 mg test item/kg body weight/day; 247 mg a.i./kg bw/day

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Main study groups:
- There was no evidence of an adverse effect of treatment in any of the tissues examined including inspection of seminal vesicles, prostate, epididymides, testes, mammary glands, ovaries and fallopian tubes, uterus, cervix and vagina.
.
-- Caecum: There was no evidence of an effect of treatment on the morphology of the caecum.
-- Liver: There were no findings to explain decreased liver weight in the rats fed 1.00% test item.
-- Incidental findings: A variety of incidental findings was recorded in rats from all groups with no evidence of an effect of treatment on their character, incidence or distribution. The findings are consistent with the normal spectrum of spontaneous lesions encountered in young laboratory rats.
Recovery groups:
- Since there was no evidence of an adverse effect of treatment in any of the tissues examined, no histological evaluation was undertaken for the recovery rats.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Details on results (P0)

Further details are given in the IUCLID section "Repeated dose toxicity".

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

not applicable

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The results from the evaluation of reproductive organs, especially organ weights of ovary and testis and histopathology of gonads from this 90 day rat feeding study with 38 day recovery revealed no indications of any substance-related effects up to and including the highest test dose of 1% in diet, corresponding to 247 mg a.i./kg bw/day.

Executive summary:

In a subchronic toxicity study according OECD guideline 408, Coco AAPB (a.i. 33.8%) was administered to 12 male and 12 female Colworth Wistar rats per dose via food at dose levels of 0.00%, 0.04%, 0.10%, 0.40% and 1.00% (corresponding to 0, 28, 71, 288 and 731 mg product/kg bw (9.5, 24, 97 and 247 mg a.i./kg bw/day)) for 90 days. Additional animals in satellite groups (control and high dose, 5 males and 5 females, each) were kept for further 32 days without treatment to detect recovery from, or persistence of, toxic effects. Concentrations in diet formulations were analytically verified and substance intake was calculated from recorded food consumption.

The substance was tolerated without any systemic effects relevant in view of an potential serious health risk for humans. Up to and including the highest dose tested of 1% in feed (corresponding to 731 mg product/ kg bw/d and 247 mg a.i./kg bw/day), there were no dose related effects on mortality, clinical signs, body weight, food consumption, water consumption, haematology, urinalysis and histopathology including inspection of seminal vesicles, prostate, epididymides, testes, mammary glands, ovaries and fallopian tubes, uterus, cervix and vagina.

The only treatment related effects ascertainable at termination of treatment but not after the recovery period were reduced food conversion efficiencies and organ weight changes in the caecum and liver. The enlarged caecum observed at necropsy in some male and female rats fed 0.40% and 1.00% Coco AAPB was reflected in the statistically significant increases recorded in the absolute and relative, full and empty caecal weights for both male and female rats fed 1.00% Coco AAPB. These animals fed 1.00% Coco AAPB also showed reduced absolute and relative liver weights, reduced abdominal fat depots corresponding to the reduced food efficiency and several possibly associated plasma and serum biochemical changes. There was no histopathological correlate for the organ weight changes in caecum and liver. All alterations were completely reversible in the recovery group after 32 days without treatment.

Enlargement of the rat caecum with or without subsequent effects on caecum weight, food conversion and nutritional status is a common and frequently response to feeding poorly-absorbable or osmotically-active substances, such as xylitol, sorbitol, sucralose or natural sugars like d-ribose, general changes in nutritional diet composition or application of compounds with effects on the caecal microflora. In the absence of histopathological changes, the rat caecum alterations are taken as physiological adaptive responses and considered to be of no toxicological significance. An increase in liver weight without any histopathological correlate is commonly not considered to reflect an adverse effect but should be considered as an adaptive metabolic response which in known to be reversible. As also in this study, the increase in liver weight was without any histopathological correlate and has been proved to be reversible, the liver weight alteration is not considered to be an adverse effect relevant in view of an potential serious health risk for humans.

Therefore, the NOEL derived from this study relevant in view of a potential serious health risk for humans is the highest tested dose of 1% in feed (corresponding to 731 mg product/ kg bw/d and 247 mg a.i./kg bw/day).