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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study. Guideline not explicitly stated in report, urinalysis not performed, no detailed clinical observations of the animals.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Guideline not stated, urinalysis not performed, no detailed clinical observations of the animals
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: aqueous formulation
Details on test material:
- Name of test material (as cited in study report): Kokosalkyldimethylbetain (Betaines, coco alkyldimethyl)
- Physical state: aqueous, clear, slightly yellow liquid
- Analytical purity: 29-33 % active substance
- Composition of test material, percentage of components: 7 % NaCl, 60-64 % water
- Lot/batch No.: 14/0, of 28 June 1990
- Storage condition of test material: room temperature
- Other: density was considered for calculation of doses

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld
- Age at study initiation: approx. 5 weeks (4 weeks at arrival)
- Weight at study initiation: males 51-65 g, females 52-72 g (at arrival)
- Fasting period before study: no data
- Housing: 2-3/cage
- Diet (e.g. ad libitum): pelleted, sterilized, nitrosamine-reduced maintenance diet Altromin 1324 DK, Altromin GmbH, Lage, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25
- Humidity (%): 36-58
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Daily preparation, shortly before application.


VEHICLE
- Concentration in vehicle: 12.5, 25.0, 50.0 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw

Application volumes were adjusted weekly to compensate for changing body weights. Therefore, doses were kept constant over the whole experimental period.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days (68 respective 69 applications, dependent on necropsy date)
Frequency of treatment:
once daily, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 125, 250, 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: according to already established toxicity data; doses were chosen to include a safety margin, determine a not cumulative-toxic dose, the toxicity range and reversibility of eventually occuring cumulative toxic effects.
- Rationale for selecting satellite groups: Satellite groups of 5 animals/sex/dose for the control and the high dose groups were included to investigate reversibility of possible compound-related effects.
- Post-exposure recovery period in satellite groups: 35 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during acclimatisation and twice daily during application period.
- Cage side observations included: mortality, symptoms of intoxication.


DETAILED CLINICAL OBSERVATIONS: No.


BODY WEIGHT: Yes, body weight and weight gain.
- Time schedule for examinations: at arrival, once during acclimatisation, weekly during application period.


FOOD CONSUMPTION: yes, weekly, groupwise, except for dates of clinical investigations.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, mean food consumption was calculated for males and females of each dose group as g/rat/week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.


WATER CONSUMPTION: Yes, mean water consumption was calculated as ml/rat/week for males and females of each dose group.
- Time schedule for examinations: weekly, groupwise, except for dates of clinical investigations.


OPHTHALMOSCOPIC EXAMINATION: Yes, with split lamp after application of Mydriaticum Roche R (Hoffmann-La Roche, Grenzach-Wyhlen).
- Time schedule for examinations: at the day before necropsy.
- Dose groups that were examined: control and high dose group.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 6 weeks of application (interim examination) and at the end of the study.
- Anaesthetic used for blood collection: yes, not further specified
- Animals fasted: No data
- How many animals: all animals, from retroorbital plexus, in EDTA-coated tubes.
- Parameters examined: red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), hemoglobin (HGB), leukocyte count (WBC), platelet count (PLT), differential blood count: band leukocytes (Bands), segmented leukocytes (Seg), lymphocytes (Lymph), eosinophils (Eo), monocytes (Mono), basophils (Baso), myelocites (My), juveniles (Ju), diverse (Div)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 6 weeks of application (interim examination) and at the end of the study.
- Animals fasted: No data
- How many animals: all animals, from retroorbital plexus, in non-coated tubes; serum was obtained from twice centrifuged blood samples
- Parameters examined: urea, creatinin (CREA), sodium (Na), potassium (K), glucose, calcium (CALC), alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), bilirubin (BILIR), chloride (CHLO), protein (PROT), cholesterol (CHOL)


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, animals were sacrificed by an overdose of ether and cardially exsanguinated after opening of the chest. Subsequently the corpse was examined, the eyes were removed (fixation according to Davidson), the hypodermis prepared, the abdomen, chest and the cranial cavity opened, the corpse was examined. Organs were examined in situ and after removal, and partially they were directly fixed in 10 % neutralised formalin (Formol-Fixx-Concentrate) after weighing. Absolute and relative weights were determined for the following organs: brain, testes, heart, liver, spleen, adrenals, kidneys, thymus. Animals of the satellite groups were treated in the same way after 35 treatment-free days.
HISTOPATHOLOGY: Yes, after fixation histological slices of the organs were prepared and stained with Hematoxylin/Eosin. The following organs were histologically examined: Aorta thoracica, eye, colon, glandular stomach, intestine, brain, urinary bladder, skin, heart, testes, hypophysis, cerebellum, liver, trachea, lung, maxillary lymph node, mesenteric lymph node, spleen, epididymides, adrenals, peripheral nerves, kidney, ovary, pancreas, prostate, seminal vesicle, thyroid, salivary gland, esophagus, skeletal muscles, thymus, uterus, forestomach, tongue.
Statistics:
Significant differences of body weights among the experimental groups were determined by the t-test according to L. Sachs, Statistische Auswertungsmethoden, 3rd ed., p.11, 212, Springer Publishers, Berlin, 1971. Significant differences of clinico-chemical and hematological parameters were determined according to Dunnett's t-test. The Steel-test was used for determination of significant differences of organ weights among the experimental groups.

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 examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
not adverse
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
not adverse
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
local effects
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
local effects
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no substance-related clinical signs in the exposed groups.

One female of the high dose group demonstrated emaciation, hunched posture, reduced activity and laboured breathing due to gavage dosing (chronic trauma of esophagus, confirmed by histopathology). One female of the mid and the low dose group each showed non-substance-related alopecia.
There was no substance-related mortality. Two males and two females of the high dose group were killed or died due to non-substance-related reasons (due to misapplication or harmed during sampling).

BODY WEIGHT AND WEIGHT GAIN
Males of the high dose group showed a slight reduction of mean body weights from week 10 until end of the study. Low dose females demonstrated a slight reduction of mean body weights at the beginning of the study and in weeks 1 and 6, high dose females showed a slight reduction only in week 1. The body weights of all other animals were always comparable with those of the control groups.

FOOD CONSUMPTION
Mean food consumption of the treated groups was comparable with that of the control groups. Some variations were observed which were not treatment-related.

WATER CONSUMPTION
Mean water consumption of the treated males was increased in a dose-related fashion. The high dose females demonstrated an increased water consumption, as well. Mean water consumption of the low and mid dose females was comparable to the control group.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related changes of the eyes in the high dose group, compared to the control group. Animals of the low and mid dose group were not investigated.

HAEMATOLOGY
At interim examination males of the mid dose group demonstrated a slight increase of leukocytes, at the final examination the high dose females showed slightly increased segmented leukocytes.

CLINICAL CHEMISTRY
At interim examination the mid dose males demonstrated slightly increased urea levels.

ORGAN WEIGHTS
High dose males showed reduced absolute gonad and brain weights, high dose females showed reduced absolute kidney weights. Mid dose males demonstrated slightly increased relative heart weights, and mid dose females demonstrated slightly reduced relative kidney weights. No other changes in organ weights were observed, and the observed differences were not considered to be substance-related.

GROSS PATHOLOGY
The only substance-related findings were white centres and/or edema in the mucosa of the forestomach in 5/8 males and 5/8 females of the high dose group. After 35 treatment-free days the high dose animals were free of substance-related pathological changes. A red centre was found at the bottom of the stomach in one female. All other findings were considered to occur spontaneously and not to be treatment-related (discolouration of the thymus, enlargement of mandibular lymph nodes and accessory spleen, and accumulation of fluid in the uterus).

HISTOPATHOLOGY: NON-NEOPLASTIC
The only substance-related findings were irritative changes in the forestomach of the high dose group, demonstrated by papillomatous hyperplasia of the mucosa, high-grade inflammation and ulcer formation. 3 males and 4 females of the mid dose group demonstrated irritative changes in the forestomach ranging from slight to moderate up to ulcer formation. The forestomachs of the low dose group were without findings except a slight irritation of the mucosa in one animal. Irritation was considered as local effect due to gavage dosing, but the effect was more pronounced in the high dose group in a dose-dependent fashion.
Irritative changes of the forestomach were also observed in 2 males and 3 females of the satellite group after 35 days without treatment.

Other findings included calcification of the Arteria pulmonalis, germinal hyperplasia of mandibular lymph nodes, hydronephrosis, accumulation of fluid in the uteri, hyperplasia of the mamma. Occasionally periorbital edema of various degrees due to retroorbital blood sampling was observed. Germinal hyperplasia of various degrees was regularly observed in animals of all groups. Histiocytic focuses with and without phagocytic activity were observed in the liver in nearly all groups. These findings were considered to be related to a spontaneously occurring bacterial infection of unknown etiology.

The high dose female which had died in narcosis demonstrated high-grade hyperemia of the lung, the male and the female which had died due to application mistakes both demonstrated lesions in the forestomach, and the exophtalmus was confirmed in the male that had to be killed after retroorbital blood sampling by the finding of high-grade periorbital inflammation.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Local irritative effects in the forestomach
Dose descriptor:
LOEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: water consumption (systemic effect)
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: water consumption (systemic effect)
Dose descriptor:
NOAEL
Effect level:
>= 145 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: water consumption (systemic effect)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Conclusion:

The NOEL after subchronic treatment with the test substance was considered to be 250 mg/kg bw/d. At this dose level only local irritative effects related to gavage dosing were observed in the forestomachs of the animals.

At 500 mg/kg bw/d, which was considered as LOEL, additionally a slight systemic effect in the form of increased water consumption occurred. The local irritative effect observed in the mucosa of the forestomach was increased in a dose-related fashion compared to the mid dose group.

There were no adverse effects on reproductive organs reported.

Applicant's summary and conclusion