Aluminium triformate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 November 1009 to .....

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP- and guideline compliant

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium triformate

EC Number:

230-898-1

EC Name:

Aluminium triformate

Cas Number:

7360-53-4

Molecular formula:

CH2O2.1/3Al

IUPAC Name:

aluminium triformate

Details on test material:

- Name of test material (as cited in study report): Aluminium Triformate
- Trade name: Altriform CFD, Novaltan Al
- Hill formula: Al (HCOO)3.3H2O
- Physical state: White powder
- Composition of test material, percentage of components: 24.8 % Aluminiumoxide, 62 % formic acid
- Purity test date: 24 June 2009
- Lot/batch No.: 128110/004
- Expiration date of the lot/batch: not stated
- Stability under test conditions:
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

other: Crl: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: About 8-9 weeks
- Weight at study initiation (first administration of the test substance: mean body weights between 378 and 384 g (males) and between 215 and 221 g (females)
- Fasting period before study: none
- Housing:
Dose Range Finding Study:
Makrolon cages Type IV (33 cm x 55 cm area, 20 cm height).
5-fold caging (animals of one group and one sex per cage).
Sanitation of cages once a week.

Main Study:
Single caging (in general, except for the mating period for and for dams with offspring).
Double caging (for the mating period, 1 male + 1 female per cage).
Group caging (1 dam plus her offspring per cage, for the post-partum period).
Makrolon cages Type III, high version (39 cm x 23 cm ground area, 18 cm height).
Sanitation of cages once a week.

- Diet (e.g. ad libitum):
Ssniff R/M-H maintenance diet for rats and mice (item V1534-3 ) ad libitum, supplied by Ssniff Spezialdiäten GmbH, 59494 Soest, Germany. Exception: Feed was withdrawn on days prior to blood sampling at 5:00 p.m., only from the animals, where blood was to be taken, and was re-offered immediately after the blood sampling.
Random samples of the feed are analysed for contaminants by the supplier. One sample is analysed also for contaminants in addition by an independent external laboratory. The limits of tolerance are derived from the "Deutsche Futtermittelverordnung" (German feed regulation).

- Water (e.g. ad libitum):
Tap water, acidified with HCl to pH >=3, from an automatic watering system, ad libitum. Random samples of the water are analysed by the "AGES", 1226 Vienna, Austria, to check, if the water fulfils the requirements for drinking water for humans (exception: the pH).

- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: Mean of temperature maxima: 21.0 °C. Mean of temperature minima: 20.9 °C.
- Humidity: Mean of humidity maxima: 47.2 %. Mean of humidity minima: 46.8 %.
- Air changes (per hr): 12
- Photoperiod: 12 (hrs dark / 12 hrs light)

IN-LIFE DATES:
Dose Range FInding Study: From: 13 November 2009 To: 20 November 2009
Main Study: From: 23 November 2009 To: 15 January 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.1 % aqueous solution of Na-carboxymethylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered after being suspended in the vehicle (0.1 % aqueous solution of Na-carboxymethylcellulose: Water pro analysis, Merck item No. 1.16754.5000; Na-carboxymethylcellulose: "CMC", high viscosity, item No. C-5013, Lot. No. 98H0328, Sigma).

Preparations of the test substance were made freshly every day shortly before the administration to the animals. Appropriate preparations were made to allow a uniform dose volume for all groups.

VEHICLE
- Justification for use and choice of vehicle: The test substance was not sufficiently soluble in water, a suspension in a mucilaginous aqueous vehicle was chosen for the preparation of the test substance.
- Concentration in vehicle: 1, 31.6 and 100 g per L
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Preparation of vehicle: The vehicle was prepared freshly in biweekly intervals, using boiled water and sterilised glassware, and stored in a refrigerator at about +4 °C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determinations of the test substance in some selected preparations for concentration and homogeneity were performed. All details are given in a separate report, which is attached to the report.
Homogeneity: Determined on Day 2.
3 samples of 2.0 mL each of the preparations for groups A, B and C were taken using the syringe plus probe by the technician at the beginning, in the middle and at the end of the dosing procedure for the group concerned.
A deviation of the individual samples from the mean of at most ± 10 % was tolerated.
Actual concentration: Determined on Day 2.
The same samples as for the determination of the homogeneity were used.
A deviation of the mean of the 3 samples from the target concentration of at most ± 10 % was tolerated.

Duration of treatment / exposure:

"Day 1" (of the entire experiment) was the day of the 1st administration of the test substance. Commencement of dosing was at the beginning of the pre-mating period (see 2.6).
"Day 0 of pregnancy" (of the given individual) was the day of proven mating.
"Day 0 post-partum" (of the given individual) was the day of birth (when parturition was complete).
All adult animals were treated with the test substance solutions or with the vehicle once a day from Day 1 onwards until the day prior to their sacrifice (maximum duration: 53 days).

Frequency of treatment:

Once daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 316 and 1000 mg per kg body weight and day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses chosen are derived from and based on the results of the Dose Range Finding Study.
Summarized results of the Dose Range Finding Study: All animals survived until their scheduled sacrifice. In all high dosed animals of both sexes unspecific signs of reduced well-being were noted in the last days of the dosing period. Body weights and - more pronounced - body weight gain of the high dosed males were reduced, when compared with the negative controls. No test substance related abnormalities were noted post mortem. No other indications for test substance related affects were found. None of the alterations observed is considered to constitute a severe toxic effect or to be life threatening.
In the Main Study, the high dose shall induce a clear toxicity, but no or at most isolated mortality. A dose of 1000 mg per kg body weight is commonly not exceeded as high dose. The low dose shall induce no toxic effect. The mid dose is interpolated geometrically.
Based on this, the doses of 100 mg and 316 mg and 1000 mg per kg body weight were selected for the Main Study in accordance with the study monitor.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, plus viability check once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to first test substance administration, then once a week, all adult animals.
Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities (e.g. lacrimation, piloerection, pupillar size, abnormal breathing, and body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations:
Adult animals (except for pregnant females): Determined on Day 1, then once a week, and at termination.
Pregnant females: On Days 0, 7, 14 and 20 of pregnancy and within 24 h after parturition (i.e. Day 0 or 1 post partum) and on Day 4 post partum.
Offspring: The total weight of the litter is determined within 24 h after parturition (i.e. Day 0 or 1 post partum) and on Day 4 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
Determined for weekly periods (except for changes in caging, e.g. at the beginning of mating or at proven mating; forming an interim endpoint), all animals (or per cage during mating period or with offspring).
- Mean daily diet consumption calculated as g food/kg body weight/day: No

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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day -2: The first 5 animals of each group. Day 15: The first 5 animals of each group.
- Anaesthetic used for blood collection: Yes (Isofluorane anaethesia)
- Animals fasted: Yes, overnight
- How many animals: Day -2: The first 5 animals of each group. Day 15: The first 5 animals of each group.
- Parameters examined:
Red blood cell count (RBC)
Haemoglobin concentration (HGB)
Haematocrit (HCT)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
White blood cell count (WBC)
Mean cell volume (MCV)
Platelet count (PLT)
Differential white blood cell count (% of the different cell species)
Prothrombin time (Quick) as an indicator of blood clotting capacity.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day -2: The first 5 animals of each group. Day 15: The first 5 animals of each group.
- Anaesthetic used for blood collection: Yes (Isofluorane anaethesia)
- Animals fasted: Yes, overnight
- How many animals: Day -2: The first 5 animals of each group. Day 15: The first 5 animals of each group.
- Parameters examined:
Alanin aminotransferase (ALT, GPT)
Albumin
Alkaline phosphatase (AP)
Aspartate aminotransferase (AST, GOT)
Bile acids
Cholesterol
Creatinine
Gamma glutamyl transferase (GT, GGT)
Glucose
Potassium (K+)
Sodium (Na+)
Total protein
Urea

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Males: Once in the last week of the dosing period.
Females: Once during the lactation period (i.e. on Days 1-4 post partum.
Offspring: Not examined.
- Dose groups that were examined: all
- Battery of functions tested: Assessment of the behaviour, the motor activities, and the sensory reactivity to different stimuli (acoustic, tactile, visual and proprioceptive) and grip strength.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adult animals were killed by inhalation of 80 % CO2 plus 20 % air and subjected to a necropsy including a gross pathological examination immediately after death on
Males: On the day after the end of the mating period.
Females: On Day 4 post partum or Day 54 (i.e. 25 days after the end of the mating period).

The following organs/tissues (if appropriate) were fixed in 4 % buffered formaldehyde, with the exception of the eyes (fixed in Davidson's fixative) and the skin and the testes (both fixed in Bouin's solution, the testes were punctured on both poles with a needle before immersion in the fixative). Organs/tissues marked with an "*" were not included in routine histopathology.

gross lesions, tissue masses or tumours
adrenal glands
aorta *
brain including cerebrum, cerebellum and pons)
caecum *
coagulating glands
epididymides
eyes
heart
kidneys
lacrimal glands *
large intestine (colon)
liver
lungs
lymph nodes (mandibular, mesenteric)
oesophagus *
ovaries
pancreas *
pituitary gland *
prostate
rectum *
salivary glands*
sciatic nerve
seminal vesicles
skeletal muscle (thigh)
skin, mammary glands *
small intestine (duodenum, ileum, jejunum; including Peyer's patches), prepared as "Swiss Roll"
spinal cord (cervical, thoracal, lumbar)
spleen
sternum with bone marrow
stomach
testes
thymus
thyroid glands
trachea.
urinary bladder
uterus
vagina with cervix

Additional examinations were performed in the females:
Counting of corpora lutea per ovary (visual).
Counting of implantation sites (visual, no staining).

The pups were killed by overdosed chloroform anaesthesia at the same time as their mothers and subjected to an external examination for gross abnormalities. No organs are weighed or preserved in the pups.

ORGAN WEIGHTS: Yes
Fresh weights of the following organs were determined of all adult animals at necropsy:
adrenal glands (both together)
brain
epididymides (both together)
heart
liver
kidneys (both together)
prostate and seminal vesicles with coagulating glands
spleen
testes (both together)
thymus
Relative organ weights were calculated by relating the absolute organ weights to the last determined individual body weight and to the brain weight.

HISTOPATHOLOGY: Yes
Groups K and C:
Histopathological examination was performed in the first 5 adult animals of groups K and C, of all fixed organs and tissues listed above, except those, labelled with a "*".
Groups A and B:
A histopathological examination of the stomachs of the first 5 animals of groups A and B was performed, as there was an indication for test substance related effects found in group C.
The tissue trimming was performed according to "Bahnemann et al.: RITA - Registry of Industrial Toxicology Animal Data - Guides for Organ Sampling and Trimming Procedures in Rats"; Exp.Toxicol.Pathol. 47 (1995), p 247 ff. with the following exceptions:
Not all possible sections, as given in the literature, were actually prepared. One section per organ (in paired organs one of each) was made with the following exceptions:
- Brain (3 sections, one at the optic chiasma, the second at the caudal border of the mammillary body, just posterior to the attachment of the pituitary and the third about 2 mm caudal to the transverse fibres of the pons). Representative regions of the brain, especially cerebrum, cerebellum and pons were included in these sections.
- Spinal cord (three sections, a cervical, a thoracal and a lumbar).
- Liver (two sections).
The small intestine (duodenum, jejunum and ileum) was fixed and trimmed to form two "Swiss Rolls" (one of the cranial and one of the caudal part), according to "Moolenbeek and Ruitenberg: The Swiss Roll, a simple technique for histological studies of rodent intestine"; Lab.Animals 15 (1981), p.57-59.
The trimmed samples of organs or tissues, as described above, were embedded in paraffin. Sections of about 5 µm were stained with haematoxylin and eosin (H&E). Evaluation of slides was performed using a light microscope Leica-DMRB.
To describe the severity of lesions, the following grades were applied, if appropriate:
minimal (1), mild (2), moderate (3), marked (4), severe (5).
The term "focal" together with a higher degree of severity also stands for "multifocal".

Other examinations:

MATING PERFORMANCE:
During the mating period, starting with the day after the commencement, all females were subjected to a daily examination (once in the morning,) for the presence of a vaginal plug. In case of absence of a vaginal plug, the females were subjected also once a day to a vaginal smear. The unstained vaginal smears were examined microscopically for the presence of sperm.
Presence of a vaginal plug or of sperm in the smears was taken as an evidence for successful mating.
The examinations for mating performance ended on an individual basis on the day of proven mating or with the end of the mating period.

Statistics:

Analysis of variance followed by the Scheffé-test: all data with means and standard deviations determined, comparison of more than two groups
t-test: all data with means and standard deviations determined, for comparison of two groups only
H-test of Kruskal and Wallis followed by the test of Nemenyi: counted events with scoring or in cases where the requirements for the analysis of variance were not fulfilled
Chi2-test: counted events
Fisher's exact test: counted events, if the Chi2-Test was not applicable

Results were analysed separately for males and females. P = 0.05 was chosen in each test. Two tailed test were used.
Numerical data have been rounded for presentation; a manual recalculation therefore may yield slightly different results to those given in the tables.
Please note: Whenever the term "significant" is used in this report, it stands for "statistically significant".

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
Animal No. 131 (female, group C) died on Day 3 from gavage injury. The test substance preparation was erroneously administered into the lungs and led to immediate suffocation. A contribution of the test substance itself to the death is not considered to have occurred.
This individual was replaced by a spare animal, given the same animal number.

Animal No. 127 (female, group B) was found dead on Day 12. A fibrinous plug within the laryngeal lumen was noted a necropsy. Local irritation of the larynx after a suspected but unwanted deposition of minute amounts of test substance preparation at gavage withdrawal is considered to be the most likely cause for this alteration. There was a contribution of the test substance to the death, though in an indirect mode.

Animal No. 136 (female, group C) was found to be in a poor health condition after birth and was euthanised on Day 2 post partum for humane reasons. A contribution of the test substance to the condition of this animal is possible.

All other animals survived until their scheduled sacrifice.

OBSERVATIONS IN LIFE
Bloated abdomen together with signs for reduced well-being, as noted in group C, may be taken as indications for a gastrointestinal irritation by the test substance.
Respiratory murmur may be a sign resulting from laryngeal irritation via erroneous contamination with test substance preparation at gavage administration.
Gavage had to be omitted on a single day for transient problems of two high dosed animals involved. Resistance to and problems with gavage insertion may be partly caused by a local irritatative effect of the test substance preparation.
None of the alterations was frequent enough to give a significant and clear indication for a test substance related effect, but all may use as additional hints for possible effects.
Alopecia, as noted in two individuals, and one case of wound/crusts of the skin are known spontaneous alterations of the strain of rats used, its presence is not related to the test substance. Transient diarrhoea is also known to occur spontaneously and not given toxicological relevance.
The same type and incidence of findings was noted as in the daily observations. No additional information may be derived from the detailed observations therefore.

FUNCTIONAL OBSERVATIONS AND GRIP STRENGTH DETERMINATION
No test substance related findings were made nor were there any significant group differences at the functional observations. All results represent a normal pattern of behaviour and normal reactions of rats of the strain and age examined.
There was no significant group difference in the grip strengths of both sexes

BODY WEIGHTS AND BODY WEIGHT GAIN
The high dosed males had significant lower body weights from Day 18 onwards until sacrifice. The body weight gain was reduced in parallel, but failed to gain statistical significance due to high standard deviation of the data.

In the body weights of the females before mating and of the non-inseminated dams no significant differences were found. The body weights and in parallel the body weight gain of the high dosed females during pregnancy and post partum were significantly reduced on several terms.

The significant effects on body weights are attributed to the test substance.


FEED CONSUMPTION
Feed consumption of the high dosed females during pregnancy (from Day 7 of pregnancy onwards) was significantly reduced. This is attributed to the test substance.
There were no other noteworthy differences or dose related trends noted in the feed consumption of both sexes.

HEAMATOLOGY
There were no significant differences nor dose related trends in the haematological parameters of both blood sampling terms, on Day -2 and on Day 15.

CLINICAL BIOCHCEMISTRY
Lower total protein and elevated urea levels in the high dosed males may be indications for an altered protein metabolism or a general catabolic status of the animals.

GROSS PATHOLOGY
Several isolated findings were made at the gross examination at necropsy. Some indicate a general poor health status (e.g. small spleen and thymus, emaciation, anaemia; all noted in the high dose group and a individual, found dead), some are known spontaneous alterations (e.g. white foci on adrenal glands, focal liver changes), but none of them gives an indication for a specific mode of action of the test substance.

ORGAN WEIGHTS
Altered organ weights of the high dosed group are attributed directly or indirectly to the lower body weights: absolute organ weights of livers, spleens, kidneys; relative organ weights of brains, spleens, kidneys.

HISTOPATHOLOGY
In almost all high dosed females a proliferation of the limiting ridge of the stomach - the borderline between forestomach and glandular stomach - was noted. The difference in incidence gained statistical significance, the only one of the histopathological findings. The proliferation of the limiting ridge is interpreted as an indication for a local irritation, more likely of the forestomach than of the glandular stomach. The indication for a local irritation is also supported by the finding of inflammation of the submucosa.

Isolated findings of alterations in the respiratory system (focal pneumonia, acute tracheitis) are interpreted as effects a severe local irritation of minute amounts of test substance preparation, deposited at gavage withdrawal in the oropharyngeal region, which were subsequently inhaled.

Haematopoiesis in the livers of the high dosed females is a sequel of the blood loss at birth, 4 days before sacrifice.

Some other isolated findings in histopathology are regarded as part of the background pathology, inconspicuous in type and incidence. None of them is related to the test substance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

REPRODUCTION DATA:

MATING PERFORMANCE

In 3 high dosed females the indications for successful mating (vaginal plug or presence of sperm) were missed, most likely due to a too gentle and cautious and thus insufficient sampling of the vaginal smears. Therefore pregnancy was detected in the individuals concerned delayed by body weight increase and by birth.

Nevertheless, no indication for an effect of the test substance on mating performance was found.

PREGNANCY, BIRTH, PERI- AND POSTNATAL PERIOD

Again, no test substance related group differences were noted.

OFFSPRING

There was no indication for a test substance related effect on the offspring.

Applicant's summary and conclusion

Conclusions:

The No-observed-effect-level (NOEL) of "Aluminium triformate" was at 316 mg per kg body weight and day in both sexes.
According to EU-Directive 2001/59/EC, the application of "R48" is not considered as necessary for "Aluminium triformate", as no severe toxic effects were noted at doses of up to 316 mg/kg.
According to EU-Directive 2001/59/EC, the application of "R60" is not considered as necessary for "Aluminium triformate", as no toxic effects on reproduction were noted at doses of up to 1000 mg/kg.

Executive summary:

Aim of the study

The study was performed to assess and evaluate the toxic characteristics of the test substance, resulting from a subacute oral administration via gavage to rats and comprises a reproduction and developmental toxicity screening test, according to OECD-Guideline 422, 22 March 1996.

A Dose Range Finding Study preceded this study.

Methods

All details given here refer to the Main Study; a survey of the methods of the Dose Range Finding Study is included into the Results of the Dose Range Finding Study (see below).

Test substance:"Aluminiumtriformate".

Test substance preparation for administration:Freshly blended with the vehicle.

Vehicle:0.1 % aqueous Na-methyl cellulose.

Route of test substance administration:Oral via gavage.

Dosing regimen:1/day for 28 consecutive days in males. 1/day until Day 4 post partum or for 54 Days in females. No test substance administration in the offspring.

Dose volume:10 mL test substance preparation or vehicle per kg body weight.

Test system (animals):Rats, Crl:CD(SD). 10 males and 10 females per group.

Groups, doses:

K (negative control group): vehicle only

A (low dose group): 100 mgper kg body weight

B (mid dose group): 316 mg per kg body weight

C (high dose group): 1000 mg per kg body weight,

The doses are derived from and based on the results of the dose range finding study. For a survey thereof, see below.

Experimental schedule:

- 2 weeks pre-mating period;

- 2 weeks mating period; then sacrifice of the males;

- maintenance of the successfully mated females throughout their pregnancy, birth, until Day 4 post partum, then sacrifice of the dams and their offspring;

- maintenance of the non-successfully mated females for 26 days after the end of the mating period.

"Day 1" (of the entire experiment) was the day of the 1^stadministration of the test substance.

 "Day 0 of pregnancy" (of the given individual) was the day of proven mating.

 "Day 0 post-partum" (of the given individual) was the day of birth.

Investigations:

- Analyses of the test substance preparations: In selected samples. For homogeneity and concentration

- Animal observations: All animals, once a day, plus a daily check for viability.

- Detailed clinical observations: All parental animals, once a week.

- Functional observations: All parental animals, once, prior to sacrifice.

- Body weights: All males and all females prior to successful mating once a week. All successfully mated females on Days 0, 7, 14, 20 of pregnancy and on Days 0 and 4 post partum. Pups as total litter weight on Days 0 and 4 post partum.

- Feed consumption: All animals, for weekly periods.

- Haematology: The first 5 parental animals of all groups, prior to first dosing (Day -2) and on Day 14.

- Clinical biochemistry: The first 5 parental animals of all groups, prior to first dosing (Day -2) and on Day 14.

- Necropsy with gross pathological examination: All males on Day 15, all successfully mated females on Day 4 post partum, and all non-successfully mated females on Day 54. No necropsy in pups.

- Organ weight determination: Selected organs in all parental animals at necropsy. Not determined in pups.

- Histopathological examination: Selected organs or tissues in the first 5 parental animals of groups K and C. Organs and tissues with suspected test substance related alterations also in groups A and B. No histopathology in pups.

- Data on reproduction and developmental performance: Results of vaginal smear examinations; duration of pregnancy; number, sex and viability of offspring. Number of corpora lutea and implantation sites in the dams. Several parameters were additionally derived from the primary data above.

 

Results

Dose Range Finding Study:
Doses of 100 mg or 316 mg or 1000 mg per kg body weight were given to groups of 5 male and 5 female rats once a day for 7 consecutive days. Investigations performed: Animal observations, body weights, feed consumption, and gross examination at terminal necropsy.
Summarized results: All animals survived until their scheduled sacrifice. In all high dosed animals of both sexes unspecific signs of reduced well-being were noted in the last days of the dosing period. Body weights and - more pronounced - body weight gain of the high dosed males were reduced, when compared with the negative controls. No test substance related abnormalities were noted post mortem.

 

Main Study:

-  Analyses of the test substance preparations: The test substance was found to be sufficiently stable in the preparations.
The concentrations and of the test substance in selected samples of the preparations were found to be within the chosen limits.

- Mortality 3 animals died or were killed in extremis before scheduled sacrifice.
A high dosed female died from gavage injury without contribution of the test substance on Day 3 and was replaced by a spare animal.
A mid dosed female died on Day 12 from laryngeal irritation, probably caused by minute amounts of test substance preparation inhaled.
A high dosed female was killed in a poor health condition on Day 2 post partum. A contribution of the test substance is possible.

- Observations in life, clinical and functional observations, grip strength determination: Bloated abdomen and unspecific signs of reduced well-being were noted occasionally in high dosed animals.

- Body weights and feed consumption: High dosed males and females had lower body weights; feed consumption was not notably affected.

- Haematology: No significant group differences were noted at both blood sampling terms.

- Clinical biochemistry: Only, but all, parameters with significant differences to the negative control group (indicated by bold letters) are given in this survey:

Day -2:

parameter (sex)	low dose 100 mg/kg (% of the negative controls)	mid dose 316 mg/kg (% of the negative controls)	high dose 1000 mg/kg (% of the negative controls)
potassium(females)	84	93	94
gamma glutamyl transferase(females)	250	150	150

The significant group differences are not given a toxicological relevance.

Day 15:

parameter (sex)	low dose 100 mg/kg (% of the negative controls)	mid dose 316 mg/kg (% of the negative controls)	high dose 1000 mg/kg (% of the negative controls)
urea(males)	97	99	130
glucose(males)	125	98	107
total protein(males)	98	94	89

Both altered urea and total protein levels may be a sequel of a catabolic status of the animals.

- Organ weight determination:
Only, but all, parameters with significant differences to the negative control group (indicated by bold letters) are given in this survey:

parameter (sex)	low dose 100 mg/kg (% of the negative controls)	mid dose 316 mg/kg (% of the negative controls)	high dose 1000 mg/kg (% of the negative controls)
brain(males, absolute weight)	99	99	94
liver(females, absolute weight)	94	96	84
spleen(females, absolute weight)	93	92	78
kidneys(females, absolute weight)	93	93	84
brain(females, organ weight/body weight ratio)	108	100	119
spleen(females, organ weight/brain weight ratio)	93	94	79
kidneys(females, organ weight/brain weight ratio)	92	95	86

All organ weight changes in high dosed animals may be attributed to the lower body weights of the animals concerned. The brain weight increase of the low dosed females is considered to be an incidental significance.

- Necropsy with gross pathological examination and histopathology: Isolated findings, indicating a poor health status, were made at the gross examinations in some high dosed animals.

Histopathologically, indications for a gastric irritation (proliferation of the limiting ridge, inflammation of the submucosa) were noted in some mid and high dosed animals, gaining statistical significance in the high dosed females.

- Reproduction data: No indications for a test substance related effect was made at any of the reproduction parameters

 

Discussion and Conclusion

The test substance caused local irritation on the mucous membranes of the stomach. An even more pronounced irratative effect was present on the respiratory system, as erroneously deposited minute amounts of test substance preparation caused obviously severe local effects. An additional indication for a poor local tolerance lies in the fact, that the administration of the test substance preparations caused obviously unpleasant sensations to the animals.

The few other effects of the test substance, especially on body weights, may be based on the local irritation of the stomachs.

There was no indication noted for a systemic toxic effect of the test substance.

There was no indication for an effect of the test substance on the reproduction.

There was no pronounced sex difference in the response to the test substance.

The effects noted were mild; they never became life threatening, when the test substance was administered properly in the route intended. The severe problems, resulting eventually in mortality, arose from either reflux of the preparations or from transfer of minute amounts to the larynx.

The No-observed-effect-level (NOEL) of "Aluminium triformate" was at 316 mg per kg body weight and day in both sexes.

According to EU-Directive 2001/59/EC, the application of "R48" is not considered as necessary for "Aluminium triformate", as no severe toxic effects were noted at doses of up to 316 mg/kg.

According to EU-Directive 2001/59/EC, the application of "R60" is not considered as necessary for "Aluminium triformate", as no toxic effects on reproduction were noted at doses of up to 1000 mg/kg.