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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 409)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Propionic acid
- Physical state: colourless, liquid
- Analytical purity: > 99%
- Stability on storage conditions at RT: guaranteed over the study period. (Analysis at start of the study, after about 10 weeks, at the end of the administration period and also at the end of the study)
- Stability in water: 14 days
- Stability in feed: 24 hours

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: in breeding at BASF facility
- Age at study initiation: 4-8 months
- Weight at study initiation: (♂ 6.0-11.6 kg) (♀ 6.6-10.5 kg)
- Fasting period before study: no
- Housing: single in kennels (6.0 m2 floor area). Recovery animals were housed 2/kennel (5.4 m2) for the duration of the recovery period.
- Diet: Dogs offered daily rations of 700g /feeding period
- Water: ad libitum; always blended water (fully demineralised water which has been adjusted to about 4° German hardness with drinking water). During collection of urine in metabolism cages animals were offered drinking water
- Acclimation period: 1-2 wks
- Comments: The animals showed no signs of disease at the time they were taken over into the study. They were lively, reacted to their environment in the usual manner and were eating well. Urinary excretion and defecation were normal


ENVIRONMENTAL CONDITIONS
- The animals were housed in kennels comprising of an inner and an outer kennel. The animals had constant access (day and night) to the outer kennels during both the adaptation and administration periods.
- The rooms were ventilated by means of a ducted ventilation system with high-pressure ventilation (the incoming air was warmed up additionally in winter).
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: test subtance was diluted in water, then mixed with feed to a paste
Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance/drinking water solutions were freshly prepared at intervals of about 7 to 12 days
- Mixing appropriate amounts with (type of food): Kliba laboratory diet "A"
- Storage temperature of food: no data
- Process: The test substance was diluted with drinking water to the desired concentration then made up with drinking water to give 1000 g of solution. These solutions were made into a paste using dry food at a ratio of 1 : 1 in each case (350 g feed + 350 g test substance (test group) or water (control)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Time schedule for examinations: at the beginning of administration, after about 6 weeks and at the end of the administration period
- Parameters measured: stability of test substance in drinking water, stability of test substance in food made to paste

Duration of treatment / exposure:
Males: 106 days
Females: 99 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
3000, 10000, 30000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
male: 214.2, 718.9, 2056.3 mg/kg bw; females 225.1, 749.2, 2071.8 mg/kg bw (calculated from food consumption)
Basis:
actual ingested
No. of animals per sex per dose:
- Control: 8
- 3000 ppm: 4
- 10000 ppm: 4
- 30000 ppm: 8
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on results of preliminary 15 day repeated dose trial
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: test for reversibility of effects
- Dose selected for recovery group: control (4/sex), 30000 ppm (4/sex)
- Post-exposure recovery period in satellite groups: 6 weeks. For organisational reasons (common start of the recovery period for the male and female dogs) there was an interval of several days for the males and an interval of one day for the females between the end of the administration period and the beginning of the recovery period during which they already received food without test substance.

Examinations

Observations and examinations performed and frequency:
MORTALITY: Yes
- Time schedule: twice daily on working days and once daily on weekend and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once/working day. Several times/day if signs occured

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: daily

COMPOUND INTAKE (if feeding study): Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for examinations: calculated on a weekly basis

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start of the study (all animals), at the end of the administration (control and 30000 ppm dose group)
- Method: KOWA-RC 2 fundus camera
- Comment: no examinations were performed after recovery period because the eyes (refracting media and fundus) of the animals were not affected when examinations were performed after the end of the regular administration period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1-2 days before first dosing, on days 37, 86 and on days 135 and 142 after first administration for the the recobvery group
- Anaesthetic used for blood collection: No
- Animals fasted: Yes (16 hours)
- How many animals: all animals
- Parameters checked: Leukocytes, erythrocytes, haemoglobin, hematocrit, mean cell volume, mean haemoglobin content per erythrocyte, mean corpuscular haemoglobin concentration, platelet, differential blood count, clotting analysis (partial thromboplastin time, prothrombin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1-2 days before first dosing, on days on days 37, 86 after first administration. On days 135 and 142 after first administration for recovery group.
- Animals fasted: Yes (16 hours)
- How many animals: all animals.
- Parameters checked: alanine aminotransferase, aspartate aminotranferase, alkaline phosphatase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: 5 day before first dosing, 34, 90 after first administration. On days 133 and 140 after first administration for the recovery animals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: volume, pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, specific gravity, sediment

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
- During the entire study period (administration and recovery periods) none of the animals showed signs related to the administration of the test substance. No deaths were observed during the approximately 3-month administration period and 6-week recovery period.

BODY WEIGHT AND WEIGHT GAIN
The body weight gain of the male and female animals of the test groups (3000 ppm, 10000 ppm and 30000 ppm) throughout the administration period and also of the male and female dogs of high dose group (30000 ppm) used in the recovery period was largely parallel that of the corresponding control group. All the differences which were found are within the usual range of variation, are statistically insignificant and are biologically irrelevant.
- Mean weight gain at the end of the administration period for males (%) = 100 (control), 81.3 (3000 ppm), 78.1 (10000 ppm), 93.8 (30000 ppm)
- Mean weight gain at the end of the administration period for females (%) = 100 (control), 94.5 (3000 ppm), 84.2 (10000 ppm), 79.9 (30000 ppm)- - Mean weight gain after recovery period for males (%) = 100 (control), 100 (30000 ppm)
- Mean weight gain after recovery period for females (%) = 100 (control), 100 (30000 ppm)

FOOD CONSUMPTION
- Loss of appetite was demonstrated by 2/16 animals of the control group, 1/8 animals in the low dose group in varying intensity and frequency. However due to the lack of a dose dependent relationship, these effects are considered to be incidental rather than substance dependent.
- In the high dose group, animals 9/16 animals showed slight (2♀+ 3♂) till marked and prolonged (4♀) lack of appetite which are considered to be test substance related. However, since body weights were not also affected, this effect is considered to be of no toxicological relevance.

COMPOUND INTAKE
- ♂: 214.2 ± 26.5, 718.9 ± 63.3, 2056.3 ± 246.5 mg/kg bw
- ♀: 225.1 ± 24.3, 749.2 ± 76.1 , 2071.8 ± 365.1 mg/kg bw

OPHTHALMOSCOPIC EXAMINATION
- The animals of the control group (0 ppm) and of the high dose group (30000 ppm) which had been examined ophthalmologically before the start of the study (all the dogs to be used in the study) and at the end of the administration period showed no signs whatsoever of damage to the refracting media and/or the fundus. Therefore, another eye examination at the end of the recovery period was dispensed with.

HAEMATOLOGY (Kruskal-Wallis Anova + Mann-Whitney u-tests * P<0 .05)
- 1000 ppm: no substance related finding
- 10000 ppm: no substance related finding
- 30000 ppm: significantly increased platelets (♀)(*21.9% over control), increase in eosinophile granulocytes (♀) (45.7% over controls)

The increase in platelet in females of the high dose group is regarded as incidental since the increased values are still within the range of biological variation. Furthermore, the unchanged blood clotting parameters (partial thromboplastin time and Quick's test) show that there is no impairment of the blood clotting system. The increase in eosinophilic granulocytes seen in the female animals of the 30000 ppm group is also considered to be an incidental finding since this occurred only in females. In addition the animals did not show any signs of allergy, skin disease or parasitic infection which in general are known to cause an increase eosinophilic values.

CLINICAL CHEMISTRY (see attachment 1 for detailed values in tables)
- 1000 ppm: no substance related finding
- 10000 ppm: Decrease in the total protein and globulin concentrations in the male animals following blood sampling at week 86 (see Tab. 101) .
- 30000 ppm: Decrease in the total protein concentration in both sexes at week 37 and 86; in the male animals also following blood sampling at week 86. Decrease in the albumin level in the male animals following blood samplings at weeks 37 and 86 and in the female animals following blood sampling at week 37. (see Tab. 100 and 101 and also 108 in attachment). Decrease in the globulin concentration in the male animals following blood sampling at week 86 (see Tab . 101). Decrease in the calcium level in the male animals following blood sampling at week 86 and in the female animals following blood sampling 1 at week 37 (see Tabs. 097 and 104 in attachment). Increase in the potassium concentration in the male animals following blood sampling at week 37 (see Tab. 096).

- 30000 ppm (recovery group): decreased alanine and aspartate aminotransferases (♀) following blood sampling at week 135 (see table 094 in attachment)

The decrease in alkaline phosphatase is an artefact as it seems to have been caused by deficiencies in the method of detection as shown by further investigation performed. Propionic acid did not reduce the enzyme activity of alkaline phosphatase in vitro when blood samples of untreated dogs were employed. Whereas addition of acetyl coenzyme A or propionyl coenzyme A lead to marked decrease by 60% or 45% compare to controls, respectively. The assumption is that alkaline phosphatase reacts with the phosphate groups of the coenzyme derivatives employed in the method of detection. Examination of the other clinico-chemical parameters that were modulated indicates that the values underlying the statistical calculations are in the range or borderline range of the control and historical values

URINALYSIS
- 1000 ppm: no substance related finding
- 10000 ppm: no substance related finding
- 30000 ppm: Increase in nitrate (♀♂) (see table 113 and 117 of attachment 1)
The increase of nitrite values is usually a signature of an infection of the urinary tract. However since this finding is not correlated by findings like increased leucocytes or bacteria presence in urine, it is considered incidental and not substance specific.

GROSS PATHOLOGY
Necropsy of dogs after the administration interval revealed no gross lesions.

HISTOPATHOLOGY:
- Examination of tissues revealed no lesions except for point-of-contact diffuse epithelial hyperplasia of the mucosa of the oesophagus in several high dose dogs but which was reversible after the 6 week recovery period. The incidence of focal epithelial hyperplasia in lower dose animals was comparable to controls.

Effect levels

Dose descriptor:
NOAEL
Remarks:
(local + systemic effects; subchronic, oral; dog)
Effect level:
ca. 733.4 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion