N,N'-propane-1,3-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionamide]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of 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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

> 20 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects at highest dose tested

Critical effects observed:

no

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1976

Report date:

1976

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to assess the toxicity of test substance, when administered in diet to dogs for 13 weeks, followed by 4-week recovery in dog/sex at 0 and 20000 ppm.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

EC Number:

245-442-7

EC Name:

N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

Cas Number:

23128-74-7

Molecular formula:

C40H64N2O4

IUPAC Name:

N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanamide]

Test material form:

solid

Details on test material:

- Purity: not reported

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Olac (western) Limited and Balbeggie Kennels
- Females (if applicable) nulliparous and non-pregnant: not reported
- Age at study initiation: Males: 27 to 31 weeks old; Females: 24 to 30 weeks old
- Weight at study initiation: Males: 7400 to 13300 grams; Females: 6300 to 10800 grams
- Housing: All dogs were housed singly in Kennels at alconbury
- Diet: Spratt's dog diet (400 grams per day) and supplements of milk (200 mL on week days only)
- Water: ad libitum
- Acclimation period: 2 months

Administration / exposure

Route of administration:

oral: feed

Details on oral exposure:

PREPARATION OF DIET: Each week, a premix containing 457500 ppm was prepared by adding 915 grams of test substance to 1085 grams of sieved dry diet. The resultant 2 kg of premix was thoroughly mixed and the diet for each dosage group prepared by serial dilution.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative samples of the diets fed to the dogs were taken during the first and seventh week of the study to be analysed for their content of test substance. These samples were extracted with chloroform. The quantitative determination of test substance in these extracts was performed by ultraviolet spectrophotometry. The estimated relative reproducibility of the method is ±10% or better. Within the limits of error of the mixing and sampling technique and the analytical method there was good correspondence between the values found and the nominal ones.

Duration of treatment / exposure:

13 weeks followed by 4-week recovery period for 1 male and 1 female from each of the control and high dosage groups.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 000 ppm

Remarks:

Male: 195 mg/kg/d; Female: 196 mg/kg/d; Male & Female: 195 mg/kg/d

Doses / concentrations 2

Dose / conc.:

10 000 ppm

Remarks:

Male: 368 mg/kg/d; Female: 413 mg/kg/d; Male & Female: 390 mg/kg/d

Doses / concentrations 3

Dose / conc.:

20 000 ppm

Remarks:

Male: 750 mg/kg/d; Female: 825 mg/kg/d; Male & Female: 787 mg/kg/d

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week

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 recorded daily
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
- Time schedule for examinations: Recorded at intervals during the experiment

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Ophthalmoscopic examinations were performed once before dosing commenced and then after dosing for 4, 8 and 12 weeks and after 4 weeks recovery

HAEMATOLOGY:
- Time schedule for collection of blood: Once before dosing, after 7, 8 and 12 weeks of dosing and after 4 weeks recovery (4 animals only)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes. Samples of blood were obtained prior to feeding on the day of examination with the animals in a fasting condition, food having been removed from the kennels approximately 16 hours previously.
- Parameters in table 1 were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Once before dosing, after 7, 8 and 12 weeks of dosing and after 4 weeks recovery (4 animals only)
- Animals fasted: Yes. Samples of blood were obtained prior to feeding on the day of examination with the animals in a fasting
- How many animals:
- Parameters in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Once before dosing, after 7, 8 and 12 weeks of dosing and after 4 weeks recovery (4 animals only)

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 5)

With the exception of the eyes, which were placed in Davidson's fluid, the fixative used for all organs was 10% buffered formalin. All tissues were stained with haematoxylin and eosin. Further sections of kidney were stained, with PAS to demonstrate the basement membrane. Additional pieces.of liver and kidney were placed in formol calcium prior to staining for fat with Oil Red O. A further small piece of liver was immediately frozen on solid carbon dioxide and subsequently treated with PAS to demonstrate glycogen.

Statistics:

To determine whether significant differences existed between mean values relating to test and control animals, the method used was analysis of variance followed by Student's ' t ' test, the results being expressed as the 'least significant difference' (LSD), that is, the least difference that had to exist between mean values for dosed and control animals for significance at specified levels of probability. The only other statistical procedure adopted was that used to describe the range of results obtained during the predosing investigation. These results have been described by the grand mean and the "95% range", the latter phrase being used to describe the mean + ('t' x standard deviation).

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

Description (incidence and severity):

In general the mean intake of test substance, expressed in mg/kg/day, tended to decrease as the animals increased in weight.

Food efficiency:

not specified

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

effects observed, non-treatment-related

Description (incidence and severity):

During both the predosing period and the first 4 weeks of dosing, water consumption was satisfactory for all groups. It was noted, however, that on the latter occasion water consumption had increased slightly in the control group, but had remained almost unchanged in the dosed groups. Consequently, it was possible to show statistically significant group differences relating to this parameter, but since the trend was also apparent from the-predosing measurements, it was probably of no toxicological significance. During weeks 9-12, water consumption had increased in all groups, but no significant group mean differences were demonstrated.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The plasma glucose level for one control male was 114 mg% and the SGPT values for one control dog and one at 10000 ppm were 86 mU/mL and 50 mU/mL respectively. These values approximated to or exceeded the laboratory's upper limits of 110 mg% and 50 mU/mL, respectively. Further blood samples were taken, and these findings confirmed (118 mg%; 72 mU/mL and 51 mU/mL).After 12 weeks of dosing, the platelet count for one female dog (10000 ppm) was 490000/cmm which exceeded the laboratory's normally accepted upper limit of 450000/cmm. A further blood sample, taken a few days later, confirmed this finding (550000/cmm).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 12 weeks of dosing, plasma glucose level of one male dog (20000 ppm), the SAP level of one male dog (control) and the SGPT level of one female dog (10000 ppm) all exceeded the laboratory's normally accepted upper limits of 110 mg%, 35 KA units and 50 mU/mL respectively. Further blood samples were analysed a few days later, and these findings confinned (113 mg%, 43 KA units and59mU/mL).

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 4 weeks, the specimen of urine produced by a control animal had a specific gravity of 1.026 which is below the laboratory's normally accepted lower limit of 1.035. A further specimen was obtained and this finding confirmed (1.030).

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The thyroids for a number of animals from all groups including the controls, were heavier than usually seen, whilst a number of dogs had liver weights that exceeded the laboratory's normally accepted upper limit of 4% of the bodyweight. Further investigation revealed that the highest values for both of these organs were recorded for animals obtained from Balbeggie Kennels, and it is, therefore, concluded that these observations represent a difference in the strain of beagle.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One Female dog (20000 ppm - Recovery) showed a linear area of erosion, approximately 10 mm x 2 mm, at the junction of the body and antrum of the stomach, a similar area of erosion in the duodenum together with three nodular haemorrhagic foci in the duodenum. Since this dog had received untreated diet for 4 weeks prior to sacrifice, it seems unlikely that these findings were related to administration of the test compound.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Trachea: A small focus of mononuclear cells were seen in the subepithelium of dogs one female (Control) and one male (5000 ppm) and also minimal infiltration of mixed inflammatory cells was noted in the epithelial lining and the corresponding subepithelium of one female dog (20000 ppm).

- Lung: Small areas of interstitial pneumonitis and emphysema were encountered in all dogs from both treated and control groups. Also occasional areas of parasitic pneumonitis were seen in one male dog (5000 and 10000 ppm) and one feamle (20000 ppm).

- Liver: Lobular architecture was within normal limits in all dogs examined. A proportion of animals from all groups, including controls showed small foci of mononuclear cells and/or eosinophils in sinusoids and periportal regions. Also parasitic granulomota or occasional areas of mixed inflammatory cells were found in one male dog each in control and 5000 ppm and one feamle dog each at 5000 and 10000 ppm.

-Kidney: Minor changes which were considered to be of no toxicological importance included the following: dystrophic mineralisation In the medullary collecting tubules of a proportion of dogs from both treated and control groups; cortical parasitic granulomota in one male dog (control), two male and one female (5000 ppm), one male and one female (10000 ppm); occasional small cortical foci or areas of chronic inflammatory cells in one female and two male dogs (control), one male (5000 ppm), two male (20000 ppm); occasional lymphoid aggregations in the peripelvic regions of one male dog (10000 ppm) and a small cortical area of tubular basophilia in one female dog (5000 ppm).

Other histopathological changes identified but not considered to be of significance included: occasional small calcified foci in the aortic media of one dog (5000 ppm) and an area consisting of contracted small blood vessels with minimal fibrosis in the outer zone media of the aorta in one dog (5000 ppm); minimal infiltration of fat cells between myocardial muscle cells in two dogs (5000 ppm); small areas of micro-follicles in the thyroids of the majority of dogs from both treated and control groups; parasitic granulomota in the thyroids of one dog in control and 5000 ppm, and 2 dogs each at 10000 and 20000 ppm and a small lymphoid aggregation in the thyroid of one dog (5000 ppm); small calcified foci in the gastric lamina propria of a proportion of dogs from all animals including the controls; a small submucosal area of mixed inflammatory cells in the small Intestine of one dog (20000 ppm); an intraluminal parasite in the small intestine of one dog (20000 ppm) and occasional small areas of parasitic inflammatory reaction in the small intestine of one dog (5000 ppm); pituitary cysts in a proportion of control and treated dogs; cryptorchid testes in one dog each at control, 5000, and 10000 ppm, and spermatocele granuloma in the epididymis of one dog (20000 ppm - recovery); occasional parasitic granulomota in the dermis of 3 control dogs and one dog at 20000 ppm, and small foci of mixed inflammatory cells In the dermis of 3 dogs (5000 ppm); occasional parasitic granulomota in the serosa of the urinary bladder one control dog. Also umbilical artery remnants in the bladder wall of 2 dogs each (Control – recovery and 5000 ppm), and small lymphoid aggregations in the subepithelium of the urinary bladder in one dog (5000 ppm); a developmental cyst in the parathyroid of one dog each (control and 20000 ppm); infiltrations of eosinophils in sinusoids of lymph nodes in the majority of dogs from both treated and control groups; occasional parasitic granulomota and/or areas of mixed inflammatory cells in the lymph nodes of 4 dogs each (10000 and 20000 ppm); minimal infiltration of polymorphs in the corneal limbus of one dog each (5000 and 20000 ppm); a cyst in the thymus of one dog (20000 ppm); foci of mononuclear cells with minimal skeletal muscle changes in the tongue of one dog (20000 ppm); a parasitic granuloma in the pancreas of one dog (10000 ppm); small foci of eosinophils between skeletal muscle cells in one dog (10000 ppm); dilatation of Graafian follicles in the ovary of one dog each (5000 and 10000 ppm); occasional small lymphoid aggregations in the oesophageal lamina propria of one dog (5000 ppm); a parasitic granuloma in the submucosa of the large intestine in one dog (5000 ppm).

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 20 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects at highest dose tested

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Dietary levels of up to and including 20000 ppm did not produce any detectable effects.

Executive summary:

The study was conducted to asses the toxicity of test substance when adminstered in diet to dogs for 13 weeks. Dietary concentrations of test substance 5000, 10000 and 20000 ppm was adminstered to dogs for 3 months followed by a 4-week recovery period for one male and one female from each of the control and high dosage level groups.

Observations for mortality, body weight, food consumption, water consumption, opthalmoscopy, organ weights, microscopic and macroscopic obsevations were done at regular intervals.

The group mean intakes for both males and females at 5000, 10000 and 20000 ppm were 195, 390 and 787 mg/kg/d, respectively. The results showed that there were no deaths or clinical signs observed in any group. There were no test substance related adverse effects in body weight, food consumption, or water consumption parameters. No opthamological abnormalities were detected due to intake of test substance. There were no test substance related effects observed in microscopic and macroscopic examinations or organ weights. Dietary levels up to and including 20000 ppm of test substance did not produce any detectable effects.