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Diss Factsheets

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1976
Report date:
1976

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Version / remarks:
(1981-05-12)
Deviations:
yes
Remarks:
substance purity not reported
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
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 animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Olac (Western) Ltd., Llandeilo, Dyfeld, Wales and Balbeggie Kennels, By Kirkaldy, Fife, Scotland
- Age at study initiation: 28 weeks
- Weight at study initiation: 9481 g (mean value)
- Housing: single housing in kennels at Alconburry
- Diet: complete dry diet (Spratt's dog diet), finely ground form, 400 g diet offered per day
- Water: ad libitum
- Acclimation period: 2 months

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Each week
- Mixing appropriate amounts with (Type of food): 915 g of test substance with 1085 g of sieved dry diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative sample of diets fed were selected in the first and seventh week. The samples were extracted with chloroform and the extracts analyzed by an ultraviolet spectrometer. Experimental reproducibility is ± 10 % or better.
Duration of treatment / exposure:
90 days (all animals in study are included) followed by a recovery period of 4 weeks (1 male and 1 female each of the control and high dose level) without treatment.
Frequency of treatment:
each morning animal gets offered 400 g of fresh diet , at the same time the old residues are removed.
Doses / concentrationsopen allclose all
Dose / conc.:
5 000 ppm
Remarks:
corresponding to 195 mg/kg bw for males and 196 mg/kg bw for females
Dose / conc.:
10 000 ppm
Remarks:
corresponding to 368 mg/kg bw for males and 413 mg/kg bw for females
Dose / conc.:
20 000 ppm
Remarks:
corresponding to 750 mg/kg bw for males and 825 mg/kg bw for females
No. of animals per sex per dose:
control group: 5 animals per sex
group of low and intermediate dose level: 4 animals per sex
group of high dose level: 5 animals per sex
Control animals:
yes, plain diet
Details on study design:
SATELLITE GROUPS:
Formation of a satellite group of 4 animals (consisting of a negative control group (1 male and 1 female dog) as well as a high dose level group (1 male and 1 female dog)). These animals had a treatment-free post exposure period of 4 weeks.
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a 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 (g food/week) for individual animals
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: during the 4-week predosing period, during weeks 1-4 and 9-12 of dosing, during the 4-week recovery period, measured on weekdays only.

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: Yes
- Time schedule for examinations: before dosing, after 4, 8, 12 weeks and after 4 weeks recovery
- Dose groups that were examined: eyes of all dogs were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before dosing, after 4, 8, 12 weeks and after 4 weeks recovery
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (16 hrs before examination food was removed)
- How many animals: all animals
- Parameters checked:
-Erythrocyte sedimentation rate (ESR)
-Packed cell volume (PCV)
-Haemoglobin (Hb)
-Red blood cell count (RBC)
-Reticulocyte count (Retics)
-Mean corpuscular haemoglobin concentration (MCHC)
-mean cell volume (MCV)
-Total white cell count (WBC) Differential count:
-neutrophils
-lymphocytes
-eosinophils
-basophils
-monocytes
-platelet count
-prothrombin index (PTI)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before dosing, after 4, 8, 12 weeks and after 4 weeks recovery
- Animals fasted: Yes (16 hrs before examination food was removed)
- How many animals: all animals
- Parameters checked:
-plasma urea
-plasma glucose
-total serum proteins
-serum protein electrophoresis and AG ratio
-serum alkaline phosphatase (SAP)
-serum glutamic - pyruvic transaminase (SGPT)
-serum bilirubin
-electrolytes (sodium, potassium)

URINALYSIS: Yes
- Time schedule for collection of urine: before dosing, after 4, 8, 12 weeks and after 4 weeks recovery
- Metabolism cages used for collection of urine: Yes (urine was collected in special containers between 5 pm and 9 am the following day)
- Animals fasted: no (wihdrawal of supply of water from the animals at midday at day of examination)
-Parameters checked:
-specific gravity
-pH
-protein
-reducing substances
-glucose
-ketones
-bile pigments
-urobilinogen
-haemoglobin
-specimen of urine was centrifugated and the deposit examined microscopically for:
-epithelial cells
-polymorphonuclear leucocytes
-mononuclear leucocytes
-erythrocytes
-organism
-casts
-abnormal constituents
grading of cell frequency according to following scheme:
- NIL
- few in some fields examined
- few in all fields examined
- many in all fields examined
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

following organs were examined macroscopically
- brain
- liver
- thymus
- thyroids
- pituitary
- spleen
- prostate/ uterus
- adrenals
- heart
- pancreas
- kidneys
- gonads
- lungs
- spinal cord

For histopathology all tissues from gross pathology were observed, additionally pieces of aorta (arch and abdominal), trachea, lymphnodes (cervical and mesenteric), gall bladder, urinary bladder, salivary gland, tongue, oesophagus, stomach, duodenum, jejunum, ileum, colon, skin, mammary gland, skeletal muscle, bone marrow, peripheral nerve, eye and optic nerve)
Statistics:
Student's t-test for analysis of variances

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical symptoms were observed.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Test substance has no adverse effect on body weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Test substance has no adverse effect on food consumption and body weight gain.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
During both the pre-dosing 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. Water consumption remained satisfactory during the recovery period. It may be concluded, therefore, that there was no definite adverse effect on water consumption.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No abnormalities were detected that were considered to be related to ingestion of the test compound.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No effects were seen that had any toxicological significance, however increased values could be seen for
-platelet count:
no values higher than the accepted limit were seen prior to beginning of the study, after 4, 8 weeks and in the recovery phase. After 12 weeks in one dog of the intermediate dose group a platelet count of 490,000/cm² was measured (accepted upper limit is 450,000/cm²). A further blood sample, taken a few days later, confirmed this finding (550,000/cm²).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No effects were seen that had any toxicological significance, however increased values could be seen for
- glucose level:
- prior to beginning of the study in one dog of intermediate dose level (118 mg% vs. 110 mg%). A further blood sample taken a few days later confirmed this finding (112 mg%).
- after 4 weeks in a dog of control group (114 mg% )
- after 12 weeks in a dog of the high dose (116 mg%)
- increased values were seen only in these animals at these time points, especially no increased values were seen in recovery animals
- increase SGPT -level:
After 4 weeks in a dog of control group and of intermediate groups were 86 mU/mL and 50 mU/mL respectively (accepted limit 50 mU/mL) measured. Both findings were confirmed by a further blood sample. Again after 12 weeks in another dog of the intermediate group the value was exceeded (59mU/mL) and confirmed by a further blood sample. Increased values were seen only in these animals at these time points, especially no increased values were seen in recovery animals, consequently this effect is to be considered as reversible.
- increased SAP-level:
after 12 weeks in a dog from control group (43 KA units vs. 35 KA units as accepted limit). Increased values were seen only in these animals at these time points, especially no increased values were seen in recovery animals, consequently this effect is to be considered as reversible.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
- after 4 weeks the specimen of urine produced by a dog of the control group had a low specific gravity (1.026 vs. 1.035 as accepted lower limit), this was confirmed by a further probe. Test samples taken after 8 and 12 weeks were in the accepted range.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes that were considered to be related to administration of the test compound.
It was, however, noted that the thyroids for a number of animals from all groups including the controls, were heavier than usually seen at this centre, whilst a number of dogs had liver weights that exceeded our normally accepted upper limit of 4% of the bodyweight this is only valid for the 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 dog of the high dose recovery group 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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No histopathological abnormality or variation from normal was encountered In the tissues examined which was attributable to treatment with the test substance.
- Trachea: A small focus of mononuclear cells was seen in the subepithelium of 2 dogs (control and low dose group) and also minimal infiltration of mixed inflammatory cells was noted in the epithelial lining and the corresponding subepithelium of one dog (high dose group)
- 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 3 dogs each one of the low, intermediate and high dose group. The above changes are not uncommon in the laboratory-maintained dog, and were considered to be of no toxicological significance.
-Liver: 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 dogs (1 of the control group, 2 of the low dose group and 1 of the intermediate group). These minor changes were considered to be of no toxicological importance.
-Kidney: No treatment-related changes were detected. Minor changes which were considered to be of no toxicological importance were recorded as follows:
dystrophic mineralisation in the medullary collecting tubules of a proportion of dogs from both treated and control groups,
cortical parasitic granulomata (1 animal of the control, 3 animals of the low dose, and 1 animal of the intermediate dose group),
occasional small cortical foci or areas of chronic inflammatory cells in dogs (3 in control animals, 1 in low dose, 2 in high dose groups),
occasional lymphoid aggregations in the peripheric regions of one dog (intermediate dose group) and a small cortical area of tubular basophilia in dog of the low dose group.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
787 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Effect level:
825 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Tables of body weights

Table 1: male dogs

Mean weekly body weight
 of male dogs [g] throughout the study

 

dietary concentration [ppm]

Test group

week

5000

10000

20000

1

9850

10225

9940

2

9850

10250

10080

3

9775

10300

10040

4

10050

10400

10050

5

10150

10625

10400

6

10425

10850

10800

7

10475

10950

10900

8

10650

11200

11060

9

10475

11200

11020

10

10525

11200

10900

11

10550

11425

11100

12

10450

11375

11060

13

10350

11325

10920

 

 

 

 

control group: 5 male & 5 female animals

test groups, low and intermediate dose: 4 male & 4 female animals

test group, high dose: 5 male & 5 female animals

recovery group: 1 male & 1 female animals were retained from control and high dose group

Table 2: Female Dogs

Mean weekly body weight
 of female dogs [g] throughout the study

 

dietary concentration [ppm]

Test group

week

5000

10000

20000

1

8900

8725

8980

2

8925

8925

9060

3

8975

8975

9100

4

9275

9225

9320

5

9525

9200

9440

6

9500

9500

9540

7

9675

9475

9580

8

9775

9600

9760

9

9950

9675

9720

10

9900

9650

9740

11

9975

9725

9920

12

10025

9600

9840

13

9900

9750

9680

 

 

 

 

control group: 5 male & 5 female animals

test group, low and intermediate dose:
4 male & 4 female animals

test group, high dose: 5 male & 5 female animals

recovery group: 1 male & 1 female animals were retained from control and high dose group

Applicant's summary and conclusion