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Description of key information

The data base consists of oral studies in rats and dogs of 28 day,  90 day and 2 year duration, together with two unreliable studies in monkeys. Although the chronic studies are relatively old they are well reported and allow a weight of evidence based conclusion on the repeated dose toxicity. However when evaluating the NOAEL levels it is important to consider that the animals suffered from infections and the effects observed consisted of an aggravation of effects also seen in the concurrent control animals. Therefore they are considered rather conservative. The main target organ after subchronic and chronic exposure was the liver in both rat and dog. The effects were observed at comparable dose levels in both species, suggesting that the interspecies differences are low. The 2 year study in dogs included a 7 month recovery period in which most of the effects were reversible at least down to levels that were not considered to impair the function of the liver. The two monkey studies are both old and show no significant toxicological effects in the animals over the test period. Overall, the studies show that no severe organ toxicity was observed and the lowest NOAEL that is used for the assessment of 4 mg/kg w/day in the 2-year rat study is considered conservative.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1964
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
5 groups of 10 CFN rats (5 per sex) received diets containing the test material at concentrations of 0, 0.01, 0.5, 1.0, 3.0 % w/w in feed for 90 days.Standard clinical, haematological and pathological parameters were investigated.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: CFN strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Carworth Farms
- Age at study initiation: 5 weeks
- Weight at study initiation: 75 to 145 grams
- Fasting period before study: no
- Housing: 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks
Route of administration:
oral: feed
Vehicle:
cotton seed oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
Appropriate concentration of test material was added to basic food meal, and the two combined in a food processor.

VEHICLE
- Justification for use and choice of vehicle (if other than water): cotton seed oil (substance was foreseen to be added to food oils at the time or in oily pharmaceutical applications).
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
Continously, 7d/week
Remarks:
Doses / Concentrations:
0, 0.01, 0.5, 1.0, 3.0 % w/w
Basis:
nominal in diet
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: General physical appearance and activity monitored weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated

HAEMATOLOGY: Yes
- Parameters examined: Haemoglobin, haematocrit, white blood cells, segmented leukocytes, lymphocytes, monocytes, eosinophils and prothrombin time.
Sacrifice and pathology:
GROSS PATHOLOGY: YES
ORGAN WEIGHTS: YES - brain, heart, lungs, liver, ovaries, testes, epididymes, kidneys, spleen, adrenals, thyroid
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related mortality or clinical effects were reported
Mortality:
no mortality observed
Description (incidence):
No treatment related mortality or clinical effects were reported
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related effects were reported
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related effects reported
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:
not examined
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):
Slight increase in liver weight
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
no clinical signs were reported.
BODY WEIGHT AND WEIGHT GAIN
There was no clear substance related change in body weights and weight gain. A large gain in weight was seen in the male rats fed the 0.01 % w/w dose, however this was observed alongside a large intake of food that was established in the pre-study analysis.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A large intake of food was observed in male rats fed the 0.01 % w/w dose, however this was established in the pre-study analysis and is not due to test material effects.

ORGAN WEIGHTS
A slight dose related increase in liver weight was observed in the rats compared to untreated controls. At the levels observed, this is not regarded as an adverse effect.

Dose descriptor:
NOAEL
Effect level:
ca. 3 other: % w/w
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance related adverse effects were observed. Some increased organ (liver) weights were observed, however these were not considered adverse..
Critical effects observed:
not specified

The effects on growth of the rats, on their average food consumption and on the mortality among them is detailed in the following table:

 Concentration of test material  Average weight gain    Average daily food intake Mortality 
 0 303.4  151.8  16.8  13.0  0/5  0/5 
 0.01 345.8  141.8  19.8  13.8  0/5  0/5 
 0.5 283.8  142.4  15.9  13.1  0/5  0/5 
 1.0 302.4  160.0  16.1  14.0  0/5  0/5 
 3.0  299.4 121.8  17.5  12.6  0/5  0/5 

The general physical appearance and activity of all of the rats were normal. The mean gain in weight of the males fed on the diet containing 0.01 % of the test material was large, and was accompanied by a correspondingly large intake of food. These animals had established this pattern prior to being fed the test material. Therefore, it is apparent that neither of these features can be accounted for by the addition of the test material to the diet. Although the average weight gain for females fed a diet containing 1.0 % of the test material was large (160 g) it should not be considered abnormal. The final weights of these animals were within the normal range, and they had a low average initial weight, up until the third week of feeding, which explains their greater average weight gain during the experiement.

The below table lists the average weights, in grams, of certain organs for each group of rats:

Conc. of test material (%) Brain Heart Lungs Liver Gonads Kidneys Spleen Adrenal Thyroid Initial body weight Final body weight Gain in body weigh
Male Rats
 0.01 2.1  1.2  1.6  15.7  3.7  2.4  0.6  0.033  0.015  127  474  +346 
 0.50 2.3 1.1  1.5  15.0  2.7  2.4  0.6  0.025  0.017  106  399  +293 
 1.00 1.9   1.1 1.4  14.8  2.9  2.4  0.6  0.031  0.014  95  403  +309 
 3.00 1.8  1.2  1.4  15.1  3.0  2.3  0.6  0.034  0.020  101  405  +304 
 0.00 1.9  1.2  1.4  14.1  3.7  2.3  0.7  0.034  0.018  108  411  +303 
Female Rats
 0.01 1.9  0.6  1.0  8.4  0.086  1.4  0.4  0.050  0.015  109  254  +145 
 0.50 1.9  0.8  1.2  10.6  0.094  1.6  0.5  0.049  0.015  108  250  +143 
 1.00 1.9  0.9  1.0  10.7  0.087  1.5  0.5  0.049  0.018  97  262  +166 
 3.00 1.9  0.7  0.9  8.7  0.082  1.4  0.4  0.049  0.016  102  225  +123 
 0.00 1.9  0.9  1.2  8.2  0.100  1.5  0.5  0.046  0.019  102  256  +153 

The above data reveal no consistent relationship between the weights of the viscera and the presence or absence of the test material in the diet. The female rats fed on diets containing 0.5 and 1.0 % of test material were found to have relatively large (weight) livers, but all animals fed on the diet containing 3.0 % had livers which weighed nearly the same as the control animals. The livers of male rats that were given 0.01 % of test material averaged more in weight than the controls, but some of the individual animals in this group were noticably heavy and one particularly light. Comprison of the weights of the livers, relative to the total weight of the individual animals, indicates that the ingestion of 0.01 % of test material under the conditions of the experiment had no effect, but that at the 0.5 % level or more there was often an increase in liver weight, as shown in the below table:

 Concentration of test material (%)  (Liver weight/Body weight) x 100 [Males]  (Liver weight/Body weight) x 100 [Females]  Average of both sexes (% of control)
 0.00 3.43  3.20  100 
 0.01 3.32  3.30  100 
 0.50 3.76 4.23  120 
 1.00 3.66  4.08  117 
 3.00 3.42   3.86 110 

The results of hematological examinations, performed just before killing the animals, show a slightly longer than normal prothrombin time for rats fed 0.01 % of the test material. This is not true of those on diets containing higher concentrations, and is therefore probably unrelated to the ingestion of the test substance.

The gross and microscopic examinations of all of the rats revealed normal viscera.

Conclusions:
In the 90-day study in rats, no treatement related adverse effects were observed. The only effect that could be related to the test substance, but that are not considered adverse, is a slight increase in liver weights in rats at concentrations at or above 0.50 %.
Executive summary:

Groups of 5 male and female rats received levels of 0.01, 0.5, 1.0, and 3.0 % of the substance in the diet for 90-days. The study revealed no treatment related adverse effects compared to concurrent controls. The only effect that could be related to the substance is a slight increase in liver weights in rats dosed at concentrations at or above 0.5 %, but this was not considered adverse. Overall, the study supports the low toxicity of the test material at dose levels of up to 3.0 % w/w in feed for male and female rats.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1975
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Version / remarks:
plus OECD 409 for non-rodent
Deviations:
yes
Remarks:
no analytical determination of concentration and homogeneity of test substance in the diet, only 3 animals per sex per dose instead of 4 recommended as minimium in OECD TG 409, no statistical analysis performed, some details missing
GLP compliance:
no
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: American Animal industries Inc. Indianapolis, Indiana
- Age at study initiation: 10 to 11 months
- Weight at study initiation: males: 6.9 to 13.75 kg average: 10.56 kg, females: 5.8 to 12.8 kg average: 9.0 kg
- Fasting period before study: no
- Housing: individyually in wash down metal two tier cages with floors of expanded metal
- Diet ad libitum for 1 h each day from a hoper amount was weighed each time
- Water ad libitum
- Acclimation period: 27 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 76 deg. F

Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diet Wayne Dog Food Krums, every 4 to 6 weeks new diet was prepared
- Mixing appropriate amounts with (Type of food): in stainless steel Hobart Food mixer . Diet was stored in polyethylene bags
- Storage temperature of food: 76 Deg. F
- Dose in the diet was adjusted weekly to the dogs body weights and food consumption in order to remain with a constant dose level

VEHICLE
- Justification for use and choice of vehicle (if other than water): Mazola corn oil to have the test item dissolved for homogenous distribution in the diet
- Concentration in vehicle:
- Amount of vehicle: 5 g of corn oil added to 100 g control diet
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
3 dogs per sex of each dose group were dosed for 6 months, 3 dogs per sex for 27 months for each dose group and controls.
Recovery groups were included in the mid dose group (7 months), but no controls were included.
Frequency of treatment:
Daily, 7 d/week
Remarks:
Doses / Concentrations:
0, 7, 35, 75 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on 90-day perstudies and dose levels envisaged for possible pharmacological application in humans
- Rationale for animal assignment: random
- Post-exposure recovery period in satellite groups: 7 months
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: general signs of illness and unusual behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
Complete physical examination at the start, after 1 yr of treatment and at the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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
- Compound intake calculated as function of weekly body weight and food consumption, was adjusted individually

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before dosing, after 2 weeks, 1, 3, 6, 12, 18, 24, 27, 27.5, 28, 28.75, 33, 34 months as approriate for the respective groups no all parameters at every time point.
- Anaesthetic used for blood collection: No data
- Animals fasted: yes
- How many animals:mostly all surviving animals
- Parameters checked: Packed Cell volume, Hb, Leukocytes, Differential Luekocytes, Prothrombin time,

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before dosing, after 2 weeks, 1, 3, 6, 12, 18, 24, 27, 27.5, 28, 28.75, 33, 34 months as approriate for the respective groups no all parameters at every time point.
- Animals fasted: Yes
- How many animals: mostly all surviving animals
- Parameters checked: otal Serum Cholesterol, Alkaline phosphatase, SGPT, SGOT, (=ASAT, ALAT) BUN, Blood sugar. free serum lipids, Na, K, thymol turbidity, protein bound iodine, total protein, Albumine, Ca, iorganic phosphate, Serum Bilirubin, Uric acid, Bromsulfalein retention

URINALYSIS: Yes
- Time schedule for collection of urine: before dosing, after 2 weeks, 1, 3, 6, 12, 18, 24
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: Color, Appearance, Specific gravity, pH, Albumin, Sugar, microscopy

NEUROBEHAVIOURAL EXAMINATION: No
OTHER:
Sacrifice and pathology:
Sacrifica by exsanguination under Nembutal anaestheasia
GROSS PATHOLOGY: Yes:
all viscera examined, organ weights: liver, heart, lungs, kidneys, spleen, brain, gonads, pituitary, adrenals, thyroid
HISTOPATHOLOGY: Yes
heart, lung, spleen, gall bladder, liver, kidney, stomach, two levels of small intestine, large intestine, pancreas, brain (cortex, brain stem, cerebellum,, spinal cord), pituitary, thyroid gland, adrenal gland, gonads, uterus, mammry glands, aorta, bone, eye lens, urinary bladder, lymph nodes, skin, muscle, fat, all gross lesins.
Fixation: formalin, embedding: parraffin, stain: hematoxilin/eosin, CNS additionally with Luxol Fast Blue for myelin and Nissl substance. Appropriate tissues fixed in alcohol for special staining techniques when required.
Statistics:
not performed
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
2 deaths occurred in the high dose group
Mortality:
mortality observed, treatment-related
Description (incidence):
2 deaths occurred in the high dose group
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
serum alkaline phosphatase, cholesterol, SGOT, lower lipid levels
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver weight increases
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
enlarged livers
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver changes
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
One female dog of the 75 mg/kg bw dose group died during month 14 of the study. The death was considered due to marked liver cirrhosis. A male dog of the same dose group died during month 21. Pathology findings revealed intra-intestinal hemorrhages.
No treatment related clincal signs were observed in the surviving dogs.
BODY WEIGHT AND WEIGHT GAIN
The dog that died during week 21 showed weight loss prior to death. The body weights and weight gains varied widely within all groups, but there were no systematic weight changes indicating a test substance related effect on body weight or body weight gain.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No differences in food consumption were observed between the groups.
HAEMATOLOGY
Packed cell volume, haemoglobin, numbers of leukocytes and differential luekocytes were comaprable between treated and control animals. Transient changes in prothrombin time in the high and mid dose groups were observed, but the valuse varied widely in all groups and as prothormbin times were normal at the later time points the chnages are not considrerd as biologically significant.
CLINICAL CHEMISTRY
Decreased cholesterol levels were observed from 3 months to the end of the study in the mid and high dose group. The levels returned to normal in the mid dose recovery group after 7 months of control diet.
A dose related increase in serum alkaline phosphatase was observed in all dose groups. The levels in the 35 mg/kg bw/d recovery group animals returned toward normal in most of the recovery group dogs, but had not reached normal levels in all dogs after the 7 month period.
SGPT levels were sporadically increased in the high dose group and some of the mid dose group animals at earlier times, but not at the end of the 27 month exposure period. Some of the values for serum urea nitrogen and sugar were above controls at some time points, they were normal after 12 months of the diet and thus the changes are not considered of biological significance. Serum free lipids were lower than controls in the high dose group only. The other dose groups were considered comparable to controls and within normal ranges.
Bromsulphalein retention varied widely within the group, but was considered elevated in all dogs of the high dose group and 5 of 6 dogs of the mid dose group. For the low dose the results were regarded questionable. The effect was reversilbelin the mid dose recovery animals.
In the high and mid dose groups slight decreases in serum total protein and albumin and questionable decreased in serum Ca-levels were observed. In the mid dose recovery group all values returned to norrmal after the recovery period. A slight decrease in albumin was observed in 4 dogs of the low dose group, but this is not considered biologically significant. Serum protein bound iodine (measured during baseline, at 24 month and 6 months post exposure) was reported to be decreased in the high dose group, but normal in the mid and low dose groups.
Thymol turbidity, Na, K, phosphate, SGOT, bilirubin and uric acid values were comparable between treated and control animals.
URINALYSIS
No differences between controls and treated animals were observed.
ORGAN WEIGHTS
At the 6 month sacrifice livers of one male and one female dog were enlarged. At this time point no clearly treatment related group differences in liver weights coudl be deduced from the data due to the wide variability within groups of liver and body weights. All high dose animals that were fed the diet for 25 months had increased liver weights, but no differences from controls were observed in the 35 and 7 mg.kg bw dose groups.
GROSS PATHOLOGY
see above for liever enlargement
HISTOPATHOLOGY: NON-NEOPLASTIC
Morphological change in the liver were observed at the 75 and 35 mg/kg bw dose levels, but no treatment related changes were observed at 7 mg/kg bw/day after 2 years of treatment. After 6 months of treatment liver changes in the high dose group were clearly more prominent than inthe controls, while no clear difference was observed for the mid and high dose group compared to controls. In the mid dose group recovery animals a reversion of the lesions within the 7 month period, but no complete recovery was observed, however the authors state that the remaining lesions may not have a functional adverse effect.
Dose descriptor:
NOAEL
Effect level:
ca. 7 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: slight changes in clinical chemistry are not regarded as treatment related effects. Histopathological changes in liver at 35 and 75 mg/kg bw after 2 years. Signs of reversibility in the 35 mg.kg bw recovery group after 7 months.
Critical effects observed:
not specified
Conclusions:
In a 2 year dietary study in dogs receiving 7, 35 or 75 mg/kg bw/ day of the test substance the most prominent effects were morphological changes in the liver that were accompanied by some changes in clincal chemistry parameters. Based on the histopathological changes in the liver and only very slight observation in clincal chemistry parameters in the low dose group, that are not considered of biological relevance, a NOAEL of 7 mg./kg bw/d can be derived. In the mid dose recovery animals the changes showed a tendency of reversibility indicating that the adverse effects were not severe and normal function of the organ could be retained after cessation of dosing.
Executive summary:

Groups of 3 male and female dogs received doses of 0 (vehicle control), 7, 35 and 75 mg/kg bw/day of 4,4'-methylene bis (tert.butylphenol) in the diet for periods of 6 months and 2 years. One mid dose satellite group was included for a recovery period of 7 months in which the animals recieved normal diets. After 6 months of exposure some clinical chemistry parameters were changed in both the 75 and 35 mg/kg bw dose groups, but the liver histopathology showed clear aggravation from controls only in the high dose group animals. After 2 years of exposure the most prominent effect was morphological changes in the liver that were accompanied by some changes in clincal chemistry parameters. Based on the histopathological changes in the liver a NOAEL of 7 mg./kg bw/d can be derived. In the mid dose recovery animals the changes showed a tendency of reversibility indicating that the adverse effects were not severe and normal function of the organ could be retained after cessation of dosing.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1961
Reliability:
2 (reliable with restrictions)
Qualifier:
no guideline available
Guideline:
other:
Principles of method if other than guideline:
A 90 day repeated dose study with test article incorporated in feed and a negative control group and positive control group (BHT) was conducted. Focused hematological examination was conducted to assure that the test article did not produce effects on clotting parameters similar to Vitamin K deficiency that had been seen with some other alkylated phenols under similar conditions. Body weights, food consumption, organ weights and histopathology of the viscera and endocrine glands were included in the study design.
GLP compliance:
no
Remarks:
Conducted prior to GLP regulations
Limit test:
no
Species:
rat
Strain:
other: CF albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Carsworth Farms
- Age at study initiation: 10 week old weanlings
- Weight at study initiation:
Control Group: 296 +/- 5 grams
0.125% Test Article Group: 292 +/- 7.2 grams
0.50% Test Article Group: 295 +/- 7.8 grams
1.25% Test Article Group: 305 +/- 6.7 grams
2.50% Test Article Group: 288 +/- 9.2 grams
1.25% BHT Control Group: 304 +/- 7.1 grams
- Fasting period before study: none
- Housing: 5 males or females per cage,
- Diet (e.g. ad libitum): Purina Laboratory Chow meal basic diet, ad libitum A purified cellulose (alphacel) was added to the varous dients to keep non-nutritive bulk uniform at 5%
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 30 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks
- Mixing appropriate amounts with (Type of food): Test article and cellulose mixtures incorprated into dry feed by being stirred by a mixer for 15 minutes


Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days of feeding
Frequency of treatment:
Daily in feed
Remarks:
Doses / Concentrations:
0.125%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.5%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.25%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2.5%
Basis:
nominal in diet
No. of animals per sex per dose:
15 male/15 female per group (control group, 3 treated groups, positive control group (BHT)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: High dose group was to be equal to the BHT positive control group, a level in the BHT group that was expected to cause toxicity. Maximum tolerance of BHT in human diet at the time of the study was 0.005%
Positive control:
Butylated hydroxytoluene (BHT); 2,6-di-tert-butyl-4-methylphenol. C15H24O (purity 99%)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: initial, weekly, terminal

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each cage determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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 data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: initial, and at 30 and 90 days for control, 0.125%, 0.50%, and 1.25% groups; initial, 30 days, 60 days and 90 days for 2.50% test group and the positive control (BHT 1.25%) groups
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes / No / No data
- How many animals: 5 males, 5 females per group
- Parameters examined: Total WBC, Polyleucocytes, Lymphocytes, Hemoblobin, Hematocrit, Prothrombin time (determined by modified micro-Kato method using whole blood)

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Organ weights: brain, heart, lungs, liver, gonads, kidneys, spleen, adrenal, thyroid
Statistics:
Difference of mean values tested by analysis of variance for 4 and 70 degrees of freedom
Clinical signs:
no effects observed
Description (incidence and severity):
No difference in clinical signs compared to negative control; one female death in 0.5% group at 2 weeks probably not test article related
Mortality:
no mortality observed
Description (incidence):
No difference in clinical signs compared to negative control; one female death in 0.5% group at 2 weeks probably not test article related
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistical differences from negative control values
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistical differences from negative control values
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No differences compared to negative control for total WBC, differential, HB, Hct, or prothrombin time
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No specific effects that could be related to test article; differences in female groups related to differences in initial and terminal weight increases compared to control
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences in treated animals compared to negative control
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At high dose, 6 male and 3 female rats had slight degree of liver degeneration
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: One rat in the 0.5% treatment group died after the second week, and had sustained progressive loss of weight, and respiratory difficulties prior to death. No other signs of illness were noted in the test article treatment animals. No significant effects on growth, food consumption or mortality compared to control were seen. In the positive control group (BHT), marked decrease in growth, in association with reduced food consumption, predominantly in males, was seen. The effectes in the BHT group were evident in the first week, and progressed to approximately the tenth week. Five of the male BHT rats and one female rat of that group died during weeks 3 to 10. Signs in the BHT rats included general weakness, which in some animals, became an acute illness with marked signs of pallor and listlessness, with large hematomas seen in some animals.

BODY WEIGHT AND WEIGHT GAIN: No effects seen in treated animals versus control animals. BHT animals showed decrease in weight gain beginning in the first week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No differences in food consumption were seen in the treated animals compared to control.

HAEMATOLOGY: Treated animals showed no differences in total white blood counts, percent polyleucocytes, percent lymphocytes, hemaglobin, hematocrit, or prothrombin times. Animals fed BHT, especially the males, showed increased prothrombin times, and a decrease in hemoglobin content, with a shift in leucocyte distribution characterized by increase in polymorphonuclear cells and corresponding decrease in lymphocytes. These changes in the BHT animals were most pronounced in the 30 to 60 day blood samples.


ORGAN WEIGHTS: differences in average visceral weights among the female treated gropus appeared to be associated with different initial and terminal body weights. Weights of most of the viscera of animals given BHT were altered compared with control weights. Nearly all of the livers of animals in this group were acutely hyperemic and dark reddish brown.

GROSS PATHOLOGY: No alterations were seen in gross pathology in rats in control or test article added diets. One of the rats receiving BHT was euthanized on day 20 of exposure, and showed a urethral injury associated with urine retention. Three males given BHT died after 39 and 52 days of exposure, and showed extensive degeneration and necrosis of the livers, with marked pneumonitis, and small lung hemorrhages. Another male died after 63 days, but decomposition obscured lesions. One female rat given BHT died after 67 days of exposure, and showed extensive necrosis and liver degeneration.

HISTOPATHOLOGY: NON-NEOPLASTIC: Animals given 0.125, 0.5, or 1.25% test article showed no microscopic pathology. The livers of animals given 2.5% test article in 9/15 males and 12/15 females showed no pathologic changes. The remainder (6 males and 3 females) showed slight degree of liver degeneration as evidenced by clumping of cytoplasmic granules at edges of hepatocytes, cellular swelling, and either one large or two small nuclei, and multiple prominent nucleoli. The degenerated cells were found sporadically throughout the liver lobules. The other viscera and endocrine glands in these 6 males and 3 females of the high dose group were normal except for a slight to moderate degree of pneumonitis.
All male rats given BHT showed degeneration of hepatic cells ranging from modreate to severe, and varying from focal to diffuse in distribution. The lesions were similar, but more severe, compared to the animals given test article at 2.5% in the diet. Four of the 14 female rats that survived to 90 days of BHT in diet had normal viscera; The other 10 had moderate to severe degeneration of the liver. In addition, some of the male and female rats given BHT had a slight to moderate degree of pneumonitis.

Dose descriptor:
NOAEL
Effect level:
ca. 1.25 other: wt % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
2.5 other: wt. % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Weight Gain, Food intake, and Mortality over 90 days feeding

Dietary Additive Group

Wt. % added to diet

Mean wt. gain (per cent)

M            F

Mean daily food intake (grams)

M            F

Mortality (deaths/group)

M            F

Control

0

+41        +26

20.6         13.8

0/15        0/15

702

0.125

+36        +29

19.6         14.9

0/15        0/15

702

0.50

+35        +26

19.8         14.6

0/15        1/15

702

1.25

+38        +18

20.5         13.7

0/15        0/15

702

2.50

+42        +23

19.3         14.9

0/15        0/15

BHT

1.25

- 3          + 4

16.2         12.2

5/15        1/15

Executive summary:

A 90-day repeated dose feeding study was conducted in male and female albino rats with antioxidant “Ethyl” 702 as the test substance, and Butylated hydroxytoluene (BHT) as the positive control substance. Test concentrations were control, 0.125, 0.5, 1.25 and 2.50 wt. percent of the diet; BHT was tested at a single concentration of 1.25 wt. percent of the diet. The purpose of the study was to examine the effects of the test article to some aspects of toxicity seen with other alkylated phenols. No grossly harmful effects were seen in the animals exposed to test substance. No significant differences were noted in the groups with respect to growth, consumption of food, or apparent physiological activity. The administration of BHT at a level of 1.25% of the diet caused death in 20% of the animals during the course of the study.

           Hematological examinations of the test animals carried out in intervals of 30 days disclosed no abnormalities in the numbers or types of leucocytes in peripheral blood, in hematocrit, hemoglobin, or prothrombin times. These findings of no effect was interpreted to mean that the test article did not cause the cause the anticoagulative effects seen in rats fed some other alkyl phenols in comparable diets. These hemorrhagic tendencies were thought to be associated with an increase in ‘prothrombin time” and with the metabolism of Vitamin K seen in rats fed some other alkyl phenols.

           At study determination, there were no adverse effects seen among the test article treated groups with respect to survival or gross pathological changes. There were significant effects in the final weights of females in the various groups (the test substance treated animals had heavier than control weights), and in the final weights of individual viscera. That is, the larger animals had heavier viscera, and vice versa, but without a uniform proportional relationship of body weight to weight of organs.

           The histopathologic examination of viscera and endocrine glands of these rats revealed no changes in the groups 0.125%, 0.5%, or 1.25% test article in the diet. Slight degeneration of the liver was observed at the dietary concentration of 2.5% test article in the diet in about 30% of the animals in the group. Comparatively, marked degeneration of the livers was found in almost all of the rats fed BHT at 1.25% in the diet.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Unknown
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No indication of Guideline followed or GLP status, however the protocol followed OECD TG 407 . No Quality Assurance signature page. No Study Director signature page. No testing dates or company information. No single animal data available.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center Charles River Laboratories
- Age at study initiation:
Rats five weeks old at start of dosing.
Stratified according to body weight, then randomized.
- Weight at study initiation: mean males: 160 g (149 to 168), mean females: 145 g (135 to 155 g)
- Fasting period before study: no
- Housing:
Individually housed.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Quarantined/acclimated for 7-8 days prior to dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature 22 plus/minus 3 degrees Celsius
- Humidity (%): 55 plus/minus 10 %.
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12 h

IN-LIFE DATES: From: To: no data

Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
pharmacopoea grade
Details on oral exposure:
Dose range-finding test: 0, 50, 100, 200, 500 and 100 mg/kg/day.
Dosing for range-finding and main study was at 5 mL/kg body weight.


- Lot/batch no of vehicle. (if required): Kozaki Pharmaceutical, M1-13
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Details not apparent from report.
Duration of treatment / exposure:
28-day
Frequency of treatment:
Once per day, 7 days per week
Remarks:
Doses / Concentrations:
0, 8, 40, 200, 1000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a dose range findign study of 14 days duration at dose levels of 50, 100, 200, 500 and 1000 mg/kg bw/day of the test substance.
- Rationale for selecting satellite groups: 5 mals and 5 females in the control and high dose group were used as recovery animals
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
No
Observations and examinations performed and frequency:
-
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General condition: Immediately before dosing, 30 minutes - one hour post-dosing, 4 hours post-dosing
- Cage side observations checked : Mortality, appearance, behaviour, and others not specified.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Detailed clinical examination: Day prior to dosing, then once weekly
Reactivity on removal, Reactivity on handling, muscle tone, skin, fur, piloerection, palpebral closure, exopthalmus, lacrimation, smudge arouind eyes or nose, salivation, blotted fur in lower abdomen with urine, blotted fur around anus with faeces, vocalization, breathing, body position, convulsion, tremor, exploration, walk, abnormal beahvior, streotypy, urination, defecation
BODY WEIGHT: Yes
- Time schedule for examinations:
before TS adminstration, dayzs 1, 7, 14, 21, 28 and day 7 and 14 of recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption : Yes, once weekly on days 5, 12, 19 26 of the main group and in addition, days 5 and 12 of reciovery period in males and days 4, 11, 18 anmd 25 of main group and in addtition days 4 and 11 of recovery period inm females.

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: after the last dosing
- Anaesthetic used for blood collection: Yes Ether for laparotomy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: RBC, HB. HT, MCV, MCH, MCHC, Ret. PT, APTT, Platelets, WBC, differential leukocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: Yes
- How many animals: all
- Parameters examined:
LDH, AST, ALT, gamma-GTP, ChE, T.P., Alb., A/G ratio, total cholesterol, T.G., Glu, BUN. Creatinine, total bilirubin, Ca, P, Na, K, Cl

URINALYSIS: Yes
- Time schedule for collection of urine:
3 h in week 4 on days 22 to 23 and week 2 of recovery period (day 8 to 9)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: appearance, pH, occult blood, protein, glucose, ketone bodies, bilirubin, urobilinogen, sediment. In addition urinary volume andensity was assessed in rats accomodated in the cage fro 18 h,

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Sensory functional examination: Week four (week 2 of recovery group)
- Dose groups that were examined:all
- Battery of functions tested: sensory activity / grip strength / motor activity / pain reaction, pupil reflex, righting reflex,
Sacrifice and pathology:
Day 27 sacrifice of main group.
Day 13 sacrifice of recovery group.
Urinalysis
Hematology
Serum chemistry
Gross necropsy
Organ weights - brain, thymus, heart, liver, kidney, adrenals, spleen, pituitary, thyroid, testis, epididymis, ovary
Histopathology - brain, pituitary, eyes, thyroid/parathyroid, spinal cord, heart, trachea, lung, liver, kidney, thymus, spleen, adrenals, stomach, intestines, genitalia, urinary bladder, sciatic nerve, mandibular and mesenteric lymph nodes, bone marrow (femur). Histopathology performed only on control and 1000 mg/kg dose groups.
Thyroid gland histopathology of all dose groups.
Statistics:
Parametric data - Bartlett's test for variance. Then, One-Way ANOVA, Student's t-test, Aspin-Welch t-test.
Non-parametric data - Kruskal-Wallis rank teset. Then, Dunnet's type test, Mann-Whitney U test.
Categorical data - Fisher's exact test or Mann-Whitney U test.
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
slight but significant from control changes in some parameters. Not considered of toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A few clinical chemical changes that were statisitcally significantly different from controls were not considered of toxicological relevance.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significantly high relative kidney weights in males =>40 mg/kg/d. High liver, ovary and thyroid weights in females rats. Male rats in 1000 mg/kg had high liver weights while females had low thymus weights.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlargement of liver accompanied by yellow hepatization in one 200 mg/kg/d female.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Slight diffuse thyroid follicular hyperplasia in 2 females (200 mg/kg/day) and 4 females (1000 mg/kg/day). Complete recovery.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities were observed in the controls and dose groups. All animals survived until termination. Clinical observations in all dose grousp were comparable to controls.
BODY WEIGHT AND WEIGHT GAIN
No significant effects on body weight or body weight gain were observed in any of the dose groups compared to controls.

FOOD CONSUMPTION
No significant effect on food consumption was observed in any of the dose groups compared to controls.

HAEMATOLOGY
A few statistically significant findings were reported:
Males at 200 mg/kg bw: slightly, but significantly decreased Hemoglobin, significantly decreased Hematocrit, MCV and MCH values.
Males at 1000 mg/kg bw: : slightly but significantly decreased MCV and MCHC, no clear dose response in these findings.
Males recovery group (1000 mg/kg) males hemoglobin and MCHC were significantly lower than control values.

Females at 200 mg/kg slightl, but significant decrease in MCHC and prothrombin time compared to controls. Significant increase in reticulocyttes compared to controls.
Females 1000 mg/kg: slight, but significant decrease in MCHC, no dose response.
Females recovery group: no significant changes.

Further findings: males of the 8 mg/kg anmd 40 mg/kg dose groups and females of the 8 and 200 mg/kg dose group had significantly reduced APTT levles, but as these findings were not observed in the higher dose groups they are regarded as chance findings. Slightly, but significantly elevated levles of WBC, lymphocytes and neutrophiles were reported in females of the recovery group.

The authors state in their discussion that most of the hematological changes were slight and within the historical background data of the institute, except the MCH, and as there were no corresponding histopathological changes the changes are regarded of low toxicological significance.
CLINICAL CHEMISTRY
Male rate of the 200 and 1000 mg/kg bw groups showed slightly, but significantly decreased creatinine levles.
Male rats of the recovery group: statistically significant increase in AST and decrease in total cholesterol.
Female rats of the 40, 200 and 1000 mg/kg bw dose groups showed statistically significant decreases in Cholinesterase activity, but without clear dose response.
Females in the 200 mg/kg bw dose group showed statistically significant increases in LDH and gamma-GTP, and statistically significant decreased levels of total cholesterol and BUN. These changes were not observed in the 1000 mg/kg bw dose group.
In females of the recovery group statisitcally significant findings were an increase in gamma-GPT and a slight increase in sodium levels.
Most of the values were, however in the historical control range or only slightly outside (AST in males, gamma-GTP in females) of the insitute and wee regarded as incidental by the authors.

URINALYSIS
No differences between treatment groups and controls were observed in all dose groups including the recovery group.
NEUROBEHAVIOUR
No differences between treatment groups and controls were observed in all dose groups including the recovery group.
ORGAN WEIGHTS
Relative, but not absolute kidney weights were slightly but statistically significantly ellevated in males of the 40, 200 and 1000 mg/kg bw dose groups. There was however no dose related increase. Relative liver weights were statistically significantly increased in males of the high dose goup only.
In the recovery group males absolute, but not relative thyroid weights were slightly, but significanly increased.

In females of the 200 mg/kg dose group absolute and relative liver weights were increased, but no such increase was observed at 1000 mg/kg. In the 200 and 1000 mg/kg females significant increases in absolute and relative ovary weights (no dose response) and absolute and relative thyroid weights were observed. The relative, but not absolute thymus weight was slightly, but significantly increased in high dose females.

In recovery group females a slight increase in relative, but not absolute thymus weigth and a slight increase in relative, but not absolute pituitary weight was reported. Both were statistically significant.

Except for the thyroid weight changes other organ changes had no histopathological correlate the changes in the weigh tof thymus and pituitary were regarded as incidental findings by the authors.

GROSS PATHOLOGY
One female of the 200 mg/kg bw dose group was reported to have a yellowish large liver.
HISTOPATHOLOGY: NON-NEOPLASTIC
The only findign that was statistically different from control incidences was a slight hyperplasia of follicular cells in the thyroid of female rats in the 200 and 1000 mg/kg dose groups. The incidence increased in a dose related manner form 2 of 5 to 4 of 5.
Dose descriptor:
NOEL
Effect level:
8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Relative high kidney weights in males and low cholinesterase in females at 40 mg/kg/d. However these effects were not considered as adverse by the authors in the absence of any histopathological changes in the 40 mg/kg bw dose group.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
ca. 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
The NOAEL of a repeated 28-day oral administration of test article was 40 mg/kg/day, as toxicologically relevant effects were only observed in females of the
200 mg/kg bw group. These effects consisted of increased absolute and relative liver weights with no accompanying histopathological changes, increased absolute and relative thyroid weights and slight diffuse hyperplasia of the follicular epithelium in 2 of 5 females of this dose group and 4 females of the 1000 mg/kg dose group. Similar findings were not observed in the recovery group and the effects were considered fully reversible.
Executive summary:

The repeated dose 28-day oral toxicity study of 2,2’,6,6’-tetra-tert-butyl-4,4’-methylenediphenol was performed using SD rats. In the study, 0 (control: olive oil used as a solvent for the test substance was administered), 8, 40, 200, and 1000 mg/kg/day of the test substance were administered. In the control group and 1000 mg/kg group, each ten female and male rats were used including each five female and male rats used as the 14-day recovery group, while in the 8, 40, 200 mg/kg groups, each five female and male rats were used.

In the 40 mg/kg or more groups, a high relative weight of the kidney was detected in the male rats and a low cholinesterase level in the female rats. However, as there was neither clear dose dependency in these changes nor relating blood biochemical and histopathological findings, the changes were judged to be of low toxicological significance. In the rats in the 200 mg/kg or more groups, a low mean corpuscular volume and a low mean corpuscular hemoglobin were detected in the male rats, while high absolute and relative weights of the thyroid gland and a diffuse hyperplasia of the follicular epithelial cells, a low mean corpuscular hemoglobin concentration, high absolute and relative weights of the ovary without organic changes were detected in the female rats.

In the recovery groups, all the changes appeared were recovered, indicating that they were reversible. There were no changes that were regarded as late toxicity.

Based on these results, it can be concluded that the NOAEL of a repeated 28-day oral administration of test article was 40 mg/kg/day, as toxicologically relevant effects were only observed in females of the 200 mg/kg bw group. Based on the toxicologically insignificant findings in the 40 mg/kg bw dose group the 8 mg/kg bw dose was regarded as a NOEL.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study is old (1970) and not conducted to regulatory guidelines or to GLP standards. Only three animals were used.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Three male rhesus monkeys received 125 mg/kg of AN-702 per kg of body weight as a 4 percent solution (w/v) in corn oil (3.1 ml/kg) by oral gavage. Treatments were given once daily preprandially for 42 consecutive days.
GLP compliance:
no
Limit test:
no
Species:
monkey
Strain:
other: Rhesus
Sex:
male
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
42 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
125 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Positive control:
None
Observations and examinations performed and frequency:
CLINICAL CHEMISTRY: Yes
- Parameters examined: Plasma cholesterol and alkaline phosphatase levels

Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified

The cholesterol values of both the treated and control animals decreased by 20 to 30 percent during the first two weeks. During the ensuing three weeks of treatment, the test animals maintained this decreased level, while the level in the control animals gradually returned to the baseline average. At six weeks all values increased. The plasma cholesterol values for individual animals are presented below:

   Cholesterol levels in mg/100 ml
 Monkey No. Baseline Values  Baseline Average  2 weeks

3 weeks

4 weeks

5 weeks 

6 weeks 

 Control Monkeys 

 4247

160 

164 

142 

160 

156 

133 

133 

129 

131 

185 

 4258

160 

156 

153 

193 

166 

127 

133 

161 

163 

208 

 4270

137 

137 

129 

146 

137 

107 

133 

142 

150 

185 

  Treated Monkeys 

4238 

 194

188 

200 

193 

194 

147 

133 

129 

125 

146 

 4246

172 

188 

165 

173 

174 

107 

145 

142 

125 

168 

 4269

137 

132 

129 

146 

136 

100 

109 

103 

100 

154 

During the six weeks of treatment there appeared to be an increasing divergence between the average cholesterol values for the control and test animals, which reached a maximum of 24 -27 percent at five to six weeks.

There was considerable variation in the plasma alkaline phosphatase values, as summarised in the table below, but these data are considered to represent normal fluctuations for this colony.

 Alkaline Phosphatase levels in King-Armstrong Units
 Monkey No. Baseline Values Baseline Average  4 weeks 5 weeks 6 weeks
 4247 98 78  122  99  32  54  70 
 4258 54  81  89  75  29  58  74 
 4270 67  62  90  73  42  86  63 
Treated Monkeys
 4238 62  60  76  66  20  40  23 
 4246 54  62  94  70  46  59  58 
 4269 45  42  50  46  38  45  36 

                                                       

Conclusions:
No significant effects are seen in the study.
Executive summary:

When the test material was administered to the male rhesus monkeys by stomach tube, both the treated animals and the controls receiving only the oil menstrum exhibited a decrease in plasma cholesterol levels. There was some divergence between the cholesterol levels of the treated animals and the controls, the levels in the treated animals averaging about 25 percent less than those of the control monkeys after five to six weeks of treatment. Based upon previous experience with fluctuations of normal cholesterol levels, we have used a decrease of 30 percent or more as indicative of a significant hypocholesterolemic effect. No effects on plasma alkaline phospatase levels were observed.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study is old (1970) and not conducted to regulatory guidelines or to GLP standards. Only two animals were used.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Two male rhesus monkeys received diet containing 125 mg of AN-702 and 3.1 ml of corn oil per 25 g of ground Purina R monkey chow for 31 days.
GLP compliance:
no
Limit test:
no
Species:
monkey
Strain:
other: Rhesus
Sex:
male
Route of administration:
oral: feed
Vehicle:
corn oil
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
31 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0.5% w/w
Basis:
nominal in diet
No. of animals per sex per dose:
2
Control animals:
yes, plain diet
Positive control:
None
Observations and examinations performed and frequency:
CLINICAL CHEMISTRY: Yes
- Parameters examined: Plasma cholesterol and alkaline phosphatase levels

Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Critical effects observed:
not specified

In the experiment, the monkeys exhibited no consistent hypocholesterolemia, as shown in the following table:

Cholesterol Levels in mg/100 ml
 Monkey No. Baseline Average  Pre-Rx  1 week  2 weeks  3 weeks  4 weeks 
Control Monkeys
 425 166 128 150 186 141 193
Treated Monkeys
 4247 156 148 93 107 130 127
 4270 137 128 121 122 146 173

The low cholesterol level seen in monkey 4247 after one week of being fed the experimental diet did not persist with feeding during successive weeks, although there was a persisting 14 percent reduction. The plasma alkaline phosphotase levels after three and four weeks of feeding the experimental diet are summarised in the below table, and show no significant change.

The plasma alkaline phosphotase levels after three and four weeks of feeding the experimental diet are summarised in the below table, and show no significant change.

Alkaline Phosphotase Levels in King-Armstrong Units
 Monkey No. Baseline Average  Pre-Rx  3 weeks  4 weeks 
Control Monkey
 4258 75 56 75 67
Treated Monkeys
 4247 99 72 54 29
 4270 73 91 72 36
Conclusions:
No significant effects are seen in the study.
Executive summary:

No significant hypocholesterolemia was observed in the small series of monkeys that received the test material in their diet for four weeks. Plasma alkaline phophatase levels were determined after three and four weeks of feeding the test marterial in the diet and no differences between the treated and control monkeys were observed.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1964
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
Groups of 6 Beagle dogs (3 per sex)received diets containing the test material at concentrations of 0, 1.0, 3.0% for 90 days. Two additional groups of dogs received radiolabelled material at concnetrations of 0.3 and 0.01% respectively. Standard cclinical, haematological and pathological parameters were investigated
GLP compliance:
no
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: pure bred, no further data
- Age at study initiation: 4 to 13 months
- Weight at study initiation: 7.3 to 8.1 kg
- Fasting period before study: no
- Housing: individually
- Diet ad libitum):
- Water ad libitum):
- Acclimation period:no data

ENVIRONMENTAL CONDITIONS: no data
Route of administration:
oral: feed
Vehicle:
cotton seed oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Solutions containing the compound up to the solubility limit were prepared, additional product added and mixesd homogenously to the diets
- Mixing appropriate amounts with (Type of food): Allied Mills Tail Wagger Dog Food Krums
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): cotton seed oil (substance was foreseen to be added to food oils at the time or in oily pharmaceutical applications).
- Concentration in vehicle: over saturated
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
Continously, 7d/week
Remarks:
Doses / Concentrations:
1 and 3%
Basis:
nominal in diet
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: maximum amount that could be homogenously mixed into the food
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: satellite groupd were fed with radiolabeled material to investigate its distribution
- Post-exposure recovery period in satellite groups: no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS:No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 2 samples before dosing, after 30, 60 and 90 d
- Parameters checked
Haemoglobin, Haematocrit, luecocyte count, differential leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:2 samples before dosing, after 30, 60 and 90 d
- Parameters examined.
electrophoresis of serum proteins, isocitrate dehydrogenase, total lipids, cholesterol free and esters

URINALYSIS: Yes
- Time schedule for collection of urine: 2 samples before dosing, after 30, 60 and 90 d
- Metabolism cages used for collection of urine: Yes
- Parameters checked:
colour, appearance, specific gravity, pH, albumin, sugar, acetone, white blood cells, red blood cells, microscopic casts, epithelial cells, crystals, bacteria
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes:
organ weights: brain, heart, lungs, liver ovaries, tested, epididymes, kidneys, spleen, adrenals, thyroid
HISTOPATHOLOGY: Yes
all organs above, haematoxilin eosin stain
Other examinations:
14C determination in tissues: brain, heart, lungs, liver, kidneys, spleen, gonads, adrenals, thyroid, fat, skin , muscle, distribution, excretion and storage in tissues determined.
Statistics:
least square linear equation for body and organ weights
Clinical signs:
no effects observed
Description (incidence and severity):
no treatment related mortality
Mortality:
no mortality observed
Description (incidence):
no treatment related mortality
Body weight and weight changes:
no effects observed
Description (incidence and severity):
effects cannot clearly be related to substance intake
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
decreased lipids and cholesterol
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
slight liver weight increase in males
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
no clinical signs were reported. 2 female animals of the 3% group died for test substance unrelated reasons, one from hepatitis on day 5 one from anaesthesia during a debarking operation.
BODY WEIGHT AND WEIGHT GAIN
There was no clear substance related change in body weights and weight gain, but the data were very heterogenous and no clear conclusion on a possible substance related effect coudl be drawn.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption did not show a substance related change. The test substance intake could be calculated from the average body weight (begin and end of the study) and the average food intake as: Radiolabled compound: 0.01%: 2.3 mg/kg bw males, 2.7 mg/kg females, 0.3%: 78 mg/kg males, 75 mg/kg females, unlabelled 1%: 270 mg/kg bw males and females, 3%: 840 mg/kg bw males, 810 mg/kg bw females

HAEMATOLOGY
No test substance related changes were observed.
CLINICAL CHEMISTRY
No substance related changes in clinical chemistry parameters, a significant dicrease in blood lipids was observed in the treated groups from 0.3% of the test substance. Total serum cholesterol was decreased in animals receiving 0.3, 1 and 3% of the test substance in the diet, the levle was reversed to normal after 90d in the 0.3% group. This effect is not considered as adverse in the absence of any histopathological changes.
URINALYSIS
No test substance related changes were observed.
ORGAN WEIGHTS
A slight dose related increase in liver weight was observed in male dogs compared to untreated controls, but not in females. In the absence of any gross pathological and microscopic findings this is not regarded as an adverse effect, but could be indicative of induction of liver metabolising enzymes.
GROSS PATHOLOGY
No test substance related changes were reported.
HISTOPATHOLOGY: NON-NEOPLASTIC
No test substance related changes were reported.
Dose descriptor:
NOAEL
Effect level:
ca. 810 - 840 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance related adverse effects were observed. The effects on blood lipids and cholesterol (decrease) and slight increases in liver weights in males were not considered of toxic, but rather of adaptive nature.
Critical effects observed:
not specified

Of the radiolabel test the following percentages could be detected in the faeces and urine at about 6 weeks of feedimg:

In faeces:

at 0.3%: 83% in males and 94% in females

at 0.01%: 106% in males 91% in females

In urine

at 0.3%: 0.8 % in males and 1.5 % in females

at 0.01%: 1.7 % in males 1.3 % in females

Concentrations in organs after the 90d feeding period were in percent of the total ingested amount:

at 0.3% in the diest

males: Fat: 2.08%, skin 1.32%, muscle: 0.48%, liver 0.09% Total retention: 4%

females: Fat: 1.5%, skin 1.02%, muscle: 0.66%, liver 0.09% Total retention: 3.3%

at 0.01% in the diest

males: Fat: 1.93%, skin 1.11%, muscle: 0.81%, liver 0.14% Total retention: 4%

females: Fat: 1.81%, skin 1.2%, muscle: 0.89%, liver 0.09% Total retention: 4%

.

Conclusions:
In the 90-day study in Beagle dogs (3 per sex per dose) no clearly test substance related adverse effects were observed. The only effects that could be related to the test substance, but that are not considered adverse are a dose dependent decrease in bloodlipids and cholesterol from 1% in the diet and a slight increase in liver weights in male dogs. No histopathological correlate was found in the microscopic evaluations. This study is supporting the low toxicity of the test material after repeated oral adminitration to dogs at dose levels up to 840 mg/kg bw/day for males and 810 mg/kg bw/day for females.
Executive summary:

Groups of 3 male and female dogs received levels of 0.01, 0.3 (radiolabeled), 1, and 3% of the test substance in the diet for 90 -days. The dose levels corresponded to approximately 2.3, 78, 270 and 840 mg/kg bw per day in males and 2.7, 75, 270, 810 mg/kg bw/day in females. The study revealed no test substance related clinical signs, no changes in gross and histopathology and no changes in heamatology and urinalysis compared to concurrent controls. The only effects that could be related to the test substance, but that are not considered adverse are a dose dependent decrease in blood lipids and cholesterol from 1% in the diet and a slight increase in liver weights in male dogs.Of the radiolabel appr. 95% was excreted in the faeces and 2 percent in the urine. About 4% was found in tissues at the end of the exposure period, with the highest concentration in fat tissue identified as unchanged test substance.

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1969
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
limited clinical chemistry, no urinalysis, limited data on test substance purity and no report onm analylsis of the diets
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Lab. Inc. Brookline Mass.
- Age at study initiation: weanling rats
- Weight at study initiation: males 394 - 436 g (av 1. week), females 248 - 269 g (av. week 1)
- Fasting period before study: no
- Housing: 3 males or 4 females per cage
- Diet ad libitum
- Water ad libitum:
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 76 +- 2 deg. F
- Humidity (%): not controlled
Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): 1 kg of corn oil was added to 19 kg of the base diet with or without the test substance. The mixture was stirred for 20 min in a Hobart mixerr (stainless steel). 5 diets with test item were prepared at a concentration of 15, 60, 100, 500, 3000 ppm. The diet was freshly perpared every week.
- Mixing appropriate amounts with (Type of food):special perparation of Rockland Mouse and Rat diet containing no more than 1% of fat. The diet was obtained fresh every month.
- Storage temperature of food: refigerator

VEHICLE
- Justification for use and choice of vehicle (if other than water): for dissolution of at least part of the substance is dissolved in cornoil (6% solubility) to ensure homogenous distribution
- Concentration in vehicle: 300, 1200, 2000, 10000, 60000 mg/kg corn oil
- Purity: Mazola Corn oil free of antioxidants
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
continuously in diet
Remarks:
Doses / Concentrations:
15, 60, 100, 500, 3000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
25, 50 controls, 4 males and 4 females were assigned to subgroups for interim kills after 13, 26, 52 and 78 weeks
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose selection based on 90-day study
- Rationale for animal assignment (if not random): were randomly assigned
.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: general appearance and behaviour

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
weekly
BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly per cage
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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 data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all for cataracts

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 week before dosing, week 13, 26, 52, 78, 104
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals:10 per dose
- Parameters examined: prothrombin time, hemoglobin, packed cell volume of erythrocytes, total and differential leukocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at scheduled sacrifice
- Animals fasted: No
- How many animals: all allocated to the respective groups
- Parameters examined: total lipids, cholesterol, alkaline phopsphatase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: liver, heart, lungs, kidneys, spleen, brain, gonads, pituitary, adrenals, thyroid
HISTOPATHOLOGY: Yes : Heart, Aorta, lungs, liver, spleen , kidney, stomach, duodenum, pylorus, small intestine (ileum), large intestine (colon), mesenteric lymph nodes, urinary bladder, prostate, testes, ovaries and uterus, pancreas, thyroids, thymus, adrenals, pituitary, brain, spinal cord, eye, bone marrow, bone, skeletal muscle, skin.
Statistics:
Performed, but no data on methods
Clinical signs:
no effects observed
Description (incidence and severity):
no test substance realted effects observed
Mortality:
no mortality observed
Description (incidence):
no test substance realted effects observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no test substance realted effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
no test substance realted effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
no test substance realted effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
no test substance realted 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):
increase in liver weight at the highest dose tested
Gross pathological findings:
no effects observed
Description (incidence and severity):
no test substance realted effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver increased fatty degenaration and bile duct proliferation at 3000 and 500 ppm, but similar lesions also in controls
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
no test substance realted effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Throughout the study several rats had various infectious diseases and died in the course of the illness. The incidence and severity of these diseases were unrelated to the content of the test substance in the diets and occurred equally in the control rats. The mortality in the different groups was comparable to controls. (see Table 1). The only test substance related clinicall sign was a greenish-yellow discoloration of the fur of the high dose rats commencing after 6 motnhs and in rats receiving 500 ppm after one year. Occasional discoloration was seen some the rats receiving 100 ppm after 15 months.
BODY WEIGHT AND WEIGHT GAIN
No test substance realted changes in body weight or body weight gain were observed in the study. Variations in body weight and body weight gain of individual animals during the cours of the study were attributed to infectious diseases or spontaneous tumors that occurred at comparable incidence in the treated and control groups and were accompanied by respective changes in food consumption.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was comaprable between treated and control animals and only influenced by diseases as mentioned above. The average test substance intake during the study was calculated for males and females (see table 2).
FOOD EFFICIENCY
Food efficiency was reported to be comparable between treated and control animals.
OPHTHALMOSCOPIC EXAMINATION
No findings were reported from the ophthalmologic examinations
HAEMATOLOGY
Mild to moderate leukopenia was reported in some females at week 52 and 78, but not after 104 weeks of feeding. The incidence in treated and control animals was comparable. Single cases of some degree of anaemia or polychromasia wer occaisonally seen during week 78 and 104 in males and females of all groups including controls with no relationship to treatment. Prothrombin time was statisitcally significantly different from controls in males of the 15 and 500 ppm group in the week before treatment started, and in the 15, 100 and 3000 ppm groups at 13 weeks. In females statistically significant differences to controls were observed in the 60, 100, 500 and 3000 ppm groups one week before treatment started and in the 500 ppm group on week 26. No statistically significant differences to control values were observed at the 26, 52, 78 and 104 week observation times. As the finding was not dose related and did not persist throughout the course of the study, it was not considered treatment related. All other haematological parameters were comparable between treated and control animals.
CLINICAL CHEMISTRY
Alkaline phosphatase levels varied widely within the groups and the only statistically significant finding was an increased level in the high dose group males in week 104. Due to the major fluctuations in the measurements this finding was not considered treatment related by the authors. No consistent treatment related effect was observed on the serum cholesterol anmd total lipid levels
ORGAN WEIGHTS
All relative organ weightsand most absolute organ weights in the treated groups were comparable to the respective control values. Single rats receiving 3000 ppm of the test substance were reproted to have enlarged livers, but the group avaerages did not reach statisitcal significance, with the exeption of females killed aftter week 13 and males killed after week 78. In females fed for 104 weeks at 15 ppm a relative liver weight statistically significantly lower than control values was reported. This is an isolated finding and in the higher dose groups no significant changes in liver weight were observed. Therefore this finding is not considered treatment related by the authors.
GROSS PATHOLOGY
No test substance related macroscopic organ changes were observed in the study.
HISTOPATHOLOGY: NON-NEOPLASTIC
All animals (treatment and control) killed at 24 months had pneumonia or interstitial nephritis or both. A total of 183 rats died during the cours of the study. Fior 52 no postmortem pathology could be done due to autolysis. For 131 postmortem pathology revealed in most instances massive pulmonary infection as the cause of death. Liver lesions were the most prominent findings and consisted of fatty degeneration of the hepatic sells and a proliferation of the bile ducts. Fatty degeneration reached from small fat vacuoles to a fatty liver. There was no special localization of the degeneated cells in the liver lobe. The incidence and severity in the different groups is given below (table 3). Two types of proliferation of the bile ducts coudl be distinguished morphologically: 1. multiplication of the bile ducts with retainment of their normal stucture occurred in small clusters. 2. A dilatation and multiplication of the bile ducts with fibrotic walls without epithelium. In some of the dilated bile ducts mucous plaques were observed. No inflammatory reaction was present. Up to 3 months of feeding no alterations in the livers were observed. Both lesions invcreased in control and treated rats from 18 months of treatment. After 24 more severe lesions were observed in the 500 and 3000 ppm dose groups. Incidence and severity was more pronounced in males than in females.
HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Neoplastic lesions observed in the study were subcutaneous or in females mammary gland adenofribroma, pituitary adenoma, one adenoma in the parathyroid, thyroid adenoma, adrenal adenoma, liver angioma that wer all benign tumors. Leukemia, Seminoma and ovary carcinoma were single findings in control animals only. No test substance related increased incidence of any tumour was observed and it can be concluded that the test substance did not induce neoplastic lesions under the conditions of this study. The findings are summarized in chapter 7.7.

The histopathology findings were confirmed in a second reading of the data with independent pathologists and reported in a supplementary report of the same laboratory dated July 9, 1971.
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased incidence and severity of hepatic lesions compared to controls at 3000 and 500 ppm
Critical effects observed:
not specified

Table 1 Mortality in the different groups

Level of TS in the diet (mg/kg diet)

Mortality males %

Motality females %

Mortality malea and females combined %

0

54.9

52.9

53.9

15

46.2

40.0

43.1

60

57.1

44.0

50.9

100

44.0

68.0

56.0

500

61.5

38.5

50.0

3000

46.2

52.0

49.0

Table 2: Average Test substance intake over the study period

Dose group mg/kg in diet

TS intake males mg/kg bw/day

TS intake female mg/kg bw/day

0

0.55

0

15

2.14

0.67

60

3.56

2.63

100

18.13

4.35

500

107.25

22.23

3000

0.55

138.43

Table 3 incidence and severity of liver lesions

Table 3.1 Fatty degeneration males

Dose group

No of months

Number of rats With no change

(%)

No rats (%)

Grade 1 severity (marginal)

No rats (%)

Grade 2 severity

slight

No rats (%)

Grade 3 severity

modrate

No rats (%)

Grade4

Severity

Severe

0

6

4 (100)

0

0

0

0

 

12

4 (100)

0

0

0

0

 

18

4 (100)

0

0

0

0

 

24

14 (62)

4 (17)

3 (13)

1 (4)

0

15

6

4 (100)

0

0

0

0

 

12

3 (75)

1 (25)

0

0

0

 

18

4 (100)

0

0

0

0

 

24

6 (43)

5 (36)

0

1 (7)

2 (14)

60

6

3 (75)

1 (25)

0

0

0

 

12

4(100)

0

0

0

0

 

18

1 (100)

0

0

0

0

 

24

7 (58)

2 (17)

2 (17)

1 (8)

0

100

6

4(100)

0

0

0

0

 

12

4(100)

0

0

0

0

 

18

4(100)

0

0

0

0

 

24

6 (43)

5 (36)

1 (7)

2 (14)

0

500

6

4(100)

0

0

0

0

 

12

0

0

0

3 (75)

1 (25)

 

18

0

0

1 (33)

2 (67)

0

 

24

5 (45)

2 (18)

1 (9)

3 (18)

0

3000

6

1 (25))

2 (50)

0

1 (25)

0

 

12

0

0

1 (25)

2 (50)

1 (25)

 

18

1 (33)

0

1 (33)

1 (33)

0

 

24

0

2 (14)

4 (29)

6 (43)0

2 (14)

Table 3.2 Fatty degeneration females

Dose group

No of months

Number of rats With no change

(%)

No rats (%)

Grade 1 severity (marginal)

No rats (%)

Grade 2 severity

slight

No rats (%)

Grade 3 severity

modrate

No rats (%)

Grade4

Severity

Severe

0

6

4 (100)

0

0

0

0

 

12

4 (100)

0

0

0

0

 

18

4 (100)

0

0

0

0

 

24

20 (84)

1 (4)

1 (4)

2 (8)

0

15

6

4(100)

0

0

0

0

 

12

4(100)

0

0

0

0

 

18

4(100)

0

0

0

0

 

24

11(73)

 

2 (13)

1(7)

 

60

6

4(100)

0

0

0

0

 

12

4(100)

0

0

0

0

 

18

3(75)

0

0

1 (25)

0

 

24

10(72)

0

3 (21)

1 (7)

0

100

6

4(100)

0

0

0

0

 

12

4(100)

0

0

0

0

 

18

4(100)

0

0

0

0

 

24

6(76)

0

1 (12)

1 (12)

0

500

6

4(100)

0

0

0

0

 

12

3(75)

0

1 (25)

0

0

 

18

1(25)

0

3 (75)

0

0

 

24

10 (63)

0

4 (25)

2 (12)

 

3000

6

4(100)

0

0

0

0

 

12

1(25)

0

1 (25)

2 (50)

 

 

18

0

1(25)

0

2 (50)

0

 

24

4 (33)

3(25)

4(33)

0

1 (9)

 Table 3.3 Bile duct multiplication males

Dose group

No of months

Number of rats With no change

(%)

No rats (%)

Grade 1 severity (marginal)

No rats (%)

Grade 2 severity

slight

No rats (%)

Grade 3 severity

modrate

No rats (%)

Grade4

Severity

Severe

0

6

4 (100)

0

0

0

0

 

12

4 (100)

0

0

0

0

 

18

4 (100)

0

0

0

0

 

24

18 (78)

3 (13)

1 (4)

0

1 (4)

15

6

4 (100)

0

0

0

0

 

12

3 (75)

1 (25)

0

0

0

 

18

4 (100)

0

0

0

0

 

24

11 (79)

3 (21)

0

0)

0

60

6

4 (100)

0

0

0

0

 

12

3 (75)

0

0

1 (25)

0

 

18

1 (100)

0

0

0

0

 

24

10 (83)

1 (8)

1 (8)

0

0

100

6

3 (75)

0

0

1 (25)

0

 

12

2 (50)

1 (25)

0

1 (25)

0

 

18

4(100)

0

0

0

0

 

24

9 (64)

1 (7)

2 (14)

2 (14)

0

500

6

4(100)

0

0

0

0

 

12

1 (25)0

2 (50)0

0

1 (25)

0

 

18

0

1 (33)0

2 (67)

0

0

 

24

6 (55)

2 (18)

2 (18)

1 (9)

0

3000

6

1 (25))

0

1 (25)

2 (50)

0

 

12

0

0

1 (25)

2 (50)

1 (25)

 

18

0

0

2 (67)

1 (33)

0

 

24

4 (35)

0

5 (28)

4 (35)

1 (17)

Table 3.4 Bile duct multiplication females

Dose group

No of months

Number of rats With no change

(%)

No rats (%)

Grade 1 severity (marginal)

No rats (%)

Grade 2 severity

slight

No rats (%)

Grade 3 severity

modrate

No rats (%)

Grade4

Severity

Severe

0

6

4 (100)

0

0

0

0

 

12

4 (100)

0

0

0

0

 

18

3 (75)

0

1 (25)

0

0

 

24

22 (92)

0

2 (8)

0

0

15

6

4 (100)

0

0

0

0

 

12

4 (100)

0

0

0

0

 

18

4 (100)

0

0

0

0

 

24

12 (80)

1 (7)

2 (13)

0)

0

60

6

4 (100)

0

0

0

0

 

12

3 (75)

0

0

1 (25)

0

 

18

1 (25)

0

2 (50)

0

1 (25)

 

24

10 (71)

2 (14)

2 (14)

0

0

100

6

4 (100)

0

0

0

0

 

12

3 (75)

0

0

1 (25)

0

 

18

4(100)

0

0

0

0

 

24

4 (50)

1 (12.5)

1 (12.5)

2 (25)

0

500

6

3 (75)

0

0

1 (25)

0

 

12

3 (75)0

1 (25)`

0

0

0

 

18

1 (25)

0

3 (75)

0

0

 

24

5 (31)

3 (19)

3 (19)

4 (25)

1 (6)

3000

6

2 (50)

0

2 (50)

0

0

 

12

1 (25)

0

0

2 (50)

1 (25)

 

18

0

1 (25)

0

3 (75)

0

 

24

4 (33)

1 (8)

2 (17)

3 (25)

2 (17)

Tumor incidences: see chapter 7.7. Carcinogenicity

 

 

Conclusions:
In a 2-year combined repeated dose-carcinogenicity dietary study in rats the test substance related effects were confined to a dose related aggravation of liver lesions identified as fatty degeneration and bile duct multiplication that were also present to a lesser extend and severity in control animals. The incidence and severity of the changes at 500 and 1000 ppm was regarded to represent an adverse effect. Therefore the NOAEL in this study is 100 mg/kg diet which corresponds to approximately 3.56 mg/kg bw in males and 4.35 mg/kg bw in females. The onset of the lesions occurred from 18 to 24 months of study duration. With regard to carcinogenicity it can be concluded that under the conditions of this study the test substance did not lead to an increased tumour incidence up to the highest dose level tested of 3000 mg/kg diet corresponding to appr. 107 mg/kg bw/day in males and 138 mg/kg bw/day in female rats.
Executive summary:

Groups of male and female CD-1 rats (25 per sex per dose, with 50 controls per sex) were fed diets containing 0 (control), 15, 60, 100, 500, and 3000 ppm (mg/Kg diet) of 4'4'-methylene bis(2,6 -di-tert. butylphenol) for a period of 2 years. This corresponded to approximate dose levels of 0, 0.55 (m)/0.67 (f); 2.14 (m)/2.63 (f), 3.56 (m)/ 4.35 (f), 18.1 (m)/22.2 (f); 107 (m) and 138 (f) mg/kg bw/day. Mortality was comparable to controls in all dose groups. No test substance related clinical signs, body weight changes, haematological or clinical chemical changes were observed during the study. A discoloration of the fur was observed among animals of the 500 and 3000 ppm dose group and single animals in the 100 ppm dose group. No clearly test substance related changes in organ weights and gross pathology were observed at all dose levels.

Test substance related effects were confined to histopathological findings of a dose related aggravation of liver lesions identified as fatty degeneration and bile duct multiplication that were also present to a lesser extend and severity in control animals. The incidence and severity of the changes at 500 and 1000 ppm was regarded to represent an adverse effect. Therefore the NOAEL in this study is 100 mg/kg diet which corresponds to approximately 3.56 mg/kg bw in males and 4.35 mg/kg bw in females. The onset of the lesions occurred from 18 to 24 months of study duration. With regard to carcinogenicity it can be concluded that under the conditions of this study the test substance did not lead to an increased tumour incidence up to the highest dose level tested of 3000 mg/kg diet corresponding to appr. 107 mg/kg bw/day in males and 138 mg/kg bw/day in female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
4 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The lowest NOAEL of the chronic studies is chosen. The NOAEL of the 2 year dog study was comparable at 7 mg/kg bw/day. The 90-day studies had NOAEL levels of 700-800 mg/kg bw/day in male and female rats respectively and 810 to 840 mg/kg bw in male and female dogs. In both species liver was the target organ. However, infections in the animals in the longer duration studies may have led to an aggravation of the effect.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The data base consists of dietary chronic and subchronic studies in rats and dogs, a 28-day oral gavage study in rats and two oral studies in monkeys. Although the chronic and subchronic studies are relatively old, they are well reported and allow a weight of evidence based conclusion on the repeated dose toxicity. However when evaluation the NOAEL levels it is important to consider that the animals suffered from infections and the effects observed consisted in an aggravation of effects also seen in the concurrent control animals. Therefore they are considered rather conservative.

In the 28-day study the most sensitive target organ with regard to histopathological changes was the thyroid. The changes were mild and consisted of slightly increased thyroid weights in the 200 and 1000 mg/kg dose groups and slight diffuse hyperplasia of the follicular epithelial cells. Increased liver weights without any histopathological changes were also observed in these dose groups. The effects were reversible in the recovery animals. 

The main target organ in the subchronic and chronic toxicity studies in rats and dogs was the liver for both species and the effects were observed at comparable dose levels in both species, suggesting that the interspecies differences are low. Interestingly no effects on the thyroid were reported in these studies, although this organ was examined as well. This indicates that the effects in the thyroid observed in the 28 day study may have been an reversible adaptive change that is not observed after longer exposures. The additional two monkey studies are unreliable but showed no significant effects throughout the test period.

The 2 year study in dogs included a 7 month recovery period in which most of the effects were reversible at least down to levels that were not considered to impair the function of the liver. This indicates that no severe organ toxicity was observed and the lowest NOAEL that is used for the assessment of 4 mg/kg w/day in the 2-year rat study is considered conservative. The NOAEL values of the 90 day studies were 700 to 800 mg/kg bw in rats and 810 to 840 mg/kg bw in dogs.

In order to reinvestigate previous findings and to address certain specific concerns, an enhanced Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) is proposed. The proposed study would be substantially augmented with additional parameters in order to investigate any potential endocrine effects.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The selection should encompass all repeated dose studies in a weight of evidence approach, but IUCLID only allows to select one study. One subacute study in the rat is available together with subchronic and chronic toxicity studies in 2 species. The latter give comaparable results with regard to target organs and effects.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

The NOAEL values in the 90 day rat and dog repeated dose dietary toxicity studies are above 700 mg/kg bw. Effects observed in the chronic and subchronic studies are confined to the liver and were indicative of an aggravation of existing infective diseases in the test animals. Furthermore the adverse effects showed signs of reversibility in a 2 year dog study that included a recovery period. It can therefore be concluded that the weight of evidence suggests that exposure to the substance did not cause significant toxic effects in experimental animals at dose levels and conditions that would warrant a classification for repeated dose toxicity. Consequently the substance is not classified for repeated dose target organ toxicity according to Regulation EC 1272/2008 and amendments.