Hexamethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

Endpoint summary

Description of key information

In rats, the substance targets the liver and activates the well described liver-thyroid axis resulting in higher thyroid activity as reflected by organ size and follicular hypertrophy. As expected, this species specific effect on the thyroid was not observed in dogs up to the highest dose levels tested. Since this mechanis is not relevant for humans, the dog study was considered as the most relevant study for risk assessment. In this 90d feeding study, adaptive liver increases were the only findings, the NOAEL was dermined at 147 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1975-04-03 to 1975-08-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non GLP but guideline compliant study. Well documented report.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

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: Oloc (Western) Ltd.
- Age at study initiation: 29 weeks
- Weight at study initiation: 11922 g (9364 - 14480 g)
- Housing: individually in kennels
- Diet: 400 mg of complete dry diet (Spratts Dog Diet) were offered daily
- Water: ad libitum
- Acclimation period: 4 weeks

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with: powdered diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative samples of dog diets containing the test substance were extracted with chloroform. The 500 ppm and 1500 ppm samples were then determined by TLC and visual comparison with standards. The 5000 ppm sample was determined by UV-spectrophotometry with the control extract in the reference beam and the test substance supplied with the samples as a reference standard. The estimated relative reproducibility of this method is ±10% or better. Within the limits of error of the sampling technique and the analytical method there was good correspondence between the values found and the nominal ones.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
500, 1500, 5000 ppm (eq. 14.7, 46.2, 148.7 mg/kg bw/d)
Basis:
nominal in diet

No. of animals per sex per dose:

4 - 5

Control animals:

yes, plain diet

Details on study design:

- Post-exposure recovery period in satellite groups: 4 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: during week days only

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before start of exposure and during week 4, 8 and 12
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once before start of exposure and during week 4, 8 and 12
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, food was withdrawn for 16 hours
- How many animals: all animals
- Parameters examined: erythrocyte sedimentation rate (ESR), packed cell volume (PCV), haemoglobin (Hb), red cell count (RBC), reticulocyte count (Retics), mean corpuscular haemoglobin concentration (MCHC), mean cell volume (MCV), total white cell count (WBC), differential count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes), platelet count, prothrombin index (PTI)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once before start of exposure and during week 4, 8 and 12
- Animals fasted: Yes, food was withdrawn for 16 hours
- How many animals: all animals
- Parameters examined: urea, glucose, total serum proteins, serum protein electrophoresis, alkaline phosphatase (AP), glutamic-pyruvic transaminase (GPT), bilirubin, sodium, potassium

URINALYSIS: Yes
- Time schedule for collection of urine: once before start of exposure and during week 4, 8 and 12
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes, food was withdrawn for 16 hours
- Parameters examined: pH, protein, reducing substances, glucose, ketones, bile pigments, urobilinogen, haemoglobin

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following organs were examined: brain, pituitary, heart, lungs, liver, spleen, pancreas, thymus, prostate/uterus, kidneys, thyroids, adrenals, gonads, aorta (arch and abdominal), trachea, lymph nodes (cervical and mesenteric), gall bladder, urinary bladder, salivary gland, tongue, oesophagus, stomach, duodenum, jejunum, ileum, colon, skin, mammary gland, skeletal muscle, bone marrow, peripheral nerve, eye and optic nerve,spinal cord

Other examinations:

ORGAN WEIGHTS: Yes
The following organs were weighed: brain, pituitary, heart, lungs, liver, spleen, pancreas, thymus, prostate/uterus, kidneys, thyroids, adrenals, gonads

Statistics:

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

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 examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs or mortality were observed in the course of the study.

BODY WEIGHT AND WEIGHT GAIN
From the figures illustrating bodyweight changes of individual animals, it can be seen that all dogs, with the exception of dog 566 (female) and 568 (female) (5000 ppm) showed an overall gain in weight during the pretreatment period. The latter animals were replacement dogs and lost weight during their first 2 weeks. However, both animals gained weight during the week prior to commencement of dietary administration. With the onset of dietary intake, no marked changes in bodyweight were seen. With the exception of dogs 539 (male), 541 (male) (Control) and 548 (female) (500 ppm) all dogs gained or maintained weight satisfactorily. These three animals all showed a slight weight loss over the 13 - week period of dietary intake. It should be noted, however, that minor fluctuations in bodyweight would be expected for dogs of this age. Examination of the group mean bodyweight graphs indicates that all groups gained weight at a similar rate. Statistical analysis of the data confirmed this observation, no significant group mean differences being demonstrated. During the 4 - week recovery period, all four animals showed an initial loss of bodyweight, with a subsequent return to normal.

FOOD CONSUMPTION AND COMPOUND INTAKE
Each dog was offered a basic ration of 2800 grams of powdered diet per week. Throughout the experimental period, appetite was good for all dogs, many animals consuming all of the food offered. During weeks 1 and 13 of treatment mean food consumption was 2625 (control), 2610 (500 ppm), 2694 (1500 ppm) and 2662 (5000 ppm) g per week for male and female animals.

WATER CONSUMPTION AND COMPOUND INTAKE
The quantities of water consumed by individual dogs during the 4 - week pre-treatment period, during weeks 1 - 4 and 9 - 12 of dietary intake, and during weeks 1 - 4 of the recovery period were measured on weekdays only. As the study progressed, the water intake increased, as would be expected for growing animals. Throughout the experimental period, the mean weekly quantities of water consumed were comparable, no significant group differences being demonstrated.

OPHTHALMOSCOPIC EXAMINATION
No abnormalities that could be associated with dietary intake were detected in male and female animals.

HAEMATOLOGY
All findings were within normal limits throughout the study period for male and female animals.

CLINICAL CHEMISTRY
Before dietary intake commenced plasma glucose levels in excess of our normal upper limit of 110 mg% were recorded for male dogs numbered 533, 537, 541 (Control); 543, 549 (500 ppm). Repeated investigations confirmed these findings.
After 4 weeks of dietary intake, male dog 541 (Control) again had a plasma glucose level in excess of 110 mg%, whilst the levels for male dogs 533, 537 (Control), 543 and 549 (500 ppm) were on this occasion within our normal limits. Female dog 542 (Control) had a serum potassium level in excess of our upper limit of 5.5 mEq/L and male dog 543 (500 ppm) had an GPT level that was above our normally accepted upper limit of 50 mU/mL. These findings were confirmed by repeated investigations. All other results were within normal limits.
Atfer 8 weeks of dietary intake male dogs 541 (Control) and 543 (500 ppm), as well as female dog 554 (1500 ppm) had GPT levels that were above our normally accepted upper limit of 50 mU/mL. Male dog 549 (500 ppm) had a plasma glucose level slightly in excess of normally accepted upper limit of 110 mg%. Repeated investigations confirmed these findings. All other findings were within normal limits.
After 12 weeks of dietary intake male dogs 541 (Control) and 565 (5000 ppm) had AP levels that were above our normally accepted upper limit of 35 KA units. Repeated investigations confirmed these findings. All other findings including GPT levels were considered to be within normal limits.

URINALYSIS
Before dietary intake commenced all specimens of urine were considered to be normal and all dogs were showing satisfactory urinary concentrations.
After 4 weeks of dietary intake the urine specimen from female dog 542 (Control) had a specific gravity of less than 1.035 which is our normally accepted lower limit. This was confirmed by a repeat investigation. All other specimens were considered to be normal and the remaining dogs were showing satisfactory urinary concentration.
After 8 and 12 weeks of dietary intake all specimens of urine were normal and all dogs were showing satisfactory urinary concentration.

ORGAN WEIGHTS
On completion of the macroscopic post mortem examination, the major organs were removed and weighed. These weights have been expressed as a percentage of the bodyweight and as a percentage of the brain weight. The organ weights for animals which were maintained for the 4 - week recovery period have been included in the study report, but excluded from the means and statistical analysis. The relative liver weights for male dogs numbered 537, 541 (Control), 565, 567 (5000 ppm) and 563 (5000 ppm - Recovery) were slightly in excess of our normally accepted upper limit of 4% of the bodyweight. The same has been observed in one female dog (No. 568) of the highest dosing group. Routine statistical analysis revealed that when expressed in grams, as a percentage of the bodyweight, and as a percentage of the brain weight, the mean liver weight for animals that had received 5000 ppm was significantly greater than that of the control mean, the differences being significant at the 1 % , 5% levels of probability respectively (LSD =99 grams, 0.63% and 88%). All other organ weights were considered to be within normal limits.

GROSS PATHOLOGY
The only findings which may possibly have been related to treatment were areas of congestion seen in the gastro-intestinal tract. Minimal congestion of the intestinal mucosa was seen in male dogs numbered 543, 547 (500 ppm); 555 (1500 ppm) and 559, 561, 565 (5000 ppm). This has also been observed in female dogs numbered 544, 546, 550 (500 ppm); 558 (1500 ppm) and 568 (5000 ppm). Similar observations were made for male dog 537 (Control - Recovery) and female dog 564 (5000 ppm - Recovery). This congestion was mainly confined to the jejunum and ileum.

HISTOPATHOLOGY: NON-NEOPLASTIC
Lungs: Minimal changes were encountered in the lungs in a proportion of control and treated animals. They included peribronchial, peribronchiolar, perivascular or interstitial foci of chronic inflammation and they were associated with several areas of pneumonitis in male dog 563 (5000 ppm, recovery). In addition, an area of bronchopneumonia was observed in female dog 558 (1500 ppm). These are common findings in the lungs of laboratory dogs and, as there was no evidence of a treatment-related effect, they were considered to have no toxicological significance.

Liver: Inflammatory cell foci, with or without eosinophils, were noted in occasional portal tracts in male dogs 539 (Control) and 547 (500 ppm). Similar effects have been observed in female dogs 536 (Control), 544, 546 (500 ppm) and 556 (1500 ppm). A focus of necrosis with acute inflammatory cell infiltration was identified in female dog 552 (1500 ppm). These changes were probably related to parasitic infestation and, thus, were considered to be of no importance.
Slight to moderate periportal glycogen depletion was observed in female dogs 540, 542 (Control), 550 (500 ppm), 556 (1500 ppm), 560, 562, 568 (5000 ppm). Since controls and animals of all test groups were similarly affected, this change was regarded as being without toxicological significance.
Varying degrees of bile duct hyperplasia with on associated inflammatory reaction were encountered in male dogs 551, 553, (1500 ppm) and 559 (5000 ppm) as well as in female dogs 560 and 568 of the highest dosing group. This change was not seen in any of the controls nor in animals receiving 500 ppm. In male dog 553 (1500 ppm) and female dog 560 (5000 ppm), the degree of bile duct hyperplasia was greater than that previously identified in untreated Beagle dogs. There was no evidence of bile duct hyperplasia in either of the two dogs receiving 5000 ppm after a recovery period of four weeks. These changes tended to be focal non-specific in nature, and probably related to parasitism. It is unlikely that they were related to treatment with the test substance. The significance of the intracytoplasmic, eosinophilic globules in hepatocytes in a single dog (female No. 552, 1500 ppm) was unknown.

Kidney: Treatment related changes were not detected. Minimal changes, commonly encountered in the kidneys of laboratory dogs, included the following:
fat deposition in the epithelium of a variable number of cortical tubules in the majority of control and treated dogs from all groups;
minimal calcification within a few medullary tubules in a proportion of control and treated dogs;
peripelvic chronic inflammation in female dogs 536 (Control), 544, 550 (500 ppm), 552 (1500 ppm) and 560, 562 (5000 ppm);
cortical foci of intestitial chronic inflammation in male dogs 543 (500 ppm) and 561 (5000 ppm) as well as in female dogs 540 (Control) and 566 (5000 ppm).

Intestine: There were no morphological findings corresponding to the mucosal congestion noted macroscopically at post mortem examination. Parasitic worms were seen within the lumen of the small intestine in male dog 539 (Control) and female dog 552 (1500 ppm) and minimal inflammatory changes, probably related to parasitic infestation, were identified in the mucosa or submucosa of the gastro-intestinal tract in female dogs 538 (Control, recovery), 544, 548, 550 (500 ppm).

No morphological change of toxicological significance was seen in any of the other tissues examined.

Dose descriptor:

NOAEL

Effect level:

148.7 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were seen in any of the animals at this dose.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

148.7 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Quality of whole database:

Acceptable and well documented study report, compliant to guideline.

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

90-day repeated dose toxicity study (Dog)

In a repeated dose toxicity study, the test substance was administered to pure bred Beagle dogs (5 animals per sex with control diet and at 5000 ppm; 4 animals per sex at 500 and 1500 ppm) (Huntingdon, 1978). The study was conducted equivalent or similar to OECD guideline 409. The dosing period was followed by a 4 week recovery period for one male and one female dog from each of the control and high dosage groups. Doses were analytically verified in testing representative samples of the dog diet. The estimated relative reproducibility was ±10 % or better. Within the limits of error of the sampling technique and the analytical method there was good correspondence between the values found and the nominal ones. None of the animals died during the course of the study. No compound related effects were seen in bodyweight, food consumption, water consumption, ophthalmoscopy, clinical chemistry and hematology. The following group mean intakes of the test substance were calculated:

Dose [ppm]	males [mg/kg bw/d]	females  [mg/kg bw/d]	male and female  [mg/kg bw/d]
500	14.7	14.7	14.7
1500	45.6	46.8	46.2
5000	150.3	147.0	148.7

 

One dog receiving the highest dose developed an oedematous swelling of the head and face on day 13, this subsiding after 24 hours. This was considered to be typical of a hypersensitive reaction but it seems unlikely that it was related to administration of the test compound. No other clinical signs were observed. During necropsy small areas of submucosal congestion were noted in the gastrointestinal tract of a number of dogs from all treatment groups, but in view of the histological findings relating to the intestine, it is unlikely that this was related to treatment. When expressed in grams, as a percentage of the bodyweight and as a percentage of the brain weight, the group mean liver weight for animals which had received 5000 ppm was significantly greater than the control mean. These findings were not correlated with histopathological findings or altered liver enzyme parameters. The higher liver weight was therefore not regarded as adverse effect. No morphological change of toxicological significance was seen in any of the tissues examined and in particular there were no findings corresponding to the mucosal congestion noted at macroscopic post mortem examination. A NOAEL of 148.7 mg/kg bw/d was determined for male and female dogs.

 

90-day repeated dose toxicity study (Rats)

In a repeated dose toxicity study the test substance was administered to 8-9 weeks old rats (15/sex/dose) in diet at dose levels of 0 (control), 2000, 10000 and 30000 ppm for 90 consecutive days (Geigy Ltd, 1970). The study was conducted equivalent or similar to OECD guideline 408. Therefore a generic food intake of 15 g/day (based on a body weight up to 250 mg) and 20 g/d (based on a body weight above 250 mg) was assumed. Based on the mean food consumption and mean body weight data given in the report, the following dose levels were calculated:

 

Dose [ppm]	males [mg/kg bw/d]	females  [mg/kg bw/d]	male and female  [mg/kg bw/d]
30000	1760	1675	1718
10000	529	592	561
2000	111	121	116

 

The general health of the animals at 2000 and 10000 ppm was good throughout the study period. Mortality was observed in the two highest dosing groups. At 10000 ppm three male animals died during week 6 and 9. At 30000 ppm six male and female animals died or had to be killed in extremis. Normal body weight gain was observed in the lowest dosing group. At 10000 ppm the female animals showed a slightly but significantly lower body weight gain compared to the control group. At 30000 ppm body weight gains were significantly lower in both male and female animals. The body weight of the male animals was 20 % lower than compared to the body weights in the control group. The reduced body weights of the female animals of the two highest dosing groups were well within 10 % of the body weight of the control group. Food consumption was normal for both male and female animals in the lowest dosing group. In the two highest dosing groups food consumption was significantly lower compared to controls. No substance related adverse effects were observed for hematology, urine analysis as well as ophthalmoscopic examination. A significant increase in alkaline phosphatase was observed in male and female animals of the highest dosing group. In all test groups there is weight increase of thyroids and livers compared to controls, as shown by statistical analysis of gross organ weights and percentage organ weights. Thyroid hypertrophy was reported starting at the lowest dose level with a dose dependent intensity increase. From 10000 ppm, liver hypertrophy and abnormal fatty infiltrates into the convoluted epithelium of the kidneys were reported in addition. At the highest dose level, the hypertrophy of the liver and the thyroids became marked. Based on these results, a LOAEL of 111 mg/kg bw/d (males) and 121 mg/kg bw/d (females) was determined.

90-day repeated dose toxicity study (Rats)

In a repeated dose toxicity study the test substance was administered to young rats (10/sex/dose) in diet at dose levels of 0 (control), 400, 2000, 10000 and 30000 ppm for 90 consecutive days (Hoechst, 1972). No food intake of the test animals was recorded in the study report. Therefore a generic value of 0.08 mg/kg bw/d was assumed equal to 1 ppm leading to dose levels of 0 (control), 32, 160, 800 and 2400 mg/kg bw/d. The study was conducted equivalent or similar to OECD guideline 408.

Throughout the experiment, the behaviour of all the animals was normal and no mortality was observed. In all animals in Groups I (30000 ppm), II (10000 ppm) and III (2000 ppm) food consumption was distinctly reduced immediately after the beginning of the experiment. From the first week onwards it was normal and comparable to that of the animals in Group IV (400 ppm) and the control group. Body-weight gain in the male rats was lower in the high dose group during the first four weeks of the experiment, when the body weights of the males were distinctly lower than those of the controls. In the males treated with 2000 and 400 ppm test substance, body weight was greater than in the controls throughout the experiment. In the females, only those in the group given 2000 ppm of test substance in the feed showed no difference in weight gain from the controls. The body weights of the females that received 30000 and 10000 ppm were significantly lower (p < 0.005) than in the control group. From the fourth week of the experiment onwards, the weights of the animals in Group IV (400 ppm) were also lower than those of the controls (p <0.01). Haematological examinations and urine analysis revealed no pathological changes, even after 90 days' treatment. Corneal opacities and changes in the teeth were not detectable. Determinations of serum enzyme activity 24 hours and 13 days after the conclusion of the experiment gave no indication of any change definitely attributable to treatment with the test substance. In the animals killed 24 hours after the withdrawal of the substance, a clear-cut, dose related increase was noted in the weights of the liver and the thyroid gland. Liver-weight was increased in the males and females of Group I (30000 ppm) and thyroid-weight in the males and females of Group I and II (30000 and 10000 ppm). In the animals killed 13 days after the withdrawal of the substance the same tendency was still discernible, but the differences between the individual groups and the controls, especially in regard to the weight of the liver and thyroid were by then so slight as to be statistically non-significant. Histological examination of the organs of the treated animals revealed that under the given experimental conditions the test substance in a dosage of 2000, 10000 and 30000 ppm led to epithelial hyperplasia of the thyroid, i.e. to hyperactivity of the thyroid. The nucleus size of the thyroid epithelia in the group given 400 ppm was not significantly different from that of the controls. A NOAEL of 32 mg/kg bw/d (400 ppm) was determined for male and females rats.

30-day repeated dose toxicity study (Rats)

In a repeated dose toxicity study the test substance was administered to Wistar rats (10/sex/dose) in diet at dose levels of 0 (control), 400, 2000, 10000 and 30000 ppm for 30 consecutive days (BASF, 1971). The study was conducted equivalent or similar to OECD guideline 407. No mortality or clinical signs were observed throughout the study period. The general behaviour of the animals of all dose groups did not differ from the behaviour in the control group. The body weight gain was significantly reduced in the two highest dosing groups of the male and female animals. In the females the reduction differed more than 10 % from the mean body weight of the control animals in the highest dosing group. No significant effects on the body weight were observed in the other dosing groups. At the beginning of the study, food consumption was reduced in the three highest dosing groups. After the first treatment week food intake of these groups had normalized and was comparable to the lowest dosing group and the control group. No compound related effects were seen in hematology, urinanalysis and gross pathology in any of the treated groups. Regarding clinical chemistry the serum GOT and GPT values were explicitly raised in the highest dosing group of both male and female animals. In the other dosing groups the clinical chemistry parameters were well within the range of variation. The absolute organ weights of liver and thyroid were significantly raised in the highest dosing group of male and female animals. The relative organ weights show a dose dependent increase of the liver weights in the three highest dosing groups in male and female animals. Furthermore an increase of thyroid weight was observed in the male and female animals at the 30000 ppm dosing group. Histological examinations revealed epithel hyperplasia in the thyroid, i.e. a thyroid activation of the 2000, 10000 and 30000 ppm dosing groups. At the lowest dosing group, the thyroidal epithel was not significantly different from that of the control group. A NOAEL of 400 ppm was determined.

10-day repeated dose toxicity study (Rats)

In a repeated dose toxicity study, the test substance was administered to Tif: RAI rats (5/sex/dose) per gavage at dose levels of 0 (control), 30, 100 and 300 mg/kg bw/d for 10 consecutive days (Ciba-Geigy, 1977). The study was aiming towards thyroid function and included measurements of T3 and T4 levels and the binding capacity of T3. No mortality was observed and no clinical signs were detected in any of the tested animals. The test animals gained weight throughout the study period. Regarding clinical chemistry, disturbances in T3 and T4 levels were observed in the highest dosing group. Cholesterol was decreased in the 30 and 300 mg/kg dosing group, indicating an increase in thyroid function. Histopathological findings included signs of focally increased activity of the thyroid showing minimal hypertrophy and hyperplasia of the epithelial layer of the follicles and focal decrease of the stainable colloid. There was also slightly increased number of mitosis in the thyroid apparent.

Conclusions

The main findings of the studies performed with rats are increases in liver and thyroid weights correlating with liver and thyroid hypertrophies. These findings are species specific and of no relevance humans. This species-specific mechanism causing the thyroidal stimulation following uptake of 2,6-substituted phenols (and similar compounds) in rats has been described in standard textbooks and is generally acknowledged by the scientific community (Davies, D.T. (1996). Thyroid Endocrinoloy. Animal Clinical Chemistry: A primer for Toxicologists. Ed: A.O. Evans and Francis). From basic research performed over several years, it became clear, that the stimulation of glycuronidyl-transferase synthesis is used to facilitate excretion of 2,6-substituted phenolic antioxidants. However, glycuronidyl-transferase is also able to promote the excretion of thyroid hormones. Since in the rat, the thyroid hormones T3 and T4 are not bound to a specific carrier protein in the plasma, increased levels of glycuronidyl-transferase (induced e.g. by 2,6-substituted phenolic antioxidants) speed up the elimination of T3 and T4. This causes a drop of the plasma concentrations of T3 and T4. By feedback mechanism, reduced levels of T3 and T4 hormone subsequently lead to a stimulation of the thyroid to produce new hormone. The chronic stimulation of the thyroid can cause hyperplasia and can lead in extreme cases to thyroid tumours. In primates, about 90% of the thyroid hormones T3 and T4 are bound to a specific protein (thyroxine binding protein) in the plasma. Due to this protein, primates are protected from significant changes of thyroid hormone levels in the plasma, even in the presence of high levels of glycuronidyl-transferase. Consequently, in primates thyroid hormone concentrations are not affected, no feedback mechanisms are induced to produce new T3 and T4 and no thyroid hyperplasia occurs. The results obtained using dogs confirms this mechanism to be rat specific. As a consequence, the NOAEL obtained in the dog study is considered the most relevant threshold for human risk assessment.

Disregarded Study:

In a repeated dose toxicity study the test substance was administered to young rats (15/sex/dose) in diet at dose levels of 0 (control), 1000, 3000 and 10000 ppm for 90 consecutive days (IBT, 1971). The study was conducted equivalent or similar to OECD guideline 408. The following group mean intakes were calculated based on weekly body weight and food consumption data:

Dose [ppm]	males [mg/kg bw/d]	females  [mg/kg bw/d]	male and female  [mg/kg bw/d]
10000	785	1060	923
3000	241	297	269
1000	80	95	88

A few deaths occurred during the study. In general there were one or two that died from each group. These deaths were ascribed to acute respiratory infections. They occurred equally in all groups including controls which indicated that they were not caused by ingestion of the compound. No untoward behavioral reactions were noted among any of the animals employed in the study. No treatment effects were observed for body weight or food consumption. No gross internal lesions were observed during necropsy. No changes were reported for hematology, clinical chemistry, organ weight measurements.

However, since this study was performed by a CRO that was known for reporting false data, this result is not taken into consideration, especially since it does not support the findings in the other available rat studies. It is therefore disregarded and not used for risk assessment.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Acceptable and well documented study report, compliant to guideline.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result, the substance does not need to be classified and labelled for repeated dose toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EC.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation EC No 605/2014.