Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2-ethylhexyl ester

Carcinogenicity

Description of key information

No carcinogenicity was observed in feeding studies with rats and mice (Huntingdon, 1974 and Ciba-Geigy, 1982) performed similar to OECD testing guidelines 453 and 451, respectively. Both studies were performed with an analogue substance.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Principles of method if other than guideline:

No report on analytical procedures and results. No analysis of the lowest dose level. No analysis of homogeneity of the initial preparation.

GLP compliance:

no

Species:

mouse

Strain:

other: Tif: MAGf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production Stein, CIBA-GEIGY LTD.
- Age at study initiation: approx. 4 weeks
- Mean group weight at study initiation: 21.1-21.5 g (males), 20.3-20.8 g (females)
- Housing: 5 per cage
- Diet (e.g. ad libitum): Pelleted, certified standard diet Nafag No. 890 Tox
- Water (e.g. ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±10
- Air changes (per hr): 15-17
- Photoperiod (hrs dark / hrs light): 10

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- the test item was weighed on a calibrated Mettler balance. The pulverised food was then homogeneously mixed with the appropriate concentrations of the compound and 30 % water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently airdried. The animals in the control group were fed with similarly pelleted food without compound.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the initiation of the study pretest feed samples were analysed for concentration and stability of the test material. The same was undertaken with the food batches applied during the test. The analytical results revealed a concentration of 82-95 % of the nominal value. The analytical results of the diet of the lowest dose groups (5 mg/kg feed) are not available for technical reasons: The limit of detection was at 5 to 10 mg/kg feed.

Duration of treatment / exposure:

24 months

Frequency of treatment:

continously

Post exposure period:

none

Dose / conc.:

5 mg/kg diet

Remarks:

corresponding to 0.6 mg/kg in males and females

Dose / conc.:

50 mg/kg diet

Remarks:

corresponding to 5.4 mg/kg in males and females

Dose / conc.:

500 mg/kg diet

Remarks:

corresponding to 58 mg/kg in males and 54 mg/kg in females

No. of animals per sex per dose:

50

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: symptoms, mortality

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, after 3 months monthly

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

- Macroscopic: complete gross examination
- Microscopic: adrenals, aorta, bone marrow, brain, caecum, colon, duodenum, epididymis, eyes, gall bladder, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (axillary and mesenteric), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder and uterus, and all nodules, tissue masses and otherwise macroscopically abnormal tissues.

Other examinations:

Liver, adrenals, brain, heart, kidneys and gonads were weighed.

Statistics:

- Uni-variate statistical comparison of the treatment group with control group (Lapage Y., Biometrika 1971) and a Trend test (Jonckheere H.R., Biometrika, 1954) considering all groups.
- Generalised Wilcoxon Test (Breslown., Biometrika, 1970)
- Generalised Savage Test (Mantel-Cox, Cancer Chemotherapy Reports 1966, 50: pp. 163-170)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical symptoms and no signs of local and/or systemic toxicity were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

The highest dose group of treated males and females showed a tendency to shortened survival time compared to the respective control group (males: 583 Vs 695 in controls; females: 667 Vs 709 in controls), however not significant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight gain in all treated male and female groups was similar to that of the respective control groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of all treated male and female groups was similar to that of the respective control groups. Based on the nominal value the mean daily intake in male and female of 5 mg/kg-feed group was 0.6 mg/kg bw. Corrected for the actual amount of substance, found by chemical analysis, the dose in 50 and 500 mg/kg- feed groups was 5.4 and 58 mg/kg bw in males and 5.4 and 54 mg/kg bw in female animals, respectively.

Food efficiency:

no effects observed

Description (incidence and severity):

Specific food consumption in relation to body weight - adressed as food conversion ratio - of all treated animal groups was similar to that of the respective control groups.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The analysis of organ weights and organ weight ratios revealed no treatment related effects.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross changes in the organs and tissues related to the treatment were noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic changes in the organs and tissues related to the treatment were noted.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Numerous benign and malignant tumours were observed in both control and treated mice. Frequency and type of the neoplasms occurring in these animals were not influenced by the treatment.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 58 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: carcinogenicity

Remarks on result:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

> 54 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: carcinogenicity

Remarks on result:

other: highest dose tested

Key result

Critical effects observed:

no

Conclusions:

No systemic toxicity or microscopic changes in the organs and tissues were observed. The only effect observed was the shorter survival time in the animals of highest dose group. Thus, the NOAEL for carcinogenicity was considered to be ≥ 54 and 58 mg/kg bw/d for the female and male animals, respectively. The test substance does not require any classification for carcinogenicity according to EU or GHS standards.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Comparable to guideline, without GLP (acceptable with restrictions); No GLP study - Limited haematology, biochemistry and urinalysis only on the control and high dose groups instead of all groups - No analysis of the accuracy of preparation of the diet were reported. Actual test article intake only calculated values - No historical data included

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Principles of method if other than guideline:

- No GLP study
- Limited haematology, biochemistry and urinalysis only on the control and high dose groups instead of all groups
- No analysis of the accuracy of preparation of the diet were reported. Actual test article intake only calculated values
- No historical data included.

GLP compliance:

no

Species:

rat

Strain:

other: CFY

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Carworth Europe, England
- Age at study initiation: 27 ± 1 days
- Mean weight at study initiation: male 123 g, female 103 g (differing from one another by no more than ± 2.5 g)
- Housing: 5 per cage
- Diet (e.g. ad libitum): powdered laboratory rat food (Spratt's Laboratory)
- Water (e.g. ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 55±5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- A premix containing 30000 ppm was prepared each week, and from this the different dietary concentrations were obtained by direct dilution with further quantities of diet. Homogeneity was achieved by mixing for ten minutes in a rotary double-cone blender; all diets were stored until use in heat-sealed, opaque polythene bags.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the diets fed to the rats have been analysed for their content of the test item by a thin layer chromatographic method.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

continously

Post exposure period:

none

Dose / conc.:

500 ppm (nominal)

Remarks:

corresponding to 22 mg/kg in males and 27 mg/kg in females

Dose / conc.:

1 500 ppm (nominal)

Remarks:

corresponding to 64 mg/kg in males and 81 mg/kg in females

Dose / conc.:

5 000 ppm (nominal)

Remarks:

corresponding to 218 mg/kg in males and 275 mg/kg in females

No. of animals per sex per dose:

50

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, all signs of ill-health and toxicity, together with any behavioural changes. Detailed descriptions were made of any skin lesions, cataracts or palpable growths, together with the progression or regression of such lesions.

BODY WEIGHT: Yes
- Time schedule for examinations: initially, then at weekly intervals for the first twelve weeks and at four-weekly intervals thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal 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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes,assessed in the first instance by inspection of the water bottles
- Time schedule for examinations: Accurate measurement of water intake was introduced during week 37 since a treatment-related effect on water consumption was suspected

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced, and after 13, 26, 52, 75 and 102 weeks
- Dose groups that were examined: control and 5000 ppm groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 13, 26, 52, 78 and 103 weeks of treatment
- Anaesthetic used for blood collection: samples of blood were withdrawn from the orbital sinus
- How many animals: 10 males and 10 females from control and 5000 ppm groups
- Parameters checked: Packed cell volume (PCV), Haemoglobin (Hb), Red cell count (RBC), Mean corpuscular haemoglobin concentration (MCHC), mean cell volume (MCV), Total white cell count (WBC), Differential count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 13, 26, 52, 78 and 103 weeks of treatment
- How many animals: 10 males and 10 females from control and 5000 ppm groups
- Parameters checked: Plasma Urea nitrogen, Plasma Glucose, Serum alkaline phosphatase (SAP), Serum glutamic-pyruvic transaminase (SGPT)

URINALYSIS: Yes
- Time schedule for collection of urine: After 13, 26, 52, 78 and 103 weeks of treatment
- Parameters checked: pH, Specific gravity (SG), Protein, Reducing substances, Glucose, Ketones, Bile pigments, Urobilin, Blood pigments, Microscopy (examined for epithelial cells, polymorphonuclear leucocytes, mononuclear leucocytes, erythrocytes, organisms, casts, abnormal constituents)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

- Macroscopic: complete gross examination
- Microscopic: adrenals, aorta, brain, caecum, colon, duodenum, eyes, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), optic nerve, ovaries, pancreas, pituitary, prostate, rectum, sciatic nerve, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, urinary bladder and uterus, and all nodules, tissue masses and otherwise macroscopically abnormal tissues. Examinations were performed in decedents and 10 males and 10 females from the control group and 5000 ppm group.

Other examinations:

The weights of the following organs were recorded: adrenals, kidneys, pituitary, brain, liver, spleen, heart, testes, thyroid

Statistics:

Student's 't' test was employed to assess the significance of intergroup differences where the data suggested a treatment-related effect.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no overt signs of reaction to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

The survival rates among treated rats were comparable with those of the controls, with the exception of females exposed at 5000 ppm where a marginally superior survival (33 vs 23 in control) was recorded during the 104 week treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Although a superior rate of bodyweight gain was recorded during weeks 24 to 52 and weeks 80 to 104 among males treated at 1500 ppm, the apparent effect was not dosage related and, therefore, was not considered to be of toxicological significance. Bodyweight gain of other treated males was comparable with that of the controls. Females receiving 5000 ppm had an inferior bodyweight gain throughout the treatment period, although the difference from the control value was not statistically significant after week 52. Bodyweight gain of other treated females was comparable with that of the confrols.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A reduction in food intake was recorded among female rats receiving 5000 ppm during the 104 week treatment period and among females receiving 1500 ppm during the first 80 weeks of freatment. During weeks 53 to 80, a significant reduction in food intake was recorded among all male and female treated rats, although among freated males there was no evidence of a dosage relation. The mean intake of test material (mg/kg bw/day) during the 104 week treatment in males were 22, 64 and 218, respectively, and in females were 27, 81, 275, respectively.

Food efficiency:

no effects observed

Description (incidence and severity):

The efficiency of food utilization among male rats was not disturbed by treatment during the period of fastest growth, i.e. weeks 1 to 24. Among female rats receiving 5000 ppm, a minor impairment of the efficiency of food utilization was recorded during weeks 9 to 16 and 21 to 24. There was, however, no marked overall between-group difference in the efficiency of food utilization among female rats during the period of fastest growth.

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

no effects observed

Description (incidence and severity):

As the water intake of male and female rats during 37 receiving 5000 ppm was found to be closely comparable with that of the confrols, further measurements were not performed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No changes attributable to treatment were observed.

Haematological findings:

no effects observed

Description (incidence and severity):

At the week 13 investigation, a small decrease in the mean haemoglobin value was recorded for male rats receiving 5000 ppm (P<0.01) and a small increase in the mean cell volume (MCV) among female rats receiving the same dosage (P<0.01). No statistically significant between-group differences were observed at the 26 week investigation. At week 52, male rats receiving 5000 ppm showed a small increase in the total white blood cell count (P<0.05) with an increase in the neutrophil value, attaining a level of statistical significance (P<0.05). At week 78 male rats receiving 5000 ppm showed a small decrease in red blood cell count (P<0.001), associated with an increase in the mean cell volume (MCV), (P<0.01), and a small decrease in the total white cell count (P<0.05) with a decrease in the lymphocyte value attaining a level of statistical significance (P<0.05). A small decrease in the mean cell volume (P<0.05) was recorded among female rats receiving 5000 ppm. No statistically significant between-group differences were observed at the week 103 investigations with the exception of a small increase in mean cell volume (MCV) among females receiving 5000 ppm (P<0.05), associated with a marginal, but not statistically significant decrease in red cell count. Since the changes recorded during the course of the study were of a small order of magnitude, lacked consistency in relation to time and sex, and all values were within the normal range for the strain of rat employed, it was considered that the differences were of doubtful biological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

At the 13 and 26 week investigations, rats receiving 5000 ppm showed a small but significant reduction in plasma glucose levels when compared with the control values. (Week 13 - males P<0.05, females P<0.001; week 26 - males and females P<0.05). At week 52, only the females of this group showed glucose values which were significantly different from those of the controls, (P<0.01). At the week 78 investigation, male rats receiving 5000 ppm showed a small reduction in plasma glucose levels (P<0.001), while females treated with the same dosage showed a small increase in plasma glucose levels (P<0.05). Although a level of statistical significance was attained by the marginal difference between SAP levels recorded among controls and treated female rats (P<0.05) the difference was not considered to be biologically significant. No statistically significant between-group differences were recorded at the week 103 investigations.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes attributable to treatment were recorded at week 13 and 52. At the 103 week investigations, the only significant between group difference observed was a decrease in the protein in the urine of male (P<0.05) and female (P<0.01) rats compared to control values.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight analysis performed on rats killed after 104 weeks of treatment revealed heavier absolute liver weight in males treated with 5000 ppm. When expressed relative to bodyweight, males and females treated with 5000 ppm had liver weights heavier than those of the controls. In the absence of any histopathological change, it was considered that these changes probably represent the effect of work hypertrophy. Increased weights of thyroids in rats treated with 5000 ppm and decreased weights of adrenals in females treated with 5000 or 1500 ppm were recorded. As histopathological examination revealed no apparent change, these changes could not be unequivocally attributed to treatment. Due to the uniformity of brain weight recorded in rats employed on this study, expressing the organ weights relative to brain weight resulted in similar differences as recorded in the absolute data. Other small differences in weights of organs from treated rats were attributed to the intergroup disparity in bodyweight and were considered to be of no toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The prime cause of death among rats dying during the course of the study showed no relation to treatment. Macroscopic examination of decedents and rats killed after 104 weeks of treatment showed pathology which was common to animals from control and treated groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No morphological abnormalities or variation from normal were seen in any of the tissues examined which was considered to be due to the administration.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The administration was not associated with any evidence of a tumorigenic effect on the spontaneous tumour incidence of the CFY rat.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 218 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: carcinogenicity

Remarks on result:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

>= 275 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: carcinogenicity

Remarks on result:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

64 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on a reduction of body weight in females and effects on organ weights reported at 5000 ppm in both sexes, the NOAEL is set at 1500 ppm. Effects on organ weights were not taken into account because of the absence of histopathological findings.

Key result

Critical effects observed:

no

Conclusions:

No changes attributable to treatment were observed in clinical or biochemical. No non-neoplastic or neoplastic effects were observed in any of the tissues examined. Based on the results, the NOAEL for carcinogenicity was considered to be ≥ 218 mg/kg bw/day (males) and ≥ 275 mg/kg bw/day (females).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

218 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Valid and reliable study with rats.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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 is not considered to be classified for carcinogenicity under Regulation (EC) No 1272/2008, as amended for the eighth timeRegulation (EU) No 2016/218.

Additional information

The read-across substance was tested for its carcinogenic potential in 2 species (rats and mice). Fifty male and 50 female rats per dose group were fed diets containing the test substance (22, 64 and 218 mg/kg bw in males, and 27, 81 and 275 mg/kg bw in females) for 104 weeks. A dietary control group was also included. Similarly mice were fed with the test substance (0.6, 5.4 and 58 mg/kg bw/ day in males; 0.6, 5.4 and 54 mg/kg bw in females) for 104 weeks. Survival in both species was similar to that of controls and no neoplasms were observed.

Summarized, no carcinogenic potential was detected by feeding maximal dose levels tested in both the species.