Iso(C10-C14)alkyl (3,5-di-tert-butyl-4-hydroxyphenyl)methylthioacetate

Endpoint summary

Description of key information

The NOAEL for the test substance (described in section 1.2) was determined at 50 mg/kg bw/day and the NOEL at 5 mg/kg bw/day in repeated dose (subacute) oral toxicity studies in rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-10-18 to 1990-05-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Centre d'Elevage Charles River (76410 Saint-Aubin-les-Elbeuf, France)
- Age at study initiation: 6 weeks
- Weight at study initiation (mean): Males: 200 g; Females: 157 g
- Housing: housed in suspended wire-mesh cages (43.0 X 21.5 X 18,0 cm) and each cage contained 2 rats of same sex and group
- Diet: A04 C pellet diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days under test conditions, following a health examination

ENVIRONMENTAL CONDITIONS
- Temperature 21 +/- 2°C
- Humidity: 50 +/- 20 %
- Air changes: about 13 cycles/hour of filtered non-recycled air
- Photoperiod: 12-hrs light, 12-hrs dark

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was diluted in peanut oil. The solutions were homogenized by a magnetic stirrer. The preparations were performed once a week by the C.I.T. Pharmacy,

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On Weeks 1 and 4, 2 separate samples of each preparation (vehicle included) were analysed in duplicate. The results showed a satisfactory correlation between obtained and theoretical concentrations. The stability of the test substance was checked in the preliminary study No, 4972 TSR (Ciba-Geigy Test No. 884143), and the analyses showed a good stability of all preparations stored at +4°C.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once a day, approximately at the same daily time (in the morning), 7 days per week

Remarks:

Doses / Concentrations:
5, 50, 500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 male and 12 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon the results of a previously conducted study: 4972 TSR (Ciba-Geigy Test No. 884143), in rats.
- Post-exposure recovery period in satellite groups: 6 males and 6 females from each group were kept for a recovery period of 15 days.

Positive control:

None

Observations and examinations performed and frequency:

MORTALITY / VIABILITY
Observations for mortality/viability were recorded at least twice a day.

DETAILED CLINICAL OBSERVATIONS
Clinical signs were recorded for each animal, at least once a day.

FOOD CONSUMPTION
The quantity of food consumed by the animals of each cage was recorded once a week, over a period of 7 days until the end of the study, including
the recovery period.
Food intake per animal and per day was calculated using the amount of food given and left in each cage. If one of the 2 animals from the same cage died, the exact time of presence of that animal in the cage was taken into consideration for the calculation of the weekly food consumption of the cage.

BODY WEIGHTS
The bodyweight was recorded for each animal once before allocation of the animals into groups, on the first day of treatment and once a week until the end of the study, including the recovery period.

CLINICAL LABORATORY INVESTIGATIONS
Blood samples were withdrawn, approximately 24 hours after treatment, in weeks 4 and 6 from the orbital sinus in animals under light ether anaesthesia. The animals were placed in metabolism cages and fasted overnight about 18 hours for urine collection.

HEMATOLOGY
In all surviving animals on week 4 and at the end of the recovery period (week 6).
Parameters checked: Erythrocytes (RBC), Haemoglobin (HB), Mean Cell Volume (MCV), Packed Cell Volume (PCV), Mean Cell Haemoglobin, Concentration (MCHC), Mean Cell Haemoglobin (MCH), Leucocytes (WBC), Thrombocytes (PLAT), Differential White Cell Count, Quick Time (QT), Activated Partial Thromboplastine Time (APTT), Methaemoglobin (METHB).

CLINICAL BIOCHEMISTRY/BLOOD CHEMESTRY
In all surviving animals on week 4. Some parameters indexes were determined at the end of the recovery period (week 6) in all surviving animals.
Parameters checked: Sodium (NA+), Potassium (K+), Chloride (CL-), Calcium (CA++), Inorganic Phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA),
Creatinine (CREAT), Total bilirubin (TOT.BIL), Total proteins (PROT), Cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Gamma glutamyl transferase (GGT), Protein electrophoresis.

URINALYSIS
The following Parameters were determined in all surviving animals on week 4: volume (VOLUME, ml), appearance (APP), , pH (PH), specific gravity (SP.GRAV), urobilinogen (UROB, U% = Ehrlich unit/100 ml), proteins (PROT), glucose (GLUC), ketones (CETO), bilirubin (BILI), blood (BLOOD), nitrites (NITR) using N-Multistix SG test strips (AMES) (Miles, 75755 Paris, France).
Microscopy of sediment after centrifugation: investigation for leucocytes (WBC), erythrocytes (RBC), hyaline (HYAL) and granular (GRAN) cylinders, ammoniaco-magnesium phosphate (AMM.PH,), calcium phosphate (CAL.PH) and calcium oxalate (CAL.OX) crystals, epithelial (EPITH.), bladder (BLAD) and kidney (KIDN) cells.

MACROSCOPIC EXAMINATION
A complete gross examination was performed on all animals including the animal that died during the study.

ORGAN WEIGHTS
In all the surviving animals at the end of the treatment and recovery periods, the following organs were weighed wet as soon as possible after dissection: adrenals, kidneys, liver, ovaries and testes. Paired organs were weighed separately, except the adrenals which were weighed together.

MICROSCOPIC EXAMINATION
All tissues required for microscopic examination were embedded in paraplast, sectioned at approximately 4 microns in thickness and stained with hemalum-eosin.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Microscopic examination was performed on the following tissue:
adrenals
brain including medulla/pons, cerebellar and cerebral cortex (*)
eyes (*)
femoral bone with bone marrow (*)
heart
kidneys
liver
lungs (*)
ovaries
spleen
stomach
testes and epididymides
thyroids and parathyroids (*)
urinary bladder (*)
kidneys
tissues marked by (*) were preserved in fixative for an eventual microscopic examination

Statistics:

The following sequence was used for the statistical tests of the clinical parameters (bodyweight and food consumption), and for the haematological and biochemical parameters, and for organ weights:
- Kolmogorov-Smirnov's test (Smirnov, 1948)
- Mann-Whitney's test (Mann & Whitney, 1947)
- Bartlett's test (Bartlett,1937)
- Fisher's test (Fisher, 1934)
- Dunnett's test (Dunnett, 1955)

Details on results:

CLINICAL SIGNS AND MORTALITY
One female of the vehicle control group died. The cause of death was accidental following a blood sample. No mortality occurred during the recovery period. Hypersalivation was observed after treatment in all males and in 3/12 females of the 50 mg/kg bw/day group and in all males and females of the 500 mg/kg bw/day group. This clinical sign was considered treatment related and was observed sooner as the dose level became higher and it disappeared during the recovery period. Additional clinical signs (area of hair loss on head and/or cutaneous lesions and/or soiled urogenital area) were observed in some treated animals. These clinical signs are commonly observed in rats of this strain and consequently were not considered to be treatment-related.

BODY WEIGHT AND WEIGHT GAIN
A slight retardation in bodyweight gain was observed in males of the 500 mg/kg/day group from the second week to the end of the treatment period resulting in lower bodyweights compared to control animals (mean 9%). This change was considered to be treatment-related. Otherwise, a very slight retardation in the bodyweight gain was observed in the males of the 5 mg/kg/day group after 3 weeks of treatment. Due to the fact that it was very slight, not statistically significant and without dose-relationship, it was considered to be of no toxicological significance. During the recovery period, the growth of the males of the 5 and 500 mg/kg/ day groups was normal, but the difference of the bodyweight between the treated and control animals was not reduced.

FOOD CONSUMPTION
A slight decrease in food consumption was observed in males of the 500 mg/kg/day group during the treatment period (mean 9%) which could be related to the slight retardation in the bodyweight gain and was considered to be treatment-related. At the end of the recovery period the observed change was very slight, punctual and concerned only 2 animals of the same cage. Consequently, it was considered to be of no toxicological significance. A slight increase in food consumption was observed in females of the 500 mg/kg/day group from Week 2 to Week 4 (mean 17%) without an effect on bodyweight gain and consequently considered to be of no toxicological significance.

HAEMATOLOGY
A moderate increase in activated partial thromboplastin time (APTT) was observed in the males of the 500 mg/kg/day group (approximately + 70%), together with a slight increase in Quick time (QT, approximately + 40%). These changes were no longer observed after the recovery period. The relationship of the above-mentioned increases in APTT and QT to the treatment cannot be ruled out. All other variations of the erythrocytic parameters were minor, not dose-related, and the individual values were within the normal range of the background data.

CLINICAL CHEMISTRY
At 500 mg/kg/day, the following variations were observed on Week 4 when compared with the control:
- a slight decrease in chloride, slight increase in bilirubin, moderate increase in cholesterol, total proteins, albumin, alpha-1 and beta globulins in both sexes.
- slight increase in creatinine and aminotransferase activity (ALAT), and a slight decrease in calcium and triglycerides were noted only in the males.
- a slight decrease in potassium and ASAT activity was noted only in the females.
- slight changes in urea were observed (increase in males, decrease in females).
Reversibility was found after a 2 week recovery period for the above mentioned modifications except for the increase in cholesterol which was still present in the females. The increases observed in cholesterol, total proteins, albumin and globulins were either above or at the upper limit of the background data, therefore, they were considered to be treatment-related. The other variations mentioned above were considered to be of no toxicological significance, since they were minor and the individual values were still within the normal range of the background data.

URINALYSIS
No abnormality that could be related to the treatment was noted.

ORGAN WEIGHTS
The analysis of organ weights at the end of treatment period revealed:
- marked increase in the absolute (76% males – 112% females) and relative (104% males – 110% females) liver weights in the animals of both sexes of the 500 mg/kg/day group
- slight decrease in the absolute and relative weights of the adrenal glands in the males of the 50 mg/kg/day group {24% and 21% respectively).After the withdrawal period, no changes in organ weights were found in the liver. A slight decrease in the absolute weight of the adrenal glands was found in the males of the 500 mg/kg/day group (20%), whereas the weight of adrenal glands in the males of the 50 mg/kg/day group was comparable with the control animals. In the absence of relevant histopathological changes in the adrenal glands, the variations noted were considered to be of no toxicological significance.

GROSS PATHOLOGY
Liver enlargement was noted in 3/6 males and 6/6 females of the 500 mg/kg/ day group sacrificed at the end of treatment. Evidence of reversibility was observed after the recovery period. The few other macroscopical findings encountered were commonly recorded in the Laboratory Rat and thus considered to be of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
The only change considered to be treatment-related was found in the liver. Centrolobular hepatic cell hypertrophy was found in both sexes of the 500 mg/kg/day group. This was on line with the liver enlargement and the increased liver weights seen in these animals. Furthermore, there was no evidence of any nuclear/ cytoplasmic degenerative/necrotic changes. Therefore, the hepatic cell hypertrophy might represent a demand for increased liver function at such a high dose level. Evidence of reversibility was found at the end of the recovery period. Slight to moderate acidophilic globule accumulation in the cortical tubular epithelium was found in the kidneys of 3/6 males of the 500 mg/kg/day group sacrificed at the end of treatment. This is most probably due to a sex-linked protein; a well-known phenomenon in the male Laboratory Rat, therefore it was considered unlikely to be treatment-related. Moderate splenic congestion was observed in 4/6 males and 3/6 females of the 500 mg/kg/day group at the end of the treatment period. The same was found in 1/6 female of the control group, 1/2 male of the 5 mg/kg/day group, 2/6 males and 3/6 females of the 500 mg/kg/day group at the end of the recovery period. This lesion can be found, in the Laboratory Rat, due to the method of sacrification. Therefore, the relationship of this change to the treatment was considered unlikely.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Executive summary:

In this subacute toxicity study, the test substance was administered daily by oral gavage to Sprague-Dawley rats of both sexes at dose levels of 5, 50 and 500 mg/kg bw/day for a period of 28 days. At the end of the treatment period, 6 males and 6 females of each group were kept for a 14-day recovery period. A concurrent control group was treated similarly with the vehicle (peanut oil) only. No treatment-related mortality was observed. Treatment-related hypersalivation was observed in some mid and in all high dose animals. This clinical sign disappeared during the recovery period. There was a slight retardation in body weight gain related to a slight decrease in food consumption in males of the 500 mg/kg bw/day group from the second week of treatment which was still present at the end of the recovery period. The hematological investigation revealed changes for activated partial thromoplastin time (about 70 % increase) and a slight increase in Quick time (about 40%) in males of the high dose group (500 mg/kg bw/day). The biochemical investigations revealed reversible increase of plasma protein levels (albumin, alpha-1 and beta globulins and total protein) among males and females receiving 500 mg/kg bw/day. Non-reversible increase in cholesterol level was noticed in the females of the high dose group. Urine analysis showed no treatment-related changes. Organ weight analysis and macroscopic pathology revealed increased liver weights and liver enlargement for both sexes in the high dose group. The microscopic examination revealed the liver as target organ. A reversible centrilobular cell hypertrophy was observed in all animals of the high dose group. This hepatic hypertrophy showed no nuclear or cytoplasmic degenerative or necrotic changes indicating an adaptive response of the liver to the xenobiotic load as confirmed by the reversibility of hepatic changes noted during the treatment-free recovery period.

In conclusion, the no observed adverse effect level (NOAEL) was 50 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

In a subacute toxicity study according to OECD guideline 407 and in compliance with GLP, a total of 96 Sprague-Dawley rats (12 males and 12 females per dose group) were dosed by gavage with 0, 5, 50 and 500 mg/kg bw/day for 28 days (C.I.T., Project No.4971 TSR, 1990). At the end of the treatment period, 6 males and 6 females of each group were kept for a 14-day recovery period. No treatment-related mortality was observed. Treatment-related hypersalivation was observed in some mid and in all high dose animals. This clinical sign disappeared during the recovery period. There was a slight retardation in body weight gain related to a slight decrease in food consumption in males of the 500 mg/kg bw/day group from the second week of treatment which was still present at the end of the recovery period. The hematological investigation revealed changes for activated partial thromoplastin time (70% increased) and a slight increase in Quick time (about 40%) in males of the high dose group (500 mg/kg bw/day). The biochemical investigations revealed reversible increase of plasma protein levels (albumin, alpha-1 and beta globulins and total protein) among males and females receiving 500 mg/kg bw/day. Non-reversible increase in cholesterol level was noticed in the females of the high dose group. Urine analysis showed no treatment-related changes. Organ weight analysis and macroscopic pathology revealed increased liver weights and liver enlargement for both sexes in the high dose group. The microscopic examination revealed the liver as target organ. A reversible centrilobular cell hypertrophy was observed in all animals of the high dose group. This hepatic hypertrophy showed no nuclear or cytoplasmic degenerative or necrotic changes indicating an adaptive response of the liver to the xenobiotic load. Based on these results, the NOAEL was set at 50 mg/kg body weight.

A follow up biochemical and ultrastructural investigation (CIBA-GEIGY Ltd. CB90/35 BY, 1991) on liver tissue prepared from of male and female rats of the above-mentioned 28-day study revealed that the test item is a “mixed-type inducer” in the liver with peroxisome proliferator properties. In particular, peroxisomal lauric acid 11- and 12-hydroxylase activities were induced during treatment in parallel with the respective cytochrome P-450 IIB proteins and ethoxycoumarin O-de-ethylase activity. Ultramorphologically, the test item caused an increase in the number and size of peroxisomes and a moderate proliferation of smooth endoplasmic reticulum membranes. In addition, some mitochondrial alterations were observed. Only minimal effects were observed at 50 mg/kg bw/day, whereas the lowest dose level of 5 mg/kg bw/day was without any effect. The increased liver weights and the biochemical and ultramorphological changes observed at the highest dose level of 500 mg/kg bw/day can be considered as an adaptation of the liver to a functional load on the investigated parameters. The hepatic peroxisome proliferation of the test item may reflect a rodent specific response. The no observed adverse effect level (NOAEL) was 50 mg/kg bw/day and the no observable effect level (NOEL) was 5 mg/kg bw/day for both sexes.

The liver was also identified as target organ in a preceding study performed with Sprague-Dawley rats. The animals were treated by gavage with the test substance at 250, 500, 1000 and 2000 mg/kg body weight for 15 consecutive days (C.I.T., Project No.4972, 1990). A dose-related focal or diffuse hepatic cell hypertrophy, on line with liver enlargement and increase in liver weights was observed in animals of all treated groups. In addition, clinical signs of bad health condition in the 500, 1000 and 2000 mg/kg/day dose levels and mortality in the two high dose groups was observed. The mortality rate was clearly dose-related and all animals of the highest dose level died. Furthermore, hypersalivation was observed in all treated animals. Retardation in bodyweight gain was noted in males of the 500 and 1000 mg/kg/day dose levels. It was related to a decrease in food consumption in males of the 1000 mg/kg/day dose level, only. The food consumption was also decreased in females of the 1000 mg/kg/day dose level. A slight anemia was observed at the 500 and 1000 mg/kg/day dose levels and methemoglobinemia was observed among all treated animals. Treatment with higher doses of the test substance also revealed the stomach as target organ. In the forestomach, hyperkeratosis together with basal cell hyperplasia and sometimes submucosal inflammatory reaction in the majority of animals of the 1000 mg/kg/day dose level as well as ulceration in 1 male of the 2000 mg/kg/day dose level were observed. In the fundic part of the stomach, mucosal exulceration and pseudomelanosis were observed in some animals of the 2000 mg/kg/day dose level. In this study, no dose level could be clearly defined as no-effect-level. However, the liver changes observed at 250 mg/kg body weight were considered adaptive as a result of an increased demand on liver function. This level can therefore be defined as the NOAEL.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP-compliant guideline study

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

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the present data, classification for repeated dose toxicity is not warranted under Directive 67/548/EEC.

 

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for repeated dose toxicity is not warranted under Regulation (EC) No.1272/2008.