4-(1,1,3,3-tetramethylbutyl)phenol

Endpoint summary

Description of key information

In a subchronic guideline study similar to OECD 408 (Bayer (1982) T7010745), 20 Wistar rats/sex/dose were exposed to 0, 30, 300, 3000 ppm 4-(1,1,3,3-tetramethylbutyl)phenol (OCT) for 90 days. The dose equals 0, 2, 23, 228 mg/kg bw/day in males based on the average bw (week 1-13). 
In a subacute GLP guideline study according to OECD 407 (Huntington Research centre (1994) SAZ 464/9424419), OCT was administered to 5 SD rats/sex/dose at 0, 15, 150, and 300 mg/kg bw/day by gavage. A similar GLP study was conducted according to a Japanese "Guidelines for 28-Day Repeated Dose Toxicity Test of Chemicals", using 6 SD rats/sex/dose at 0, 15, 70, and 300 mg/kg bw/day (Matsuura 1992). The study is in Japanese. Only abstract and tables are translated into English.  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August - November 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study OECD 408; individual animal were recorded but are not part of this study report

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

: Higher number of animals used (20 per sex and dose; OECD recommends 10); no ophthalmological examination; sensory reactivity not examined separately.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Züchter Winkelmann, Borchen
- Strain. BOR: WISW (SPF Cpb)
- Age at study initiation: 28 to 35 days
- Weight at study initiation: mean value 69g (male/female)
- Housing: Makronlon cages type II on dust free wood granulate
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +- 2°C
- Humidity (%): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Isooctylphenol was mixed into the rat feed .

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Altromin R-Pulverfutter (source: Altromin GmbH, Lage)
- Storage temperature of food: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

stability of test substance in feed was verified analytically during the test period.

Duration of treatment / exposure:

3 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 30, 300, 3000 ppm (0, 2.2, 22.5, 227.9 mg/kg/d in males)
Basis:
nominal in diet

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: no data
- Rationale for selecting satellite groups: random

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: after 1 and 3 month

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 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 (body weight gain shown in graphical form: bodyweight plotted against time for each dose group and sex)

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 and 3 months after begin of administration
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 5 per sex per dose group
- Parameters checked stated in section "any other information on materials and methods" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1 and 3 months after begin of administration
- Animals fasted: Yes
- How many animals: 5 per sex per dose group
- Following parameters were examined: blood glucose, partial thromboplastin time.

URINALYSIS: Yes
- Time schedule for collection of urine: after 1 and 3 month; 16 hours
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked stated in section "any other information on materials and methods" were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Folgende Organe wurden gewogen: Schilddrüse, Thymus, Herz, Lunge, Leber, Milz, Nieren, Nebennieren, Hoden bzw. Ovarien, Gehirn.

HISTOPATHOLOGY: Yes
Nachstehende Organe wurden in Bouin'scher Flüssigkeit fixiert: Aorta, Augen, Darm (Duodenum, Jejunum, Ileum, Colon, Rectum, Caecum), Femur, Gehirn, Harnblase, Herz, Hoden Hypophyse, Leber, Lunge, Lymphknoten, Magen, Milz, Nebenhoden, Nebennieren, Nieren, Oesophagus, Speicheldrüsen, Ovarien, Pankreas, Prostata, Samenblase, Schilddrüse, Skelettmuskulatur, Thymus, Thrachea und Uterus sowie zusätzliche Leberproben (Lobus sinister) von je 5 Tieren/ Dosis/Geschlecht wurden für den Fettnachweis in Formol-Calcium fixiert.
Für die histopathologische Auswertung wurde das in Bouin'scher Flüssigkeit fixierte Material von männlichen und weiblichen Ratten der Kontroll- und der Dosisgruppe 3000 ppm Isooctylphenol in Paraplast eingebettet, geschnitten und mit HE gefärbt. Zusätzlich wurden die PAS-Reaktion für Nierenschnltte und die Oil-Red-0-Färbung für Leberschnitte (Formalinfixierung) angewandt. Die Entkalkung der Knochen erfolgte in einer EDTA-
Lösung. Das Material wurde in der Abteilung Histopathologie unter der Eingangsnummer 1559 registriert.
Zur histologischen Auswertung gelangten die nach oben genannten fixierten Organe von je 5 männlichen und 5 weiblichen Tieren der Kontroll-
gruppe und der höchsten Dosisgruppe mit Ausnahme des N.ischiadicus. Die Auswahl der Tiere erfolgte anhand einer Randomliste.

Other examinations:

no data

Statistics:

Arithmetic means, standard deviation s, confidence intervals: 95% & 99%.
U-Test according MANN, WHITNEY and WILCOXON, level of significance: 5% & 1%.
Randomization according “Subprogram Randu" from the "Scientific Subroutine Package" (IBM-370).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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
Während der Versuchszeit unterschieden sich die Ratten, die Isooctylphenol in Dosierungen von 30 bis 3000 ppm im Futter erhielten, im Aussehen und Verhalten nicht von den Kontrolltieren. Bei den Ratten der Dosierungen 30 bis 3000 ppm im Futter ergaben sich keine Unterschiede in der Lebhaftigkeit und Fellbeschaffenheit sowie im Fress- und Trinkbedürfnis.
Sterblichkeit: 1 Tier in Dosisgruppe 30 ppm und 1 Tier in Dosisgruppe 3000 ppm. Die Sterblichkeit war nicht behandlungsbedingt erhöht.

BODY WEIGHT AND WEIGHT GAIN
Die männlichen und weiblichen Ratten der Versuchsgruppe 30 ppm nahmen etwa wie die Kontrolltiere an Körpergewicht zu. Die Körpergewichtszunahme war nach 300 ppm vorwiegend bei Männchen leicht verzögert (~10%) (signifikant bei männlichen Tieren in der 3. und 10. Versuchswoche) während bei 3000 ppm die Gewichtszunahme gegenüber den Kontrolltieren deutlich verzögert war.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Die männlichen und weiblichen Ratten die den Wirkstoff bis zur Dosis 3000 ppm im Futter erhielten, nahmen in etwa so viel Pulverfutter auf wie die Kontrolltiere.

FOOD EFFICIENCY
In der 3000 ppm Gruppe waren die Körpergewichtszunahme und das Organwachstum behandelter Tiere deutlich verzögert.

WATER CONSUMPTION
Die Wasseraufnahme unterschied sich bei behandelten männlichen Tieren nicht bedeutsam von der der Kontrollgruppe. Die weiblichen Ratten der Dosierung 3000 ppm tranken ca. 28% mehr Wasser als die Kontrollweibchen.

OPHTHALMOSCOPIC EXAMINATION
nicht untersucht

HAEMATOLOGY
In der Gruppe bis 300 ppm ergaben sich keine Hinweise auf behandlungsbedingte Beeinflussung hämatologischer Parameter. Am Versuchsende waren Hämoglobingehalt sowie Hämatokritwert weiblicher Tiere der Dosisgruppe 3000 ppm signifikant gegenüber den Kontrollweibchen vermindert. Da dieser Befund nur zu einem Versuchszeitpunkt bei einem Geschlecht auftrat und die Werte innerhalb des altersgemäßen Normalbereichs weiblicher Tiere lagen, wird er als Zufallsbefund angesehen. Histopathologisch ergaben sich keine Hinweise auf eine behandlungsbedinqte Beeinflussung der Hämatopoese

CLINICAL CHEMISTRY
Die Thyroxinkonzentration bei weiblichen Tieren der Dosisgruppe 3000 ppm war gegenüber den Kontrollweibchen erhöht. Nach 3000 ppm wurde bei männlichen Tieren eine gegenüber den Kontrolltieren geringfügig erniedrigte Na-Konzentration gestellt. Diesem Befund wird jedoch, da der Wert innerhalb des altersgemäßen Streubereichs der Tiere lag, keine toxikologische Bedeutung beigemessen. In allen übrigen untersuchten Parametern unterschieden sich behandelte Ratten nicht bedeutsam und dosisabhängig von den Kontrollratten.

URINALYSIS
Die Befunde der Harnuntersuchungen nach 1 und 3 monatiger Versuchszeit bei jeweils 5 männlichen und 5 weiblichen Ratten/Dosisgruppe erbrachten keine auf die Behandlung zurückzuführenden Unterschiede zwischen Kontrolltieren und behandelten Ratten bis zur Versuchsgruppe von 3000 ppm.

NEUROBEHAVIOUR
nicht untersucht

ORGAN WEIGHTS
Dem zwar signifikant, aber nur geringfügig erniedrigten Schilddrüsengewicht männlicher Tiere wird, da es zudem nur bei einem Geschlecht erhoben wurde, keine toxikologische Bedeutung beigemessen. Die zum Teil signifikant erniedrigten Organgewichte behandelter Tiere ab 300 ppm sind als Folge der verzögerten Körpergewichtsentwicklung der Tiere anzusehen und wurden daher nicht als toxikologisch bedeutsam gewertet.

GROSS PATHOLOGY
Bei der Sektion der Überlebenden Tiere am Versuchsende wurden keine dosisabhängigen pathologischen Befunde erhoben.

HISTOPATHOLOGY: NON-NEOPLASTIC
In allen mikroskopisch untersuchten Organen sind keine dosisabhängig gehäuften Veränderungen aufgetreten.

Dose descriptor:

NOAEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: reduced body weight gain in week 3 and 10 only and small changes in organ weights

Dose descriptor:

LOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weight gain; changes in organ weights

Critical effects observed:

not specified

Compound consumption per dose group:
male
	30 ppm	300 ppm	3000 ppm
g food/animal	1533	1509	1470
mg compound/animal	46,0	452,7	4410,0
mg compound/animal/day	0,5	5,0	49,0
mean bw (g); week 1-13	233	224	215
mg compound/kg bw/day	2,2	22,5	227,9
female
	30 ppm	300 ppm	3000 ppm
g food/animal	1112	1077	1007
mg compound/animal	33,4	323,1	3021,0
mg compound/animal/day	0,4	3,6	33,6
mean bw (g); week 1-13	149	144	135
mg compound/kg bw/day	2,5	24,9	248,6

Conclusions:

Feeding 300 ppm (22.5 mg/kg/d) 4-(1,1,3,3-tetramethylbutyl)phenol for 90 day causes no adverse effects in Wistar rats under conditions of this study.

Executive summary:

In a subchronic toxicity study 4-(1,1,3,3-tetramethylbutyl)phenol (93.1%) was fed to 20 Wistar rats/sex/dose at dose levels of 0, 30, 300, 3000 ppm for three months.

No treatment related mortality or clinical signs of toxicity were reported. No significant reduction in food consumption was reported.

In the high dose group mean body weight gain was significantly reduced. Water uptake was increased in females of the 3000 ppm dose group. Haematological parameters in all treated male rats were unaffected. A decrease in haemoglobin and haematocrit was reported among females at the high dose level at the end of the testing period. This was observed only at one particular time and in one sex. The values were in the normal range for females of this age. Thus this effect is considered to be incidental. No histopathological indications of dose-related effects on haematopoiesis were observed.

The thyroxin (T4) content was increased in females in the highest dose group after one month, but decreased to control levels after 3 month. This effect can be traced back to considerably increased thyroxin concentrations in two female test animals and might be dose-related. Histopathologically the thyroids of randomly selected animals of this dose group were normal. Body weight gain was slightly reduced in male rats in the 300 ppm dose group. Some significant reduced organ weights indicate a minor impairment of growth. Pathological-anatomical and histopathological investigations as well as clinical and chemical effects and organ weight indicated no adverse effects on liver and kidney up to dosages of 3000 ppm.

The LOAEL is 3000 ppm, based on delayed body weight gain, and associated reduced organ weights. The NOAEL is 300 ppm.

This subchronic toxicity study in the Wistar rats is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rats.  

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1991

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: GLP; Guidelines for 28-Day Repeated Dose Toxicity Test of Chemicals (Japan); no details on study period; only summary available in English.

Qualifier:

according to guideline

Guideline:

other: Guidelines for 28-Day Repeated Dose Toxicity Test of Chemicals (Japan)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: male 128~148g, female was 118~146g.
- Fasting period before study: no data
- Housing: polycarbonate cage which (265W×426D×200H mm)
- Diet (e.g. ad libitum): ad libitum(Pellet for laboratory animal (MF: The Oriental Yeast Industrial Corporation)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20~25℃
- Humidity (%): 40~70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

olive oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day

Remarks:

Doses / Concentrations:
0 (vehicle), 15, 70, 300 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: yes

FOOD EFFICIENCY: no data

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: no data

Sacrifice and pathology:

Treminal kill: Male and Female, days 29 or 43
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

effects observed, treatment-related

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: Salivation was observed on test substance administration in the medium- and high-dose females and males. No mortalities.

BODY WEIGHT AND WEIGHT GAIN: Body weight gain was reduced in the high-dose males.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The test substance did not cause any consistent changes in food consumption.

FOOD EFFICIENCY

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Water intake was increased in males and females of the high dose group.

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY: no effects

CLINICAL CHEMISTRY: For the high dose group, blood chemical analyses showed an increase in Na for females and males, a decrease in cholesterol and an increase in BUN and triglyceride for females, and a decrease in albumin in females. A/G ratios were lowered in the medium- and high- dose.

URINALYSIS: Increased urine volume was evident in females and males at the high dose. Urinalysis showed decreases in specific gravity and in Na, Cl and K.

NEUROBEHAVIOUR

ORGAN WEIGHTS: Slight but statistically significant increases in kidney weights were found in the high-dose males and females and in liver weights in the high-dose females.

GROSS PATHOLOGY: The high-dose males and females showed grayish kidney patch as gross findings.

HISTOPATHOLOGY: The high-dose males and females showed regeneration of renal tubules as microscopic findings.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Salivation and increase in Na in both sexes; decreases in cholesterol and A/G (Albumin/Globulin) ratio and increases in BUN and triglyceride, in females

Critical effects observed:

not specified

All changes attributable to the test substance disappeared or tended to recover after discontinuing the administration.

Conclusions:

No adverse effects were observed at 15 mg/kg/day. The NOAEL for repeated dose toxicity was 15 mg/kg/day. However, changes attributed to p-tert-octylphenol disappeared or tended to recover after discontinuation of administration.

Executive summary:

A repeated dose toxicity study was performed to evaluate the effect of p-tert-octylphenol. The study followed the Guidelines for 28-Day Repeat Dose Toxicity Test of Chemicals (Japan). p-tert-Octylphenol was administered by oral gavage, once daily to groups of six SD rats/sex/dose for twenty-eight consecutive days at dosage levels of 0, 15, 70 and 300 mg/kg/day. Olive oil was used as vehicle and control.

The test substance caused salivation in both sexes, a decrease in the body weight gain in males and an increase in water intake in both sexes of the high dose group. Blood chemical analyses revealed an increase in Na in both sexes, and decreases in cholesterol and the A/G ratio and increases in BUN and triglyceride, in females of the medium- or high-dose group. The high dose group demonstrated increased urine volumes and urinalysis showed decreases in specific gravity, Na, Cl and K. Increases in organ weights of the kidney and liver were found in the high dose group. Pathological examination revealed greyish kidney patches and regeneration of renal tubules. However, changes attributable to p-tert-octylphenol disappeared or tended to recover after discontinuation of administration. The NOEL for repeated dose toxicity test was 15 mg/kg/day.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21/04/1994-05/08/1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 407)

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))

Version / remarks:

Annex to Directive 92/69/EEC, OJ No. L383A, 29.12.92, Part B, Method B

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., England, 13 April 1994
- Age at study initiation: 28 ± 1
- Weight at study initiation: 79-101 g
- Fasting period before study:
- Housing: metal cages with wire mesh floors in groups of five according to sex
- Diet (e.g. ad libitum): Special Diet Services rat and Mouse maintenance Diet ad libitum except as noted under clinical pathology
- Water (e.g. ad libitum): drinking water ad libitum except as noted under clinical pathology
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 46-65%
- Air changes (per hr): 19 per hr
- Photoperiod (hrs dark / hrs light): 12 hrs artifical light (7.00-19.00) in ecah 24 hrs period


IN-LIFE DATES: From: 21.4.1994 To: 24.5.1994

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A 5% w/v was prepared by grinding the appropriate amount of test substance in a pestle and mortar with a small amount of the vehicle to form a smooth paste. This was then gradually made up to volume by addition of the vehicle and mix, if necessary, using a high shear homogeniser. Further formulations (3.0% and 0.3% w/v) were prepared in a similar manner. Formulations were prepared freshly every day.
Prior to dosing the test substance formulations for group 2 and 4 were mixed by inversion (x 20) and subsequently mixed using a magnetic stirred occurred for a period of at least 10 minutes before dosing commenced.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:


VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil is use. The test substance is stable in vehicle during storage and forms a homogeneous suspension.
- Concentration in vehicle: 5%, 3%, and 0.3% w/v
- Amount of vehicle (if gavage): 5ml/kg bodyweight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical study was performed by HRC Department of Analytical Chemistry at HRC. The results are presented in the Formulation Analysis Report:
- Determination of concentrations of test substance in dose formulation analysed for day 1 of the study.
- Determination of chemical stability in corn oil formulations.
- Validation of the method of analysis for the determination of test substance in corn oil formulations
- Determination of the physical stability of the test substance in corn oil formulations.
Formulation Analysis Report:
Mean results were within +2%/-1%. At nominal concentrations of 1 mg/ml and 200 mg/ml, the test substance produces a homogeneous suspension in the corn oil formulation, which can be maintained for up to 1 hour while magnetically stirred and successfully resuspended following storage at ambient temperature for 4 hours.
At nominal concentrations of 1 mg/ml and 200mg/ml the test substance is chemically stable in the corn oil formulation during storage (ambient temperature during the day, refrigeration overnight) for 24 hours.
Procedural recovery obtained during method validation and determination of stability indicate that the analytical method is both accurate and precise: a mean procedural recovery value of 98.1% ± 1.91% CV (n = 8) was obtained for 1 mg/ml and 98.2% ±
0.99% CV (n = 8) for 200 mg/ml. Results for the analysis of test samples were corrected for the appropriate mean procedural recovery value at analysis.
A typical calibration standard graph confirmed the linearity of detector response for the test substance over the concentration range 2 -10 µ/ml. The absence of a peak at the characteristic retention volume for the test substance in the control sample chromatogram demonstrates the specificity of the HPLC assay.
The analytical results confirm that the doses were accurately formulated for Day 1 of the toxicity study. The results also confirm that specimen formulations were homogeneous and stable for a period representing the time from preparation to completion of dosing.

Duration of treatment / exposure:

Preliminary study: 7 days
Main study: 29 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 15, 150, 250 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000, 500, 250, 0 mg/kg/day (preliminary study concentrations)
Basis:
actual ingested

No. of animals per sex per dose:

Preliminary study: 3
Main study:5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose was selected on the basis of a preliminary oral toxicity investigation (HCR Schedule no. SAZ/463 Appendix 6) performed at this HRC. The low and intermediate dosage levels were selected on the basis of the key dosages relative to EEC labelling requirements.
- Rationale for animal assignment (if not random): two days prior to the start of the treatment, each animal was weighted and 40 rats were randomly allocated to for groups, each group with 5 males and 5 females. This allocation was carried out using a computer program, so that the weight distribution within each group was similar and the initial group mean bodyweights were approximately equalised.

- A preliminary study was conducted (HRC Schedule no. SAZ/463) to select the high dosage. Groups of three rats (3 males and 3 females) were treated with the test substance formulated in corn oil at concentrations of 20%, 10% and 5% at a dosage volume of 5 ml/kg/day to achieve dosages of 100, 500 and 250 mg/kg/day. A further group of six rats (3 males and 3 females) received the vehicle, corn oil, at the same dose volume and acted as a control. The study started the 24 of March 1994 and the animals were treated once daily for seven consecutive days, following which all surviving rats were killed at day 8, 31 of March 1994. See results below in "Remarks on results".

- The low and intermediate dosage levels were selected on the basis of the key dosages relative to the EEC regulations.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 3 times a day

BODY WEIGHT: Yes
- Time schedule for examinations: on day one and consequently at weekly intervals

FOOD CONSUMPTION AND COMPOUND INTAKE: the quantity of food consumed was measured weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily monitoring by visual appraisal. Gravimetric measurement was performed during week 3 following a suspected treatment-related effect seen during the first half of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination (day 27 week 4)
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination (day 27 week 4)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes The macroscopic appearance of the tissues of all rats was recorded. (see table 3)
HISTOPATHOLOGY: Yes (see table 3)

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.

The following sequence of statistical tests was used for bodyweight gains, organ weight and clinical pathology data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of values different from the mode were analysed by Fisher's exact test followed by Mantel's test for a trend in proportions. Otherwise:
Barlett's test was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1% level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out followed by Williams' test for a dose related response.
If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test).
Covariate analysis of organ weight data (with final bodyweight as covariate) was also epiformed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10% level of significance.
Significant differences between control animals and those treated with the test substance were expressed at the 5% (*P < 0.05) or 1% (**P < 0.01) level.

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):

effects observed, treatment-related

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortalities.
Increased salivation following dosing, with associated wet fur, was noted for the majority of rats on most occasions throughout the study, for animals treated at 250 or 150 mg/kg days. This finding was occasionally accompanied by post-dose res/brown perioral staining. A post-dose increase in salivation was also seen for one female rat treated at 15 mg/kg/day, on one occasion during week 3. These finding are commonly seen in rat orally dosed studies and are considered to be a result of the unpalatability of the test substance, and therefore not toxicologically important.
Likewise, greasy fur, noted for rats of all groups including the control in the latter half of the study, was related to the administration of the vehicle (corn oil) and was not to be of toxicological importance.
Slight red/brown staining of the urogenital region was noted for one female rat from the high dosage group, on one occasion during week 3. At the incidence and severity seen in this study, this finding was considered to be a chance occurence and not related to treatment

BODY WEIGHT AND WEIGHT GAIN
Bodyweights and bodyweight gains for all groups of treated rats were comparable to controls.

FOOD CONSUMPTION
Slightly higher than control food consumption was recorded for male and female rats treated at 250 mg/kg/day and for female rats treated at 150 mg/kg/day. For remaining groups of rats, food consumption values were comparable to controls.


WATER CONSUMPTION
Markedly higher water consumption was recorded during week 3 for male and females rats receiving 250 mg/kg/day. Slightly higher than control water consumption was also recorded for female rats treated at 150 mg/kg/day.

HAEMATOLOGY
There were no differences from control in haematological parameters measured, that were considered to be treatment-related.
Statistically significantly lower than control mean corpuscular haemoglobin concentration (MCHC) was recorded for all groups of treated female rats. The magnitude of this difference from control was minor and a treatment-related effect was not suspected.
The thrombotest value was statistically significantly higher than control for female rats treated at 250 mg/kg/day. There was overlap between the groups for individual values and this small difference from control was considered to have arisen by chance.

CLINICAL CHEMISTRY
Cholesterol levels were statistically significantly lower than control for male rats treated at 250 mg/kg/day.
Urea nitrogen levels were statistically significantly higher than control for male rats treated at 250 mg/kg/day. Individual values were within the expected range for rats of this age and strain and the magnitude of this difference from control was small. A treatment-related effect was not suspected.
Minor differences from control in protein levels for female rats (lower total protein at 15, 150 or 250 mg/kg/day, lower albumin at 150 or 250 mg/kg/day and lower A/G ratio at 250 mg/kg/day) were recorded. there was considerable overlap of individual values between the groups and a treatment-related effect on protein was considered unlikely.
Statistically significantly lower than control calcium levels were recorded for female rats treated at 150 or 250 mg/kg/day. All individual values for these parameters were within the expected range for rats of this age and strain, and these differences from control were not considered to be related to treatment.

ORGAN WEIGHTS
Liver and kidney weights (bodyweight adjusted) were statistically significantly higher than control fro female rats treated at 250 mg/kg/day.
All other organ weights were comparable for control and treated rats.

GROSS PATHOLOGY
The macroscopic examination performed at termination revealed no changes attributable to treatment with test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver: Minimal centrilobular hepatocyte enlargement was seen in female rats receiving 250 mg/kg/day. This change correlates with the higher weights recorded for this treatment group. No change was seen in female rats from any other dose group nor in any of the treated male rats.
Kidney: An increased incidence of basophilic epithelium in proximal convoluted tubules was seen in the kidneys of male and female rats receiving 250 mg/kg/day and in male rats receiving 150 mg/kg/day. In a number of affected rats occasional mitoses were noticed in the basophilic epithelium. An increased incidence of interstitial inflammation was noted in male rats receiving 250 mg/kg/day.
Microscopic changes were considered to be associated with the increased kidney weights recorded for female rats receiving 250 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Kidney: Basophilic epithelium occasionally with mitotic figures in proximal convoluted tubules in males; Higher food and water consumption in females; lower cholesterol levels in females.

Dose descriptor:

dose level: dose level:

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Study summary:

A study was perform to assess the systemic toxicity of 4(1,1,3,3 -tetramethyl-butyl)phenol to the rat. The method followed was that outlined in Annex V, Part B, Method B7 in the EEC Directive 79/831/EEC and OECD Guideline for Testing of Chemicals No. 407 "Repeated Dose Oral Toxicity - Rodent: 28 -days or 14 day study".

4(1,1,3,3 -Tetramethyl-butyl)phenol, a white solid intended for use as an industrial intermediate, was administered by oral gavage, once daily to groups of five male and five female rats for twenty-nine consecutive days at dosage levels of 15, 150 and 250 mg/kg/day. The test substance was prepared as suspensions in corn oil at concentrations of 0.3, 3.0 and 5% w/v. A further group of rats (five males and five females) was held as a control, receiving the vehicle alone.

Bodyweights, food and water consumption and clinical observations were recorded during the study. Blood samples for clinical investigations were taken on Day 27 and all animals were killed and examined macroscopically on Day 30. Histopathological examination of the tissues was then initiated.

Food consumption:
Slightly higher than control food consumption was recorded for male and female rats treated at 250 mg/kg/day and for female rats treated at 150 mg/kg/day.

Water consumption:
Markedly higher than control water consumption was recorded for male and female rats receiving 250 mg/kg/day and to a lesser extent for females treated at 150 mg/kg/day.

Cholesterol:
Levels were lower than control for female rats treated at 250 or 150 mg/kg/day.

Liver and kidney:
Organ weights (bodyweight adjusted) statistically significantly higher than control for female rats treated at 250 mg/kg/day. Minimal centrilobular hepatocyte enlargement in female rats receiving 250 mg/kg/day. Basophilic epithelium occasionally with mitotic figures in proximal convoluted tubules in male and female rats receiving 250 mg/kg/day and male rats receiving 150 mg/kg/day. Interstitial inflammation in kidneys of male rats receiving 250 mg/kg/day.
No treatment-related effect was detected in female rats receiving 150 mg/kg/day or male and female receiving 15 mg/kg/day.

Other findings:
No treatment related differences from control were noted for the remaining parameters measured, namely clinical signs, bodyweight gains, haematology and macroscopic pathology.

Conclusion:
The finding in the kidney at the high and intermediate dosages was considered to be an adverse effect and to represent the potential of the test substance to cause serious damage to health. Accordingly, the R48 (harmful) risk phrase is proposed. There were no treatment-related effects on the low dosage of 15 mg/kg/day and this therefore represents the no-observed effect level (NOEL) for 1(1,1,3,3 -tertramethyl-butyl)phenol in the rat when administered orally for 29 days.

  

Results of preliminary study:

Clinical signs and mortality: There were no mortalities. For animals from the high dosage group (1000 mg/kg/day) a slight to moderately wet urogenital region was seen intermittently in one female, less frequently in two females and on and isolated occasion for one male. The associated finding of a slight to moderate yellow staining in this region was also observed for all three females. These signs are considered to be attributable to a lack of grooming. Slightly abnormal gait (walking on toes) was seen for one high dosage female (no. 23♀) on day 6. This sign was only observed immediately following dosing and was transient; it was considered in this case to represent discomfort following dosing. Slight piloerection was seen occasionally in two females and one male receiving 1000 mg/kg/day, also being observed infrequently in one rat of each sex treated at 500 mg/kg/day and one male and two females receiving 250 mg/kg/day. This commonly seen in rats following handling and/or dosing and at severity and incidence seen in this study, is not considered toxicologically important. A slight to moderate increase in salivation immediately following dosing was commonly observed through the study in rats of both sexes from all treated groups and was commonly associated with wet fur. In addition, red/brown perioral staining was seen following dosing for all rats from the high dosage group. These signs are commonly observed in oral gavage studies and are considered to be a result of the unpalatability of the test substance and therefore, not of toxicological importance. Slightly greasy fur was seen during the first half of the study for one female treated at 250 mg/kg/day; this was considered to be linked to administration of the vehicle (corn oil) and was not considered to be toxicologically important.Bodyweight and weight gain: Lower bodyweight gains were recorded for female rats dosed at 500 or 1000 mg/kg/day and, to a lesser extent, males dosed at 1000 mg/kg/day over days 1 to 4. recovery was evident at day 8 with gains from day 4 to 8 for all groups being comparable to that of controls. However, there was variation between the groups in percentage of control gain over this period (day 4 to 8) from 82% to 112%, this was considered to reflect the small numbers of animals in each group. The apparent effect on bodyweight for these rats was transient and recovery was evident in the latter half of the week. Food consumption: Food consumption values were lower than control for male rats treated at 1000 mg/kg/day. Food consumption values for other groups were comparable to controls. Organ weights: Slightly higher than control kidney (for two animals) and liver weights were recorded for females receiving 1000 mg/kg/day despite lower than control terminal bodyweights. Organ weights for remaining animals were comparable to controls. Gross pathology: The glandular region of the stomach was noted to be thin and semi-transparent in one male receiving 500 mg/kg/day and one female receiving 250 mg/kg/day. At the incidence seen in this study, this finding was considered to be incidental. No other abnormalities were noted.

Conclusions:

15 mg/kg/day represents the NOAEL for 4(1,1,3,3-tetramethyl-butyl)phenol in the rat when administered orally for 29 days. Findings in the kidneys at the high and intermediate dosages were considered to show the potential of the test substance to cause serious damage to health. The authors conclude that labelling with R48 (harmful) risk phrase is required according to the EEC Council Directive 92/69/EEC Annex VI, Part II (D) as described in commission Directive 93/21/EEC.

Executive summary:

In a subacute toxicity study 4(1,1,3,3-tetramethyl-butyl)phenol (98,7%.) was administered to 5 Sprague-Dawley rats/sex/dose by gavage at dose levels of 0, 15, 150, 250 mg/kg bw/day.

No treatment related effects were noted for clinical signs, bodyweight gains, haematology and macroscopic pathology. Adjusted liver and kidney weights were statistically significantly higher than control for female rats treated at 250 mg/kg/day. Basophilic epithelium occasionally with mitotic figures in proximal convoluted tubules was reported in male and female rats receiving 250 mg/kg/day and male rats receiving 150 mg/kg/day. Interstitial inflammation was reported in kidneys of male rats receiving 250 mg/kg/day. 

The LOAEL is 150 mg/kg bw/day, based on epithelial changes in the kidney. The NOAEL is 15 mg/kg bw/day.

This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study (OECD 407) in rat.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

22.5 mg/kg bw/day

Study duration:

subchronic

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

Feeding 300 ppm 4 -(1,1,3,3 -tetramethylbutyl)phenol (OCT) (93.1%) to 20 Wistar rats/sex/dose for 90 day causes no adverse effects (Bayer 1982). Based on the mean body weight, 300 ppm equals 22.5 mg/kg bw/day in male rats and 24.9 mg/kg bw/day in female rats, respectively. No treatment related mortality or clinical signs of toxicity were reported at the highest dose of 3000 ppm. No significant reduction in food consumption was reported. Water uptake was increased in the highest dose group, but only in females. Haematological parameters in all treated male rats were unaffected. A decrease in haemoglobin and haematocrit was reported among females at the high dose level at the end of the testing period. The values were in the normal range for females of this age. Thus this effect is considered to be incidental. No histopathological indications of dose-related effects on haematopoiesis were observed. The thyroxin (T4) content was increased in females in the highest dose group after one month, but decreased to control levels after 3 month. This effect can be traced back to considerably increased thyroxin concentrations in two female test animals and might be dose-related. Histopathologically the thyroids of randomly selected animals of this dose group were normal. Pathological-anatomical and histopathological investigations as well as clinical and chemical effects and organ weight indicated no adverse effects on liver and kidney up to dosages of 3000 ppm. Body weight gain was slightly reduced in male rats in the 300 ppm dose group (significant only in week 3 and 10). Some significant reduced organ weights were attributed to the delay in body weight gain and were not considered to be toxicological effects. At 3000 ppm body weight gain was clearly delayed in both sexes. This delayed body weight gain and associated changes in organ weights are base for an NOAEL of 300 ppm (~ 22.5 mg/kg bw/day).   In a 28 day study 0, 15, 150, 250 mg/kg bw/day OCT (98,7%.) was administered by gavage to 5 SD rats/sex/dose (Huntington Research Centre Ltd., 1994). No treatment related effects were noted for clinical signs, bodyweight gains, haematology and macroscopic pathology. Adjusted liver and kidney weights were statistically significantly higher than control for female rats treated at 250 mg/kg/day. Basophilic epithelium occasionally with mitotic figures in proximal convoluted tubules was reported in male and female rats receiving 250 mg/kg/day and male rats receiving 150 mg/kg/day. Interstitial inflammation was reported in kidneys of male rats receiving 250 mg/kg/day. A LOAEL of 150 mg/kg bw/day was determined based on epithelial changes in the kidney of male rats. The NOAEL was 15 mg/kg bw/day. The subacute NOAEL of 15 mg/kg bw/day was supported by an additional 28 day study (Matsuura, 1992). The study is available in Japanese only (Klimisch 4). The abstract and tables are translated into English. The study followed the Guidelines for 28-Day Repeat Dose Toxicity Test of Chemicals. OCT (98.24%) was administered daily by gavage to groups of six SD rats/sex/dose for 28 day at dosage levels of 15, 70 and 300 mg/kg/day. The test substance caused salivation in both sexes, a decrease in the body weight gain in males and an increase in water intake in both sexes of the high dose group. Blood chemical analyses revealed an increase in Na in both sexes, and additional aberrations in females of the medium and high-dose group. The high dose group demonstrated increased urine volumes and urinalysis showed decreases in specific gravity, Na, Cl and K. Increases in organ weights of the kidney and liver were found in the high dose group. Pathological examination revealed greyish kidney patches and regeneration of renal tubules. However, changes attributable to OCT disappeared or tended to recover after discontinuation of administration.

Justification for classification or non-classification

No severe systemic toxicity was reported in rats following subchronic and subacute repeated oral exposure to 4 -tert-octylphenol.