Registration Dossier

Administrative data

Description of key information

A key 28-day toxicity study was conducted in Wistar rats by oral gavage at 15, 100 and 600 mg/kg/day. A NOAEL of 100 mg/kg/day was established. At 600 mg/kg/day serum changes were noted (increased total protein in males and females; increased albumin in males; decreased urea in females; decreased inorganic phosphate and increased calcium in females). Enlarged caecum was noted in males and females and incidentally occurring findings included enlarged liver, enlarged kidneys and swollen limiting ridge in the stomach. Increased liver weights were noted in males and females and increased kidney weights were noted in males only. A microscopically observed very slight increase of hepatocellular hypertrophy in the centrilobular area of the liver was noted in males and females and increased slight accumulations of eosinophilic inclusion bodies were noted in the renal cotex of males.

A key 90-day toxicity study with 28-day recovery period was conducted in the Han Wistar rat at 0, 150, 500, 1600 or 4000 ppm, corresponding to 0, 10, 33, 107 and 270 mg/kg bw/day in males and 0, 11, 39, 124 and 298 mg/kg bw/day in females. A NOAEL of 270 mg/kg/day was established, which is the highest dose given.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02/07/1991-12/08/1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (OECD 407)
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd. Basel, Switzerland
- Age at study initiation: approximately 6 weeks
- Housing: animals were housed 5 to a cage (same sex) in stainless steel suspended cages with wire mesh floors.
- Diet (e.g. ad libitum): free access to standard pelleted laboratory animal diet (Kliba, Klingeltalnuehle AG, 4303, Kaiseraugst, Switzerland). each batch was analysed for contaminants and results were examined and archived.
- Water (e.g. ad libitum): tap water, ad libitum. Results of chemical and contaminants analyses are examined and archived quarterly.
- Acclimation period: at least 7 days. Veterinary examination was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
- Identification: earmark and tattoo
- Randomisation: computer-generated random algorithm according to body weight with all animals ± 20% of the sex mean



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
- Method: the test substance was heated to approximately 50°C and subsequently weighed into a glass flask on an analytical balance and the vehicle (w/w) added. Adjustment was made for specific gravity of vehicle. If necessary test substance formulations were re-heated to approximately 50°C in order to form a suitable suspension.
- Frequency of test substance formulation: daily immediately prior dosing
- Homogeneity of test substance in vehicle: by stirring. Homogeneity during treatment was maintained using a magnetic stirrer.
- Storage instructions for test substance formulation: at ambient temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations: samples of formulations prepared during week 1 were analysed to check accuracy of preparation. based on changes in appearance of previously heated and dispensed test substance, possible instability was suspected. Therefore samples were taken from visually normal or different appearing test substance and analysed for the actual contents, in order to check stability of test substance under storage conditions.

Analysis of dose preparations: Analytical report (appendix 2) Subacute 28-day oral toxicity with 2,6-bis(1,1-dimethylethyl)-phenol by daily gavage in the rat. Determination of the accuracy of preparation of 2,6-bis(1,1-dimethylethyl)-phenol in polyethylene glycol and the stability of test substance under storage conditions. The concentrations were determined using a HPLC method. Test substance formulations in polyethylene glycol formed a solution at all test concentrations. Analysis of the accuracy of dose preparations revealed values within the range -4 to -2% of nominal, which was considered for this type of formulation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, approximately the same time each day, 7 days per week.
Remarks:
Doses / Concentrations:
0 mg/kg b.w./day
Basis:
other: oral gavage, 5 ml/kg body weight
Remarks:
Doses / Concentrations:
15 mg/kg b.w./day
Basis:
other: oral gavage, 5 ml/kg body weight
Remarks:
Doses / Concentrations:
100 mg/kg b.w./day
Basis:
other: oral gavage, 5 ml/kg body weight
Remarks:
Doses / Concentrations:
600 mg/kg b.w./day
Basis:
other: oral gavage, 5 ml/kg body weight
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: a 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 100 mg/kg/day) to provide basis for selection of dose levels for a study of longer duration.
Animals receiving 1000 mg/kg/day showed hunched posture, piloerection, diarrhoea, bradypnoea and laboured respiration. After 5 days of oral treatment, males receiving 1000 mg/kg/day showed low body weight gain and females receiving 1000 mg/kg/day showed body weight loss, which were both associated with decreased food consumption. An irregular grey-white appearance of the forestomach was noted in 2/3 males and 3/3 females receiving 1000 mg/kg/day, in 1 female accompanied with thickening of the limiting ridge (junction between glandular stomach and forestomach). A small increase of absolute and relative liver weights were noted in males and females receiving 1000 mg/kg/day. Among animals receiving 200 or 50 mg(kg/day, changes occurred incidentally and were within normal background variation for rats of this age and strain.
Based on these observations and following consultation of the sponsor, treatment levels for a study of 28 days duration were selected
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No
- Time schedule:
- Cage side observations checked in table [No.?] were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily. Severity of observations were graded.


BODY WEIGHT: Yes
- Time schedule for examinations: weekly and on the day preceding termination, prior to overnight fasting.
- body weight (g) and body weight gain (%)


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- 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:No


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): subjective appraisal was maintained during the study, but not quantitative investigation introduced as no effect was suspected.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: both eyes were examined following instillation of tropicamide solution (5mg/ml) during the last week of treatment.
- Dose groups that were examined: all animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of termination, immediately prior to post mortem examination, between 8.00 and 10.00 a.m.
- Anaesthetic used for blood collection: Yes (identity) light ether anaesthesia
- Animals fasted: Yes (overnight), but water was provided
- How many animals: all rats/sex/group
- Parameters checked in table were examined.


CLINICAL (BIO)CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of termination, immediately prior to post mortem examination, between 8.00 and 10.00 a.m.
- Animals fasted: Yes (overnight), but water was provided
- How many animals: all rats/sex/group
- Parameters checked in table were examined.


URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see information below)
HISTOPATHOLOGY: Yes (see information below)
Statistics:
The following statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
The exact Fisher-test was applied to the ophthalmoscopic examination data.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no clinical signs of toxicity or behavioural changes noted over the 29 day observation period that were considered to be related to treatment with the test substance.
No mortality occurred during the study period.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the 4 week study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no differences in food consumption before or after allowance for body weight between treated and control animals.

FOOD EFFICIENCY


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)


OPHTHALMOSCOPIC EXAMINATION
There were no differences between control and treated animals, upon opthalmoscopic examination at week 4, that could be attributed to the treatment with the test substance.

HAEMATOLOGY
haematological parameters of treated rats were considered not to have been affected by treatment.

CLINICAL CHEMISTRY
Changes in clinical biochemistry parameters of the serum were noted in total protein, albumin, inorganic phosphate, calcium and urea.

URINALYSIS


NEUROBEHAVIOUR


ORGAN WEIGHTS
Toxicological significant differences between organ weights of treated and control animals were noted in the liver and kidney. At 600 mg/kg/day male and females had around 30 to 40% heavier livers than control animals whilst a 20% increase in relative (% of bodyweight) liver weight was noted in males dosed at 100 mg/kg/day.

GROSS PATHOLOGY
The principal change noted at macroscopic examination was enlarged caecum. Other changes, including enlarged liver, enlarged kidneys and swollen limiting ridge in the stomach, were incidentally occurring and considered to be of possible toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic alterations that were considered to be treatment-related were noted in the liver and kidneys.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)


HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; body weight; food consumption; ophthalmoscopic examination; haematology; clinical chemistry; gross pathology; organ weights; histopathology.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Increased relative liver weight in males with no accompanying microscopic changes. Reduced serum urea in females. Macroscopically enlarged caecum in males. These changes were not of toxicological significance.
Critical effects observed:
not specified

Clinical signs

Excessive salivation was noted in control animals and treated animals. Among control animals and animal receiving 15 mg/kg/day excessive salivation occurred incidentally in a few animals. All animals receiving 100 or 600 mg/kg/day showed intermittent periods of excessive salivation over the study period. However, this finding is frequently noted in rats of this age and strain following oral treatment via a stomach tube or may be associated with a bad taste or possible irritant effect of the test substance. Therefore no toxicological significance was attached to this finding.

Alopecia was noted in 1 control female (shoulder region) and in 4 out of 5 females receiving 100 mg/kg/day (cheek region). In 3 females receiving 100 mg/kg/day incidental wounds or scabs were also noted on the cheek. Alopecia is a common phenomenon in rats of this age and strain. Furthermore, given the increased incidence in 1 cage and the incidental occurrence of wounds and scabs it is very likely that this finding occurred at a higher incidence as a result of increased grooming and scratching activity rather than as a result of treatment.

Clinical Biochemistry

Statistically significantly increased total protein was noted in males and females receiving 600 mg/kg/day when compared with control animals. In males receiving 600 mg/kg/day, albumin levels were also noted to be statistically significantly higher than controls. No differences were noted between control animals and animals receiving 100 or 15 mg/kg/day, in total protein albumin levels.

Inorganic phosphate was noted to be statistically significantly decreased compared to controls in females receiving 600 mg/kg/day, but not in males receiving 600 mg/kg/day. Inorganic phosphate levels in animals receiving 100 or 15 mg/kg/day remained in the same range as control animals.

Urea values were noted to be statistically significantly decreased, in comparison with control females, in females receiving 600 or 100 mg/kg/day. Urea values in females receiving 15 mg(kg/day or treated males remained in the same range as control animals.

Among females receiving 600 mg/kg/day, statistically significantly increased calcium levels were noted when compared to control females. Females receiving 100 or 15 and treated males were not thus affected.

Macroscopic examination

Macroscopically observed enlarged caecum was noted in 4/5 males and all females receiving 600 mg/kg/day. Enlargement of the caecum was also noted in 2/5 males receiving 100 mg/kg/day. females receiving 100 mg/kg(day or animals receiving 15 mg/kg/day did not show changes of the caecum.

Enlarged liver and enlarged kidneys (accompanied with green appearance of the kidneys) was noted in 1 male receiving 600 mg/kg/day. Another male receiving 600 mg/kg/day had a thickened limiting ridge (junction between glandular stomach and forestomach).

A cyst, noted in the left uterus left horn of 1 female receiving 15 mg/kg/day, was considered within normal background variation for rats of this age and strain and not to be of biological significance.

Organ weights

Liver weights of males and females receiving 600 mg/kg/day were statistically significantly increased in comparison with controls before and after allowance for body weight. in males receiving 100 mg/kg/day, relative liver weights were also statistically significantly higher than controls. There were no differences noted between liver weights of control animals and those of females receiving 100 mg/kg/day or animals receiving 15 mg/kg/day.

Relative kidney weights were statistically significantly increased in males receiving 600 mg/kg/day when compared to control males. Realtive kidney weights of females receiving 600 mg/kg/days or animals receiving 100 or 15 mg/kg/day remained similar to kidney weights of control animals.

Microscopic examination

Among 4/5 and 5/5 females receiving 600 mg/kg/days, very slight hepatocellular hypertrophy was noted in the centrilobular of the liver.

In the renal cortex of males receiving 600 mg/kg/day, slight accumulations of tubular eosinophilic inclusion bodies were encountered in more rats and at a high degree than among control males.

There were no treatment-related microscopic changes noted in animals receiving 100 or 15 mg/kg/day. The small number and the degree of changes recorded, remained within the normal incidence of background variation.

There were no histopathological correlates noted in the caeca, which were notice to be macroscopically enlarged.

Analysis of the test substance and preparations

The stability of the test substance under the storage conditions applied in this study (room temperature and in the dark) was confirmed as satisfactory. All test substance formulations were solutions and the actual concentrations remained within -4 to -2% of nominal concentrations, which was considered to be acceptable for this type of formulation.

Discussion

In this 28 -day oral toxicity study in rats, organs that appear to be affected by treatment were the liver and kidneys. The liver of animals receiving 600 mg/kg/day and of males receiving 100 mg/kg/day were noted to be heavier than controls. Upon microscopic examination, these livers showed slight hepatocellular hypertrophy, which may be responsible for the increase in liver weights. There were no changes in liver enzymes noted in the serum, therefore, the increased liver weights and related increase in the number of liver cells may be attributed to an increased work hypertrophy. As this is a common phenomenon in rats, in order to metabolise a xenobiotic agent, these findings were considered not to be a change of toxicological significance.

Increased kidney weights were only noted in males receiving 600 mg/kg/day and may be associated to the histophatologically observed increase in tubular accumulations of eosinophilic inclusion bodies in the kidneys. There were no changes in serum biochemical parameters of males that may be possibly related to kidney damage. Changes in serum electrolyte levels(i.e. decreased phosphate and increased calcium) and decreased urea levels in females receiving 600 mg/kg/day did not show any corroborative pathological changes. These changes were not considered to be of toxicological significance.

Increased total protein values, and in male also increased albumin values, were noted in animals receiving 600 mg/kg/day. As there were no associated disturbances in nutritional status or water balance noted during the study, these increased serum protein values were considered not to represent a clear sign of toxicity.

At termination of the study animals receiving 600 mg/kg/day and a few males receiving 100 mg/kg/day showed and enlarged caecum at macroscopic examination. there were however, no microscopic changes noted that could be related to the macroscopically enlarged caecum. The toxicological significance therefore must remain unclear.

From the results presented in this study, a definitive No Observed Adverse Effect Level of 100 mg/kg/day was established.

Conclusions:
Dose levels were 0, 15, 100 and 600 mg/kg/day. Treatment related changes were observed in the blood, liver and kidney. At 600 mg/kg/day males and females had around 30 to 40% heavier livers than control animals and these weight increases were accompanied by very slight centrilobular hepatocellular hypertrophy. Although relative liver weight (% of bodyweight) in males at 100 mg/kg/day was approximately 20% heavier than controls this was not accompanied by microscopic changes and liver weight in females was comparable to controls. The increased liver weight and centrilobular hypertrophy noted at 100 and 600 mg/kg/day was considered to be an adaptive change and of no toxicological significance. Reduced serum urea (17%) was present in females at 100 mg/kg/day and also noted in females at 600 mg/kg/day (33%); this is not considered to be a toxicologically relevant finding as a pathological change is normally reflected by an increase in serum urea levels. At necropsy examination enlarged caecum was noted in 100 and 600 mg/kg/day males. No microscopic changes were noted that could be related to the enlarged caecum noted macroscopically and therefore this finding was not considered to be of toxicological significance.
The NOEL in this study was determined to be 15 mg/kg/day. However, it may be considered that the findings at 100 mg/kg/day were not adverse in that they would not be considered to affect the performance of the whole organism or the ability of the organism to respond to an additional environmental challenge. The NOAEL in this study was therefore, 100 mg/kg/day.
Executive summary:
A subacute 28 -day toxicity study was performed to assess the repeated dose toxicity of the test material, 2,6 -bis(1,1 -dimethylethyl), in the SPF-bred Wistar rat. The method used followed that described in the OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 407, "Repeated Dose Oral Toxicity - Rodent: 28 or 14 day study", Paris Cedex, May 12, 1981 and EEC Directive 84/449/EEC, Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.7: "Sub-acute Toxicity - Oral". Official Journal of the European Communities No. L251, September 1984.

The results may be used as a basis for classification and labelling.

Following a range-finding study, the test substance was administered daily by gavage to 4 groups of ten animals (five males and five females) at concentrations of 0 (control), 15, 100 and 600 mg/kg/day.

All animals were subjected to daily clinical observation. Body weight and food consumption were measured weekly and on the day before necropsy. During week 4 of treatment, both eyes of all animals were examined. On the day of termination blood was collected from each animal for clinical laboratory investigations. Subsequently a necropsy examination was performed, macroscopic observations and organ weights were recorded. A histopathological examination was performed on adrenals, heart, kidneys, liver, spleen and stomach.

At 15 mg/kg/day no treatment-related changes were detected.

At 100 mg/kg/day decreased serum urea was noted in females only. Increased relative liver weights were noted in males only. Enlarged caecum was noted in 2/5 males at macroscopic examination.

At 600 mg/kg/day an increased serum total protein level was noted in males and females and increased serum albumin level was noted in males only. Decreased serum urea was noted in females only. Decreased inorganic phosphate and increased calcium was noted in the serum of females only. Enlarged caecum was noted in 4/5 males and 5/5 females at macroscopic examination. Incidentally occurring findings included enlarged liver, enlarged kidneys and swollen limiting ridge in the stomach. Increased liver weights were noted in males and females and increased kidney weights were noted in males only. A microscopically observed very slight increase of hepatocellular hypertrophy in the centrilobular area of the liver was noted in males and females and increased slight accumulations of eosinophilic inclusion bodies were noted in the renal cotex of males only.

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of 100 mg/kg/day was established.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2013 - 25 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant with current guidelines and GLP compliant
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
other: Han Wistar Crl: WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: approximately 7-9 weeks
- Weight at study initiation: 197-255 g and 141-187 g for males and females, respectively
- Fasting period before study: n/a
- Housing: Main study animals housed 2 per cage by sex; Recovery study animals housed 2 or 3 per cage by sex.
- Diet (e.g. ad libitum): Rat and Mouse (modified) No. 1 Diet SQC Expanded (Special Diets Services, Witham, Essex) with test item at appropriate concentrations
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70%,
- Air changes (per hr): minimum of 10 air changes per hour
- Photoperiod (hrs dark / hrs light): A 12 hour light/dark cycle

IN-LIFE DATES: From: To: From 16 Dec 2013 until 14 Apr 2014
Route of administration:
oral: feed
Vehicle:
other: Rat and Mouse No.1 Maintenance Diet SQC Expanded (Ground)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Formulated diets were prepared weekly, split into daily aliquots, and dispensed daily.
- Mixing appropriate amounts with (Type of food): Rat and Mouse No.1 Maintenance Diet SQC Expanded (Ground)
- Storage temperature of food: Stored in a freezer set to maintain -20°C

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by UPLC with UV detection using a validated analytical procedure (Charles River Analytical Procedure No. 3135).
Analyses peformed from samples collected on Day 1, and during Weeks 6 and 12 of treatment
Duration of treatment / exposure:
13 Week exposure with 4 week recovery
Frequency of treatment:
Daily dietary exposure
Remarks:
Doses / Concentrations:
0, 10, 33, 107 and 270 mg 2,6-di-tert-butylphenol/k/day in males and 0, 11, 39, 124 and 298 mg 2,6-di-tert-butylphenol/kg/day for females corresponding to dietary inclusion levels of 0, 150, 500, 1600 and 4000 ppm.
Basis:
actual ingested
No. of animals per sex per dose:
Five groups of 10 male and 10 female Han Wistar Crl:WI(Han) rats were assigned to the Main study. A further two groups of 5 male and 5 female animals were assigned as a Control and High dose recovery groups.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected with the Sponsor after evaluation of the results obtained in a 14 day dose range finding study in rats performed at Charles River (Study No. 525883). In Charles River Study No. 525883, animals were fed diets containing 2,6-di-tert-butylphenol at 300, 3000, 6000 or 10000 ppm for at least 14 consecutive days. Although there were no unscheduled deaths or treatment related clinical observations noted throughout treatment, lower body weight gains were observed in animals that received 6000 or 10000 ppm. This was associated with an initial reduction in food consumption, considered to be a palatability effect. There were no gross pathology findings or organ weight changes noted in any treatment group. The dose levels selected for this study (Charles River Study No. 525862), A 13 Week Dietary Toxicity Study with a 4 Week Recovery Period were 150, 500, 1600 and 4000 ppm (approximately mg/kg-bw/day equivalent of 10, 33, 110 and 275) in an attempt to produce graded responses to the test item. The high dose level was expected to produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The intermediate dose levels were expected to produce minimal to moderate effects and confirm a NOAEL, while the low level was expected to produce no observable indications of toxicity.
- Rationale for selecting satellite groups: to evaluate reversibility of any findings
- Post-exposure recovery period in satellite groups: 28-days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked early morning and as late as possible each day for viability. Additionally, all animals were examined at frequent intervals throughout the day for any signs of ill health or reaction to treatment, starting immediately post dose, with particular attention paid to the first hour after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week, starting during the pretrial period, all animals received a detailed clinical examination including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly, commencing during the pretrial period, and on the first day of scheduled necropsy

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 g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretrial, during Weeks 12 and 17 (Week 4 of recovery period).
- Dose groups that were examined: All animals during pretrial, all animals in the control and high dose group during Week 12 of treatment and all recovery study animals during Week 17.

HAEMATOLOGY and COAGULATION: Yes
- Time schedule for collection of blood: Week 14 and Week 17
- Anaesthetic used for blood collection: Yes : Isoflurane
- Animals fasted: No
- How many animals: All main and recovery study animals during Week 14 and all recovery study animals during Week 17
- Parameters examined: Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red Blood Cell Distribution Width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells, Activated partial thromboplastin time, Fibrinogen and Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 14 and Week 17
- Animals fasted: No
- How many animals: All main and recovery study animals during Week 14 and all recovery study animals during Week 17
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine Kinase, Total bilirubin, Urea , Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium,
Chloride, Free (T3) Triidothyronine, (T4) Thyroxine and Thyroid Stimulating Hormone (TSH)

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 and Week 17
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones and Blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Pretrial, during Weeks 12 and 17 (Week 4 of recovery period).
- Dose groups that were examined: All animals during pretrial and during Week 12 of treatment and all recovery study animals during Week 17.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Pain perception and landing foot splay
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
All statistical tests were two-sided and performed at the 5% significance level. Males and females were analysed separately. Pairwise comparisons were only performed against the control group (Group 1). Body weight, food consumption, selected functional observation battery and motor activity data, haematology, coagulation, clinical chemistry and selected urinalysis data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e., pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test). Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above. In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data was subjected to a rank transformation prior to analysis. In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data was subjected to a rank transformation prior to analysis. The results of the analysis were reported indicating the level of statistical significance (p<0.05, p<0.01 and p<0.001) of each pairwise comparison
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):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
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: There were no unscheduled deaths. There were no clinical signs attributed to treatment with 2,6-di-tert-butylphenol.

BODY WEIGHT AND WEIGHT GAIN: There was an effect on body weights in males receiving 4000 ppm of 2,6-di-tert-butylphenol, when compared with controls.
For males receiving 4000 ppm 2,6-di-tert-butylphenol, body weights were lower throughout the treatment period; achieving statistical significance from Day 0. Although the group was statistically lower on the first day of treatment, the pattern continued throughout the treatment period and resulted in a statistically significant lower group mean body weight change over the treatment period (Day 0-91) when compared with the control group (p<0.01). The difference in weight gain from the controls was approximately 16%. Group mean body weights and body weight gain of animals receiving 150, 500, 1600 and in females receiving 4000 ppm 2,6-di-tert-butylphenol were similar to controls. During the recovery period, group mean body weight change for animals that received 4000 ppm was similar to the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The overall mean achieved dosages were 0, 10, 33, 107 and 270 mg 2,6-di-tert-butylphenol/k/day in males and 0, 11, 39, 124 and 298 mg 2,6-di-tert-butylphenol/kg/day for females corresponding to dietary inclusion levels of 0, 150, 500, 1600 and 4000 ppm.

There was an effect on food consumption in males receiving 4000 ppm 2,6-di-tert-butylphenol where a slightly lower amount of food consumed throughout the treatment period was observed when compared with controls; achieving statistical significance on Day 7 and 14 (p<0.05). This effect could represent a slight palatability effect of the treated diet. Food consumption values for animals receiving 150, 500 or 1600 ppm and in females receiving 4000 ppm 2,6-di-tert-butylphenol were similar to controls. In the recovery period, food consumption in animals that received 4000 ppm was similar to controls.

FOOD EFFICIENCY: The differences in food consumption for males receiving 4000 ppm were reflected in the food utilisation, which was statistically significantly lower overall for the duration of the study (Weeks 1-13) when compared with the controls.

WATER CONSUMPTION: Visual inspection indicated no observable differences between groups throughout the treatment period.

OPHTHALMOSCOPIC EXAMINATION: There were no findings during ophthalmoscopic examinations that could be attributed to 2,6-di-tert-butylphenol.

HAEMATOLOGY: There were no toxicologically significant treatment-related differences in haematology or coagulation parameters following administration of 2,6-di-tert-butylphenol at dose levels up to 4000 ppm. At the end of the treatment period, lower white blood cells counts as a result of lower circulating lymphocytes (p<0.01) with higher fibrinogen levels (p<0.001) were observed in males receiving 4000 ppm, while higher fibrinogen levels were observed in females that received 1600 ppm (p<0.05) or 4000 ppm (p<0.01), when compared with controls. However, inspection of the data indicated that although there was a high degree of inter-animal variation, broadly the values for the animals receiving 2,6-di-tert-butylphenol were similar to the controls. There were also no differences in associated parameters, therefore, these differences were considered not to be related to treatment. No noteworthy differences were noted at the end of the recovery period, indicating any differences were more likely to be adaptive and shown to be reversible.

CLINICAL CHEMISTRY: There were no treatment-related inter-group differences in clinical chemistry parameters following administration of 2,6-di-tert-butylphenol at dose levels up to 4000 ppm. Minor inconsistent differences which achieved statistical significance at the end of the treatment period were observed in males (higher cholesterol and lower creatinine levels at 4000 ppm) and females (lower aspartate aminotransferase activity, higher cholesterol, lower triglycerides, lower globulin and higher calcium levels at 4000 ppm) when compared to the controls. Inspection of the data indicated that although there was variation within the data, broadly the values for the animals receiving 2,6-di-tert-butylphenol were similar to controls and there were also no differences in associated parameters or indices. These differences were considered not to be related to treatment. On completion of the recovery period, the differences described above were not evident, however aspartate aminotransferase and creatine phosphokinase activities were statistically significantly lower in males that had received 4000 ppm. Due to the magnitude and direction of change these differences were considered to be incidental and of no toxicological significance.

URINALYSIS: Urinary pH was lower in males that received 4000 ppm, while specific gravity was lower in females that received 4000 ppm achieving statistical significance. The toxicological significance of these changes are unclear in the absence of associated changes in both clinical chemistry parameters or in histopathological changes in the kidneys.

NEUROBEHAVIOUR: There were no treatment-related differences in the functional observation battery parameters following administration of 2,6-di-tert-butylphenol at dose levels up to 4000 ppm. All of the behaviours exhibited and observations were considered to be typical for rats of this age and strain on this type of study conducted at the Test Facility.

There were no treatment-related differences in the quantitative functional observation parameters following administration of 2,6-di-tert-butylphenol at dose levels up to 4000 ppm. Minor differences in landing foot splay, fore grip strength and tail flick that achieved statistical significance were observed in females at 500 ppm when compared with the controls. These isolated differences had no association with any other indices measuring effects on movement, the values recorded were broadly similar to controls, and as such was considered to be incidental and not treatment related.

There were no notable inter-group differences in motor activity (basic or fine movements or X and Y ambulations) following administration of 2,6-di-tert-butylphenol at dose levels up to 4000 ppm.

ORGAN WEIGHTS: In both sexes, the mean covariant and relative liver weights were higher in animals treated with 4000 ppm 2,6-di-tert-butylphenol, when compared with controls. In males, there was a decrease in the absolute, covariant and relative mean spleen weights and an increase in relative mean kidney weights in the group treated with 4000 ppm 2,6-di-tert-butylphenol compared with controls. There were no other test item-related organ weight changes between treated and control groups. Occasional organ weight differences between treated and control groups were considered incidental; as there were no patterns, trends or correlating data to suggest these differences were toxicologically relevant, they were considered unrelated to administration of 2,6-di-tert-butylphenol.

In females only, there was a statistically significant increase in relative liver weights at 4000 ppm 2,6-di-tert-butylphenol compared with controls at Day 120. The difference in female liver weight appears to be due to the lower control values relative to bodyweight (3.067 at Day 120 compared to 3.407 at Day 91). Additionally the female liver weight following a treatment-free period was also lower when compared to Day 91 values (3.706 vs 3.437). As such it is considered that the differences indicate a reversible effect of treatment on the liver. Other test item-related organ weight changes noted at the terminal euthanasia were not observed at the end of the recovery period (Day 120). Occasional organ weight differences between treated and control groups were considered incidental. As there were no patterns, trends or correlating data to suggest these differences were toxicologically relevant, they were considered unrelated to administration of 2,6-di-tert-butylphenol.

GROSS PATHOLOGY: No test item-related gross findings were noted. The findings observed were of similar incidence in control and treated animals, and of the nature commonly observed in rats of this strain and age. These findings were therefore incidental and considered unrelated to administration of 2,6-di-tert-butylphenol.

HISTOPATHOLOGY: NON-NEOPLASTIC: No test item-related microscopic findings were noted. There was no histological correlation with the mean weight changes observed in the liver, spleen and kidney in the group treated with 4000 ppm 2,6-di-tert-butylphenol. The findings observed were of similar incidence in control and treated animals, and of the nature commonly observed in rats of this strain and age. These findings were therefore incidental and considered unrelated to administration of 2,6-di-tert-butylphenol.

There was no histological correlation with the mean relative weight changes observed in the liver in females previously treated with 4000 ppm 2,6-di-tert-butylphenol. No test item-related microscopic findings were noted. The findings observed were of similar incidence in control and treated animals, and of the nature commonly observed in rats of this strain and age. These findings were therefore incidental and considered unrelated to administration of 2,6-di-tert-butylphenol.



Dose descriptor:
NOAEL
Remarks:
4000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Critical effects observed:
not specified
Conclusions:
Under the conditions of this study, the daily oral (dietary) administration of 2,6-di-tert-butylphenol up to 91 consecutive days was associated with slightly lower body weights, body weight gain and food consumed for males that received 4000 ppm. These findings recovered after a 28-day treatment free period. Additionally, liver weight increases, in the absence of histopathological correlate were considered to be an adaptive change and of no toxicological significance and were reversible following a treatment-free period.
Based on these findings the no observed adverse effect level (NOAEL) was considered to be 4000 ppm for both males and females equating to 270 mg 2,6-di-tert-butylphenol/k/day in males and 298 mg 2,6-di-tert-butylphenol/kg/day for females.
Executive summary:

The objective of this study was to assess the toxicity of 2,6-di-tert-butylphenol treatment in the rat after oral administration by the diet for at least 13 weeks (91 consecutive days) and to evaluate the reversibility of any findings. Groups of 10 male and 10 female Han Wistar Crl:WI(Han) rats were assigned to the study and fed diets containing 0, 150, 500, 1600 or 4000 ppm of 2,6-di-tert-butylphenol for a minimum period of up to 91 consecutive days. A further 5 male and 5 female animals were assigned as a recovery group to those receiving 0 or 4000 ppm of 2,6-di-tert-butylphenol and were retained for a further 28 days after the end of the treatment period to evaluate reversibility of any findings. The following observations and end points were assessed: viability, clinical observations, body weights, food consumption, achieved dosage, food utilisation, a visual assessment of water consumption and ophthalmoscopy examinations. All animals received a detailed functional observation battery (including motor activity) assessment during pretrial and Weeks 12 of treatment, as well as all designated recovery study animals during Week 17 (Week 4 of recovery period). Blood samples for haematology, coagulation and blood chemistry investigations and urine samples were collected from all animals towards the end of the treatment period and at the end the recovery period. All main study animals were euthanized after completion of at least 91 days of treatment with all recovery study animals euthanized after completion of a subsequent 28-day treatment-free period, and underwent a detailed necropsy examination with selected organs weighed. Tissues from all control and high dose (4000 ppm) animals were subjected to a comprehensive histological examination, with gross lesions (where appropriate) examined from low and intermediate dose animals. There were no unscheduled deaths, and no treatment-related clinical observations noted during the observation periods. Lower body weight gain was observed in males that were treated with 4000 ppm. This was associated with slightly lower food consumption. The overall mean achieved dosages were 0, 10, 33, 107 and 270 mg 2,6-di-tert-butylphenol/k/day in males and 0, 11, 39, 124 and 298 mg 2,6-di-tert-butylphenol/kg/day for females corresponding to dietary inclusion levels of 0, 150, 500, 1600 and 4000 ppm. There were no test substance-related effects on the movement, general behaviour or the physiology of the animals and haematology, coagulation and clinical chemistry were unaffected by treatment. There were no eye changes that were related to treatment. Very slight differences were, however, observed during pretrial in landing foot splay (higher in males that received 150 or 1600 ppm) and hind grip strength (higher in females that received 1600 ppm) and during week 12 in females that received 500 ppm (lower foot splay, fore grip strength and tail flick) when compared with the controls. Urinary pH was lower in males that received 4000 ppm, while specific gravity was lower in females that received 4000 ppm. Higher liver and kidney weights were noted in males and females that received 4000 ppm, while spleen weights were lower in males that received 4000 ppm. On completion of the treatment free period, higher relative liver weights were recorded in females that received 4000 ppm 2,6-di-tert-butylphenol. There were no gross or microscopic findings that were considered related to treatment with 2,6-di-tert-butylphenol. All findings observed throughout treatment generally recovered after a treatment-free period of 28 days which indicated all differences were adaptive and reversible and of no toxicological significance. Under the conditions of this study, the daily oral (dietary) administration of 2,6-di-tert-butylphenol up to 91 consecutive days was associated with slightly lower body weights, body weight gain and food consumed for males that received 4000 ppm. These findings recovered after a 28-day treatment free period. Additionally, liver weight increases, in the absence of histopathological correlate were considered to be an adaptive change and of no toxicological significance and were reversible following a treatment-free period. Based on these findings the no observed adverse effect level (NOAEL) was considered to be 4000 ppm for both males and females equating to 270 mg 2,6-di-tert-butylphenol/kg/day in males and 298 mg 2,6-di-tert-butylphenol/kg/day for females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
270 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Reliable
System:
hepatobiliary
Organ:
liver

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

Repeated dose toxicity oral: Test methods and results:

Three studies are available to provide information regarding the toxicity of 2,6 DTBP in rats following repeated dosing. These are a 90 day oral dietary toxicity study in rats, a 28 day oral (gavage) toxicity study in rats and a preliminary reproduction toxicity screening toxicity (oral gavage dosing) study in rats. The 90-day study is the key study for the determination of the NOAEL and the classification and labelling, however the findings of the remaining studies support the conclusions from the 90-day study.

Subchronic 90 Day Oral Dietary Toxicity Study with 2,6-bis (1,1 dimethylethyl) -phenol in the rat, study number 525862, Robertson, B., 2014.

The method used followed that described in the OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 408, "Repeated Dose 90-Day Oral Toxicity in Rodents", September 21, 1998 and Regulation (EC). No 440/2008, B. 26: "Sub-Chronic Oral Toxicity Test".

Groups of 10 male and 10 female Han Wistar Crl: WI(Han) rats were assigned to the study and fed diets containing 0, 150, 500, 1600 or 4000 ppm of 2,6-di-tert-butylphenol for a minimum period of up to 91 consecutive days. A further 5 male and 5 female animals were assigned as a recovery group to those receiving 0 or 4000 ppm of 2,6-di-tert-butylphenol and were retained for a further 28 days after the end of the treatment period to evaluate reversibility of any findings.

The following observations and end points were assessed: viability, clinical observations, body weights, food consumption, achieved dosage, food utilisation, a visual assessment of water consumption and ophthalmoscopy examinations. All animals received a detailed functional observation battery (including motor activity) assessment during pretrial and Weeks 12 of treatment, as well as all designated recovery study animals during Week 17 (Week 4 of recovery period). Blood samples for haematology, coagulation and blood chemistry investigations and urine samples were collected from all animals towards the end of the treatment period and at the end the recovery period.

All main study animals were euthanized after completion of at least 91 days of treatment with all recovery study animals euthanized after completion of a subsequent 28-day treatment-free period, and underwent a detailed necropsy examination with selected organs weighed.

Tissues from all control and high dose (4000 ppm) animals were subjected to a comprehensive histological examination, with gross lesions (where appropriate) examined from low and intermediate dose animals.

There were no unscheduled deaths, and no treatment-related clinical observations noted during the observation periods.

Lower body weight gain was observed in males that were treated with 4000 ppm. This was associated with slightly lower food consumption.

The overall mean achieved dosages were 0, 10, 33, 107 and 270 mg 2,6-di-tertbutylphenol/kg/day in males and 0, 11, 39, 124 and 298 mg 2,6-di-tert-butylphenol/kg/day for females corresponding to dietary inclusion levels of 0, 150, 500, 1600 and 4000 ppm.

There were no test substance-related effects on the movement, general behaviour or the physiology of the animals and haematology, coagulation and clinical chemistry were unaffected by treatment. There were no eye changes that were related to treatment.

Very slight differences were, however, observed during pretrial in landing foot splay (higher in males that received 150 or 1600 ppm) and hind grip strength (higher in females that received 1600 ppm) and during week 12 in females that received 500 ppm (lower foot splay, fore grip strength and tail flick) when compared with the controls; this was of no toxicological significance.

Urinary pH was lower in males that received 4000 ppm, while specific gravity was lower in females that received 4000 ppm.

Higher liver and kidney weights were noted in males and females that received 4000 ppm, while spleen weights were lower in males that received 4000 ppm. On completion of the treatment free period, higher relative liver weights were recorded in females that received 4000 ppm 2,6-di-tert-butylphenol.

There were no gross or microscopic findings that were considered related to treatment with 2,6-di-tert-butylphenol.

All findings observed throughout treatment generally recovered after a treatment-free period of 28 days which indicated all differences were adaptive and reversible and of no toxicological significance.

Under the conditions of this study, the daily oral (dietary) administration of 2,6-di-tertbutylphenol up to 91 consecutive days was associated with slightly lower body weights, body weight gain and food consumed for males that received 4000 ppm. These findings recovered after a 28-day treatment free period. Additionally, liver weight increases, in the absence of histopathological correlate were considered to be an adaptive change and of no toxicological significance and were reversible following a treatment-free period.

Based on these findings the no observed adverse effect level (NOAEL) was considered to be 4000 ppm for both males and females equating to 270 mg 2,6-di-tert-butylphenol/k/day in males and 298 mg 2,6-di-tert-butylphenol/kg/day for females.

Subacute 28-day oral toxicity with 2,6-bis(1,1-dimethylethyl)-phenol by daily gavage in the rat, Reijnders, J.B.J, 1991.

The method used followed that described in the OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 407, "Repeated Dose Oral Toxicity - Rodent: 28 or 14 day study", Paris Cedex, May 12, 1981 and Regulation (EC). No 440/2008, B. 7: "Repeated Dose (28 days) Toxicity (Oral).

Following a range-finding study, the test substance was administered daily by gavage to 4 groups of ten animals (five males and five females) at concentrations of 0 (control), 15, 100 and 600 mg/kg/day.

All animals were subjected to daily clinical observation. Body weight and food consumption were measured weekly and on the day before necropsy. During week 4 of treatment, both eyes of all animals were examined. On the day of termination blood was collected from each animal for clinical laboratory investigations.

Subsequently a necropsy examination was performed, macroscopic observations and organ weights were recorded. A histopathological examination was performed on adrenals, heart, kidneys, liver, spleen and stomach.

Treatment related changes in the blood, liver and kidney were observed at 100 and 600 mg/kg/day. At 600 mg/kg/day male and females had around 30 to 40% heavier livers than control animals and these weight increases were accompanied by very slight centrilobular hepatocellular hypertrophy. Although relative liver weight (% of bodyweight) in males at 100 mg/kg/day was approximately 20% heavier than controls this was not accompanied by microscopic changes and liver weight in females was comparable to controls. The increased liver weight at 100 (males only) and 600 mg/kg/day accompanied by slight centrilobular hypertrophy at 600 mg/kg/day was considered to be an adaptive change and of no toxicological significance. Reduced serum urea (17%) was present in females at 100 mg/kg/day and also noted in females at 600 mg/kg/day (33%). This is not considered a toxicologically relevant finding as a pathological change is normally reflected by an increase in serum urea levels.

The NOEL in this study was determined to be 15 mg/kg/day. However, it may be considered that the findings at 100 mg/kg/day were not adverse in that they would not be considered to affect the performance of the whole organism or the ability of the organism to respond to an additional environmental challenge. The NOAEL in this study was therefore, 100 mg/kg/day. Definitions on NOAEL and adverse effects are extracted from the Ecetoc technical report No 85, Recognition of, and Differentiation between, Adverse and Non-adverse Effects in Toxicology Studies.

Preliminary reproduction toxicity screening test with 2,6-bis(1,1-dimethylethyl) -phenol, Becker H., Biedermann K. (1992), RCC, Research and Consulting Company Ltd. and RCC Umweltchemie AG, Itingen, Switzerland, SI Group, Report No. 321794)

In this study male and female rats were dosed during a preparing and pairing period and until the day before sacrifice (around 43 days) at 0, 30, 150 and 750 mg/kg/day in order to investigate the effects on male and female reproductive performance. Marked toxicity as manifested by clinical signs of sedation, ataxia, lateral/ventral recumbancy, tremors, clonic spasms and hunched posture were recorded in both sexes at 750 mg/kg/day. There were no effects on general tolerability (clinical signs, bodyweight change, food intake), reproduction parameters or pup viability at 150 mg/kg/day.

The 90 d study is the study with the highest relevance for this endpoint. In addition the results shown there are in line with the findings of the other two studies. In the 28 d study slight effects in the liver and the kidneys have been seen at 100 mg/kg bw/d which are comparable to those seen in the 90 d study at the highest dose. In the latter study it has been shown that these effects are reversible in a short recovery period and therefore are not adverse. The fact that the liver effects in the 28 d study are a bit more pronounced at 100 mg/kg bw/d as compared to those seen in the 90 d study at 270 mg/kg bw/d might very well be a result of the bolus dosing in the former study. The continous exposure dietary exposure of the latter is nevertheless a better mimic for the continous exposure of worker and especially the general public.

The findings in the reproduction toxicity screening test with an exposure period of at least 43 days support the findings of the 90-day study. At very high dose of 750 mg/kg there were notable symptoms in male and female rats (increased food consumption in the male rats and reduced food consumption in the female rats). These were acute effects after bolus administration of a very high dose. Further, marginally reduced body weight gain in the males and slightly reduced body weight gain in the females were noted.

Justification for classification or non-classification

Repeated dose toxicity oral

No classification is needed in accordance with EU CLP (Regulation (EC) No. 1272/2008).

There were no changes in liver enzymes noted in the serum and no histopathological correlate, therefore, the increased liver weights may be attributed to an increased work hypertrophy. As this is a common phenomenon in rats, in order to metabolise a xenobiotic agent, these findings were considered not to be a change of toxicological significance.

Whilst increased kidney weights were noted in the 28-day and 90-day studies there were no changes in the blood and no histopathological correlate, in addition this finding was not seen after the 28 day recovery period. Therefore, this finding was not considered to be of toxicological significance.

The NOEL in the 90-day study was the highest dose administered which was equivalent to 270 mg/kg bw/day in males and 298 mg/kg bw/day in females and it was considered that the findings in these animals were not adverse in that they would not be considered to affect the performance of the whole organism or the ability of the organism to respond to an additional environmental challenge. The NOAEL for this study was therefore, the lower of the male and female values, 270 mg/kg bw/day.