Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]

Endpoint summary

Description of key information

AO-26: Toxicity Study by Oral Administration to Sprague Dawley Rats for 4 Weeks

The administration of AO-26 to Crl:CD (SD) rats at dose levels up to 1000 mg/kg/day was well tolerated during the in-life phase of the study. Pathology investigations, however, indicated test article related changes in the liver and heart of males and females receiving 100, 300 or 1000 mg/kg/day and minimal treatment-related changes in the kidneys of females receiving 1000 mg/kg/day. The microscopic changes observed in the livers and hearts were considered to be adverse, therefore a no-observed adverse effect level (NOAEL) could not be established on this study.

AO-26: Reproductive/Development Toxicity Screening Study in Sprague-Dawley Rat by Oral Gavage Administration

Treatment ofAO-26 to parental animals at 25, 50 or 100 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was generally well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, estrous cycles, mating performance, fertility or macropathology. The clinical condition, body weight and survival of the subsequent F1 offspring were also unaffected by parental treatment.

Adverse findings were limited to an increase in heart weight for males that received 50 or 100 mg/kg/day and this correlated with histopathology which showedmyocardial degeneration associated with or without inflammatory cell infiltrate which was seen in the heart of both male and females that received 25, 50 or 100 mg/kg/day.

It was therefore concluded that the No-observed-adverse-effect-level (NOAEL) for reproductive performance was 100 mg/kg/day (the highest dose in this study); however a NOAEL for parental toxicity was not established.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See section titled any other information on material and methods to see further details on deviations.

GLP compliance:

yes (incl. QA statement)

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
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation:47 to 54 days old.
- Weight at study initiation:Males: 243 g to 290 g Females: 184 g to 206 g
- Housing:Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
- Diet (e.g. ad libitum):Rat and Mouse No. 1 Maintenance Diet (pelleted diet).
- Water (e.g. ad libitum):Non-restricted Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period:12 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19-23ºC
- Humidity (%):40-70%
- Air changes (per hr):Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light):Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: From:Day 1 To: Day 29

Route of administration:

oral: gavage

Details on route of administration:

Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth. Treated at Constant doses in mg/kg/day.Volume dose was 5 mL/kg body weight. The control group received vehicle at the same volume dose as the treated groups. They were treated once daily at approximately the same time each day.Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
AO-26 was prepared for administration as a series of graded concentrations in the vehicle. The required amount of test material was weighed out starting with the lowest concentration. Small amounts of the final volume of vehicle were added to the test material and mixed. The suspension was poured into a measuring cylinder, which had been wetted with vehicle. The mixing container was thoroughly rinsed with vehicle and this was added to the cylinder. The suspension was made up to the required volume with the remaining vehicle, transferred to a beakerand mixed using a magnetic stirrer until it appeared homogenous. The suspension was transferred to the final containers, via syringe, whilst magnetic stirring. The mid and high concentrations were formulated in ascending order using the same method .All formulations were prepared weekly and refrigerated (nominally 2-8ºC) and protected from light until required for use. Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 10 and 200 mg/mL were analysed to assess the stability and homogeneity of the test substance in the liquid matrix. The results from these assays demonstrated that AO-26 produced an homogenous suspension and was stable in the liquid matrix at concentrations of 10 and 200 mg/mL for four days when stored at ambient temperature (nominally 20 ºC) and for up to 15 days when stored refrigerated (nominally 2-8ºC).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A representative sample of test formulation was accurately weighed and dissolved using ultrasonic vibration in a suitable volume of THF. The extract was diluted, initially using THF and finally using MeOH, to provide a solution containing AO-26 at an expected concentration within the range 2 μg/mL to 4 μg/mL. The concentration of AO-26 in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, linearity of detector response, precision of injection, method accuracy and precision. The homogeneity and stability was confirmed for AO-26 in corn oil formulations at nominal concentrations of 10 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 4 days and refrigerated storage for up to 15 days. The mean concentrations of AO-26 in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 males and 25 females.

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the
occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.


DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations:The weight of each animal was recorded one week before treatment commenced (Day -7), on the day that treatment commenced (Day 1), weekly throughout the study (last scheduled body weight was recorded on Day 28) and before necropsy. More frequent weighings were instituted for one animal displaying ill-health (Control male No. 19 had abnormal breathing in Week 3), so that the progress of the observed condition could be monitored.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION:No


HAEMATOLOGY: Yes
- Time schedule for collection of blood:Blood samples were collected after overnight withdrawal of food on Day 29.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 50

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 4 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing. The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the vibrissae). The animal’s reaction was recorded as:
1 No reaction or ignores probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Abnormally fearful or aggressive reaction

Touch response
The animal was stroked gently on the nape of the neck with a blunt probe and the reaction recorded as:
1 No reaction or ignores probe
2 Normal awareness and reaction e.g. turns towards or moves away
3 Abnormally fearful or aggressive reaction

Auditory startle reflex
The animal’s response to a sudden loud noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalisation without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalisation
4 Exaggerated response e.g. excessive vocalisation, body movement or
aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Force Indicators. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 4 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System, with hardware supplied by Pearson Technical
Services and software developed and maintained by Huntingdon Life Sciences. Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Not specified

HISTOPATHOLOGY: Not specified

Statistics:

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

The following data types were analysed at each timepoint separately:
Grip strength and motor activity
Body weight, using gains over appropriate study periods
Haematology and blood chemistry
Organ weights, absolute and adjusted for terminal body weight

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The detailed physical examinations and arena observations conducted each week during the treatment period did not reveal any unusual changes in the appearance and behaviour of the animals except for one male receiving 1000 mg/kg/day that had chromodacyorrhea (red staining around the eyes) in Weeks 3 and 4.
Signs associated with dosing in the test-article treated animals were confined to chin rubbing seen in one male receiving 1000 mg/kg/day in Week 1. Chin rubbing is often seen in oral studies where the material is administered by gavage and is attributed to the animal trying to clean residual material from the muzzle. It has no toxicological significance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In general the bodyweight gains of the AO-26 treated animals were similar to the gains of the controls, throughout the treatment period. One male and one female receiving 1000 mg/kg/day had lower than control bodyweight gains and this influenced the group mean results . The overall bodyweight gains (Day 1 to 28) of males receiving 1000 mg/kg/day were lower than the controls (84% of control). This first became apparent in Week 2 and continued in Weeks 3 and 4. This was due to Animal No. 2 that had lower than control gains in each week of treatment. For females receiving 1000 mg/kg/day the group mean bodyweight gain was slightly lower than control in Week 1 but this was due to Animal No. 42 that had a bodyweight loss of one gram in this week compared with gains of between 24 and 40 grams for the other females. Animal No. 42 also had a bodyweight loss of 3 grams in Week 4. These results indicate that there was a degree of individual susceptibility to treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Males receiving 1000 mg/kg/day consumed less food than the controls in all weeks of treatment. The mean amount of food eaten over the treatment period was 7% less than that eaten by the controls. Females receiving 1000 mg/kg/day consumed more food than the controls in all weeks of treatment with the mean amount eaten being 10% greater. The food consumption of animals receiving 100 or 300 mg/kg/day was considered to be unaffected by treatment.

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

not examined

Description (incidence and severity):

Water consumption was assessed visually. No difference was apparent in the amount of water drunk by the test-article treated animals when compared with the controls and, therefore, no actual measurements were made.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the blood sampling occasion after 4 weeks of treatment marginally low reticulocyte counts were seen in males receiving 1000 mg/kg/day (0.75X control); this was primarily due to low values for animals 2 and 5. There was some variability in individual differential white blood cell counts but values were generally within expected ranges for rats of the age and strain. The coagulation investigations revealed, when compared with the control groups, statistically significant increases in prothrombin times in all groups of AO-26 treated males (up to 1.33X control) and females (up to 1.16X control), with a dose response clearly evident in each sex and with males being affected to a greater extent than females.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

When compared with the controls, blood chemistry investigations at the end of the treatment period revealed the following changes in males and females in all test-article treated groups: high asparate amino transferase activities (up to 2.4X control in males and 3.0X in females), high plasma urea concentrations (up to 2.45X control in males and 1.72X in females) and low cholesterol concentrations (dose-related; down to 0.56X control in males and 0.45X in females). In addition, low concentrations of bile acids were evident in males receiving 1000 mg/kg/day (0.16X control). All other inter-group differences, including those that attained statistical significance, were minor, lacked dose relationship or were seen in one sex only and were therefore considered incidental. This included the statistically significant apparent increases in plasma glucose concentrations in all AO-26 treated male groups where there was no clear dose-relationship; the highest result being reported for one male receiving 100 mg/kg/day (Animal No. 7) and the results for males receiving 1000 mg/kg/day were considered normal. Statistically significant apparent increases in glucose concentration were also reported for females receiving 100 or 300 mg/kg/day but, with a lack of effect seen in the high dose group and the greatest effect reported in the low dose animals, this finding was attributed to normal biological variation and not treatment.

Urinalysis findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with the controls, liver weights, both absolute and adjusted for terminal bodyweight, were high in females given 100 mg/kg/day and in males and females given 300 or 1000 mg/kg/day. In terms of the adjusted liver weights the differences from control were 1.18 and 1.24X for males receiving 300 or 1000 mg/kg/day, respectively and 1.19, 1.30 or 1.37X for females given 100, 300 or 1000 mg/kg/day, respectively.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pale colouration of the liver was seen in the majority of treated animals.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength assessments were performed (before dosing) on all animals on one occasion in Week 4. There were no treatment-related findings recorded for any of the responses tested (approach, touch and pinch response, auditory startle reflex and grip strength).

Motor activity scores showed considerable inter and intra-group variation but the differences were not considered associated with treatment with AO-26. At the investigation in Week 4, high beam (rearing activity) and low beam (cage floor
activity) total scores were increased for males given 1000 mg/kg/day with low beam scores also slightly increased for males given 300 mg/kg/day. Statistical significance (p<0.05) was attained for the group mean total high beam score count for the high dose males and this was influenced by an increase in the high beam count for the 24 to 30 minute recording interval. The total counts recorded for low beam activity did not attain statistical significance when compared with the control. The low beam totals were influenced by statistically significant increases in scores at the 30 to 36 minute interval and also the 54 to 60 minute interval. Review of the individual results revealed that the highest total low beam score was 1342 and was recorded for Animal No. 8 receiving 100 mg/kg/day with the second highest score being recorded for a control male, Animal No.16 with a score of 1217. More males receiving 300 or 1000 mg/kg/day had total scores greater than 1000 than in the low dose and control group and this resulted in the higher mean results. No similar trends were evident in the females and given that there was no consistent or clear pattern to these changes in motor activity in the males they are considered not to be related to
treatment.

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with AO-26 were seen in the liver and heart of males and females and in the kidneys of females.

Liver
Periportal hepatocellular vacuolation was seen in the livers of all treated females and in the majority of treated males; there was a dose-related increase in severity in both sexes. The vacuolation was microvesicular in appearance and stained positive with Oil Red O, indicating the presence of fat.

Heart
Diffuse myocardial degeneration was observed in all treated animals. Vacuolation wasobserved in the majority of degenerate myofibres and was considered to be a component of the degeneration rather than representing fat accumulation, as the vacuoles stained negative with Oil Red O. A minimal inflammatory cell infiltrate was associated with the myocardial degeneration in all treated males and in some females receiving 100 or 300 mg/kg/day.

Kidneys
Minimal vacuolation of the tubular epithelium was observed in all females receiving 1000 mg/kg/day. Sections of kidney from two Group 4 females were stained with Oil Red O. The tubular vacuoles observed in the H&E stained sections stained positive with Oil Red O, indicating the presence of fat.

Other effects:

not specified

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

cardiovascular

Organ:

heart

liver

Treatment related:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Conclusions:

The administration of AO-26 to Crl:CD (SD) rats at dose levels up to 1000 mg/kg/day was well tolerated during the in-life phase of the study. Pathology investigations, however, indicated test article related changes in the liver and heart of males and females receiving 100, 300 or 1000 mg/kg/day and minimal treatment-related changes in the kidneys of females receiving 1000 mg/kg/day. The microscopic changes observed in the livers and hearts were considered to be adverse, therefore a no-observed adverse effect level (NOAEL) could not be established on this study.

Executive summary:

The systemic toxic potential of AO-26 (an antioxidant for plastics) to Crl:CD(SD) rats by oral (gavage) administration was assessed over a period of 4 weeks. Three groups, each comprising five male and five female rats, received AO-26 at doses of 100, 300 or 1000 mg/kg/day at a volume-dose of 5 mL/kg body weight. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose.

The following investigations were undertaken during the study: clinical condition, signs associated with dosing, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (visual observations), haematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology.

Results

No animals died prematurely. The appearance and behaviour of the animals were not affected by treatment except for one male receiving 1000 mg/kg/day that had chromodacyorrhea (red staining around the eyes) in Weeks 3 and 4. Sensory reactivity findings, grip strength values and motor activity were considered to be unaffected by treatment.

In general, the bodyweight gains of the AO-26 treated animals were similar to the gains of the controls, throughout the treatment period. There were, however, low gains for one male and one female receiving 1000 mg/kg/day. Slightly low food consumption was seen in males receiving 1000 mg/kg/day and slightly high food consumption was seen in females receiving 1000 mg/kg/day in all weeks of treatment. No effects of treatment were seen on water consumption. Haematological findings related to treatment with AO-26 comprised slightly low reticulocyte counts in two males receiving 1000 mg/kg/day and extended mean prothrombin times in all groups of AO-26 treated males and females.

Biochemical changes in the blood plasma that were attributed to treatment comprised moderately high asparate amino transferase activities, moderately high plasma urea concentrations and a dose-related reduction in cholesterol concentrations in all test-article treated groups (males and females) with low concentrations of bile acids in males receiving 1000 mg/kg/day. Absolute and adjusted liver weights were high in females given 100 mg/kg/day and in males and females given 300 or 1000 mg/kg/day. The macroscopic examination performed after 4 weeks of treatment revealed pale colouration of the liver in the majority of test-article treated animals. Histopathological findings related to treatment were seen in the liver and heart of males and females and in the kidneys of females. In the liver, periportal hepatocellular vacuolation was seen in all test-article treated females and in the majority of treated males. The vacuolation was microvesicular in appearance and stained positive with Oil Red O, indicating the presence of fat. Diffuse myocardial degeneration (minimal/slight) was observed in the hearts of all test-article treated animals. Vacuolation was observed in the majority of degenerate myofibres but stained negative with Oil Red O. A minimal inflammatory cell infiltrate was associated with the myocardial degeneration in all treated males and in two females receiving 100 and in three females receiving 300 mg/kg/day. In the kidneys, minimal vacuolation of the tubular epithelium was seen in all females given 1000 mg/kg/day; these vacuoles stained positive with Oil Red O, indicating the presence of fat.

Conclusion

The administration of AO-26 to Crl:CD (SD) rats at dose levels up to 1000 mg/kg/day was well tolerated during the in-life phase of the study. Pathology investigations, however, indicated test article related changes in the liver and heart of males and females receiving 100, 300 or 1000 mg/kg/day and minimal treatment-related changes in the kidneys of females receiving 1000 mg/kg/day. The microscopic changes observed in the livers and hearts were considered to be adverse, therefore a no-observed adverse effect level (NOAEL) could not be established on this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

cardiovascular

Organ:

heart

Justification for classification or non-classification

During the 28 day oral repeat dose study pathology investigations, indicated test article related changes in the liver and heart of males and females receiving 100, 300 or 1000 mg/kg/day and minimal treatment-related changes in the kidneys of females receiving 1000 mg/kg/day. As these effects were considered adverse and seen at all dose levels an NOAEL value could not be established.

To classify the substance for repeat exposure we have had to review the data from the OECD 421 Reproductive/Development Toxicity Screening Study in Sprague-Dawley Rat by Oral Gavage Administration as this study was conducted at lower dose levels 25, 50 or 100 mg/kg/day.

Even though no effects were seen for reproductive toxicity, adverse findings were noted in the parental animals which were limited to an increase in heart weight for males that received 50 or 100 mg/kg/day and this correlated with histopathology which showed myocardial degeneration associated with or without inflammatory cell infiltrate which was seen in the heart of both male and females that received 25, 50 or 100 mg/kg/day.

Adverse effects were noted at all dose levels including the lowest dose at 25 mg/kg/day. For classification purposes the LOAEL value is concluded at 25 mg/kg/day.

According to the CLP criteria the threshold value for STOT RE category 1 classification is C ≤ 30 therefore AO-26 is classified as STOT RE Category 1-H372: Causes damage to organs (Heart) through prolonged or repeated exposure (state route of exposure if it is conclusively proven that no other routes of exposure cause the hazard).