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Reference
Endpoint:
basic toxicokinetics
Type of information:
other: written assessment based on available information
Adequacy of study:
key study
Study period:
May 2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non-GLP assessment report
Objective of study:
other: Assessment of toxicokinetic behaviour
GLP compliance:
no
Conclusions:
In conclusion, there is little evidence that the substance is absorbed and distributed via ingestion, dermally or when inhaled. Changes in the liver in the 28 day repeated dose study indicates the substance is distributed throughout the body. In the event of absorption, the substance would likely be metabolised by oxidative pathways which will then be excreted via the urine. Unchanged substance will be excreted directly in the faeces. Consequently, the substance is considered to have low bioaccumulation potential.
The substance is not considered to pose a hazard overall.

Description of key information

Introduction


This assessment of the toxicokinetic properties of ANOX 1315/BF (A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate) is based on the results obtained for the toxicological end‑points listed below with simultaneous reference to relevant physico-chemical data:



  • Acute oral toxicity

  • Acute dermal toxicity

  • Acute inhalation toxicity

  • Skin irritation

  • Eye irritation

  • Skin sensitisation

  • Repeated Dose Toxicity Study

  • Reproduction / Developmental Toxicity Study

  • Bacterial reverse mutation test

  • In vitro chromosome aberration test

  • Mouse Lymphoma Assay


Toxicological Profile


ANOX 1315 is a UVCB phenolic antioxidant substance. The substance has a molecular weight range of > 400, a water solubility of 0.33mg/L at 25 °C, a partition coefficient (n-octanol/water) (log Kow) of > 3.56 and a vapour pressure of 166.3 Pa at 20 °C.


A single-dose oral toxicity of ANOX BF was performed according to the acute toxic class method in Sprague-Dawley Cr1:CD (SD) BR rats. ANOX BF did not cause mortality at a dose level of 5000 mg/kg bw.  Two male and three female treated rats only showed transient piloerection 2 hours after treatment.There were no effects on body weights or body weight gains that could be attributed to treatment with the test substance. There was no evidence of the macroscopic observations in the animals dosed at 5000 mg/kg bw and terminated on Day 14.Under the conditions of this study, the acute oral LD50 value of ANOX BF was found to be above 5000 mg/kg bw in male and female rats.


An acute inhalation toxicity was performed on male and female Sprague-Dawley rats in accordance with OECD 403 test guidelines. Animals were exposed to 4.07 mg/ litre -1 and 7.53 mg/ litre -1 aerosol as a single dose for 4 hours, the particle size determination indicated 94.0% and 93.9% of the test aerosol particles to be <3.5 µm for groups 1 and 2 respectively.  There were no mortalities, clinical signs or affects to body or lung weight following exposure to the test article. No abnormalities detected at post mortem observations were considered to be indicative of a reaction to treatment with the test the substance. The lung : body weight ratio values were generally all within normal limits. LC50 was determined to be >7.53 mg/L air for male and female rats.


An acute dermal toxicity study was also performed with ANOX BF in male and female Sprague Dawley Crl:CD (SD) BR rats.  Animals received a single dermal patch administration ANOX BF at the dose of 2000 mg/kg and exposed for 24 hours. Observations took place over 24 hours. During which time no deaths occurred as a result of treatment. No clinical signs or behavioural alterations were observed in any animal. At the application site, no dermal reactions were detected. No significant changes in the body weight gain were observed. No changes were found at the gross pathology examination performed at the end of the observation period. The acute dermal median lethal dose (LD50) was determined to be greater than 2000 mg/kg in male/female rats.


 


Skin irritation for ANOX BF was assessed using an in vivo skin irritation test, exposing New Zealand White rabbits to 0.5mL of the test substance for 4 hours. Observations that took place over 72 hours no deaths occurred as a result of treatment, nor were observed untoward clinical signs or behavioural alterations. At the application site, a very slight erythema (graded 1) was observed on all treated animals only 1 hour after patch removal. Therefore, ANOX BF was considered not corrosive irritating to rabbit skin.


An in vivo eye irritation study was conducted on New Zealand White rabbits where 0.1mL of the test substance was instilled into one the eye of the test animal where the other eye served as a control, following 24 hours the eyes were rinsed with water. At the examination performed at 1 hour after treatment in all treated rabbits, slight redness of the conjunctivae (graded 1) was noted. At the subsequent observations carried out 24, 48 and 72 hours after treatment in all rabbits no changes were evident in this tissue. Based on this study ANOX BF was determined not to be corrosive or irritating to rabbit eyes.


Sensitisation potential of ANOX BF was assessed with the use of a Buhler test as described by Klecak. Animals tested did not show any positive reactions to the test substance, therefore indicating a lack of sensitisation potential.


 


A 28 day repeat dose oral toxicity study was carried out in rats treated with test article ANOX – BF,  according to OECD test guideline 407. ANOX BF was administered by oral route as a suspension in corn oil once a day for 4 consecutive weeks to groups of male and female Sprague Dawley Crl:CD (SD) BR rats at the doses of 10, 100 and 1000 mg/kg/day (groups 2,3 and 4. respectively). Under the conditions of the study no clinical changes were seen at the two lowest dosages. At the highest dose, episodes of salivation were observed just after treatment in some rats of both sexes starting from the third week of the study up to the end of the administration period. No clinical changes were seen during the recovery period. Body weight and food intake were unaffected at any dose. No changes of significant toxicological relevance were observed in either sex at any dose. Urine analysis showed a slight increase in the specific gravity of urine in treated males and a slight increase in frequency of Leukocytes in individual males and females of the high dosed group. The latter change was still observed in some recovered males at the end of the withdrawal period. Centrilobular hypertrophy of the liver was noted, however is regarded as a metabolic functional change, rather than a toxic change and is considered adaptive. NOEL was determined to be 10 mg/kg/day.


 


The substance was assessed in a Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats. ANOX 1315 did not induce a biologically significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment . In conclusion, the substance has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.


In chromosome aberration assays using Chinese hamster V79 lung cells, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 500, 150, 50, 15, 5, 1.5 and 0.5 mg/ml (second experiment used: 15, 5: 1.5,0.5,0.15 and 0.05 mg/ml) with and without metabolic activation.  A further assay incorporating a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) was also performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. No observed an incidence of polyploid cells or endoreduplications statistically different from the control group, either in the presence or in the absence of metabolic activation.


None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values. In conclusion, ANOX BF did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, ANOX BF was considered as not clastogenic in this test system.


An in vitro mammalian cell assay was also performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of ANOX  BF to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).


ANOX 1315 was tested in the L5178Y/TK+/-Mouse Lymphoma Mutagenesis Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the concentration range for the mutagenesis assays. The initial and extended treatment mutagenesis assays were used to evaluate the mutagenic potential of the test article. Ethanol was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in ethanol at 500 mg/mL, the maximum concentration tested.


 


In the preliminary toxicity assay, the maximum concentration of ANOX 1315 in treatment medium was 5000 µg/mL. Visible precipitate was present at concentrations ≥150 µg/mL in treatment medium with a 4-hour exposure and ≥250 µg/mL in treatment medium with a 24-hour exposure. No visible precipitate was present at concentrations of ≤50 µg/mL in treatment medium with a 4-hour exposure and ≤ 75 µg/mL with a 24-hour exposure. Selection of concentrations for the mutation assay was based on solubility profile. Substantial toxicity, i.e., suspension growth of ≤50% of the solvent control, was not observed at any concentration with or without S9 activation. 


Based on the results of the preliminary toxicity assay, the doses chosen for treatment of the initial mutagenesis assay ranged from 5 to 150 µg/mL for both the non-activated and S9-activated cultures. Visible precipitate was present at concentrations ≥150 µg/mL in treatment medium. No visible precipitate was present at concentrations of ≤125 µg/mL in treatment medium. The concentrations chosen for cloning were 50, 75, 100, 125, and 150 µg/mL with and without S9 activation. One non-activated cloned culture exhibited a mutant frequency between 55 and 99 mutants per 106clonable cells over that of the solvent control. There was no dose-response trend.


Toxicity in the cloned cultures, i.e., total growth of 150% of the solvent control, was not observed any concentration with or without S9 activation.


Based on the results of the preliminary toxicity assay, the doses chosen for treatment of the extended treatment assay ranged from 5 to 250 µg/mL for non-activated cultures with a 24-hour exposure. Due to a calculation error, the actual doses ranged from 3.75 to 187.5 µg/mL. Visible precipitate was present at concentrations 275 µg/mL in treatment medium. No visible precipitate was present at concentrations of ≤37.5 µg/mL in treatment medium. The concentrations chosen for cloning were 3.75,7.5, 18.75,37.5 and 75 µg/mL. No cloned cultures exhibited mutant frequencies between 55 and 99 mutants per 106clonable cells over that of the solvent control. There was no dose-response trend. Toxicity in the cloned cultures was not observed any concentration with or without S9 activation.


The trifluorothymidine-resistant colonies for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS positive control yielded the expected increase in small colonies, verifying the adequacy of the methods used to detect small colony mutants.


Under the conditions of this study ANOX 1315 was concluded to be negative in the L5178Y/TK+/-Mouse Lymphoma Mutagenesis Assay.


Toxicokinetic parameters


Absorption


Oral


Specific data on absorption or tissue distribution are not available. However, the absence of systemic toxicity recorded after administration of high dosages (1 x 5000 mg/kg orally or 1 x 2000 mg/kg dermally, 28 x 1000 mg/kg orally) to rats suggests a possibility of restricted bioavailability and/or absorption from the GI tract. The absence of evident toxicity to rats given repeated, high doses of the substance in the 28-day and reproduction studies shows low biological activity. The minor liver changes seen were characteristic of adaptive changes related to metabolism of the substance.


 


Specific data on absorption or tissue distribution are not available. However, the absence of systemic toxicity recorded after administration of high dosages (1 x 5000 mg/kg orally or 1 x 2000 mg/kg dermally, 28 x 1000 mg/kg orally) to rats suggests a possibility of restricted bioavailability and/or absorption from the GI tract The absence of evident toxicity to rats given repeated, high doses of the substance in the 28-day and reproduction studies shows low biological activity. The minor liver changes seen were characteristic of adaptive changes related to metabolism of the substance.


As a worst case, for risk assessment purposes the oral absorption of the test substance is set at 100%


 


Dermal


The results obtained from the dermal toxicity study, skin irritation and skin sensitisation studies do not provide any evidence to imply that there will be significant absorption via the dermal route.  The n-octanol/water partition coefficient of the test substance was determined to be > 3.56 and the molecular weight is moderate to high (>400) (<100 being optimal for dermal update > being too large) therefore the dermal absorption of the substance is expected to be limited based on the log Kow value and molecular weight. At log Kow values above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum is likely to be high. Maximum dermal absorption is often associated with values of log Kow between +1 and +2 (ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals). Monograph No, 20; Percutaneous absorption. August 1993). In addition, the substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. The test substance has a water solubility of 0.33mg/L at 25 °C therefore dermal uptake is likely to be low.  In conclusion, dermal absorption of the substance is expected to be low.  In the absence of significant absorption, due consideration of the absorption needs to be carefully considered. 


As a worst case, for risk assessment purposes the dermal absorption of the test substance is set at 100%


 


Inhalation


An acute inhalation study is available. However when tested in rats at 4.07 mg/ litre -1 and 7.53 mg/ litre -1 there were no mortalities, clinical signs or affects to body and lung weight following exposure to the test article. No abnormalities detected at post mortem observations were considered to be indicative of a reaction to treatment with the test substance. Indicating a lack of bioavailability or absorption when inhaled.


 


The substance has a predicted low vapour pressure (166.3 Pa at 20 °C) therefore a significant inhalation exposure to vapours is not expected. Moderate log Kow values (between -1 and 4) are favourable for absorption directly across the respiratory tract epithelium by passive diffusion. The test substance has a moderate log Kow value (>3.56) therefore it may be taken up by micellular solubilisation particularly as the substance is poorly soluble in water (0.33 mg/l at 25 °C).


As a worst case, for risk assessment purposes the inhalation absorption of the test substance is set at 100%. This is due to the fact that whilst the absorption is unknown, and no effects were noted, it is appropriate to consider worst case for the purposes of the assessment.


 


 


Distribution


Specific data on tissue distribution are not available. However, the absence of systemic toxicity recorded after administration of high dosages (1 x 5000 mg/kg orally or 1 x 2000 mg/kg dermally, 28 x 1000 mg/kg orally) to rats suggests a possibility of restricted bioavailability and/or absorption from the GI tract The absence of evident toxicity to rats given repeated, high doses of the substance in the 28-day and reproduction studies shows low biological activity. The minor liver changes seen were characteristic of adaptive changes related to metabolism of the substance. Marked enterohepatic circulation may occur, as well as limited accumulation in fatty tissue (or, to lesser extent, in the liver) after long-term administration.


ANOX BF/1315 has a moderate molecular weight of > 400, a partition coefficient which (>3.56) and is insoluble in water which indicates that the substance has the potential to distribute throughout the body and into cells, however the low solubility and the moderate molecular size will hinder the diffusion of ions through aqueous channels and pores. Data from the 28-day and reproduction studies shows low biological activity. The minor liver changes seen were characteristic of adaptive changes related to metabolism of the substance.


 


Metabolism and excretion


Absorbed material is readily metabolised by oxidative pathways leading to excretion (maybe in conjugated form) in the urine any unchanged (non-absorbed) material is excreted directly in the faeces.


 


Conclusion


In conclusion, there is little evidence that the substance is absorbed and distributed via ingestion, dermally or when inhaled. Changes in the liver in the 28 day repeated dose study indicates the substance is distributed throughout the body.  In the event of absorption, the substance would likely be metabolised by oxidative pathways which will then be excreted via the urine. Unchanged substance will be excreted directly in the faeces. Consequently, the substance is considered to have low bioaccumulation potential.


The substance is not considered to pose a hazard overall.


 


Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
100
Absorption rate - inhalation (%):
100

Additional information