6-tert-butyl-2,4-xylenol

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

calculation (if not (Q)SAR)

Remarks:

Migrated phrase: estimated by calculation

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A written assessment of toxicokinetic behaviour is considered appropriate for the substance. No toxicokinetics study is available, and it is not considered appropriate to conduct this test when a written assessment can be conducted.

Materials and methods

Objective of study:

other: Assessment of toxicokinetic behaviour

Principles of method if other than guideline:

Written assessment based on toxicological profile.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

6-tert-butyl-2,4-xylenol

EC Number:

217-533-1

EC Name:

6-tert-butyl-2,4-xylenol

Cas Number:

1879-09-0

Molecular formula:

C12H18O

IUPAC Name:

2-tert-butyl-4,6-dimethylphenol

Details on test material:

Not applicable

Test animals

Species:

other: Not applicable

Administration / exposure

Details on exposure:

Not applicable

Duration and frequency of treatment / exposure:

Not applicable

Doses / concentrations

Remarks:

Doses / Concentrations:
Not applicable

No. of animals per sex per dose / concentration:

Not applicable

Positive control reference chemical:

Not applicable

Details on study design:

Not applicable

Details on dosing and sampling:

Not applicable

Statistics:

Not applicable

Results and discussion

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

Not applicable.

Any other information on results incl. tables

Toxicokinetic parameters such as uptake, distribution, metabolism and excretion form the essential toxicological profile of a substance. An approximate indication of the toxicokinetic pattern can be gained from the physico-chemical properties (solubility in solvents, log Pow, hydrolytic stability) and the results of basic toxicity testing of the test article.

The assessment of the toxicokinetic properties of the substance given below is therefore based on the results obtained for the substance and for analogues that are read across to. The following toxicological endpoints are utilised in this assessment:

 

·         Acute oral toxicity in rats (2 x studes)

·         Acute dermal toxicity in rats (2 x studies)

·         Acute dermal toxicity in rabbits (2 x studies)

·         In vivo skin irritation in rabbits

·         In vivo eye irritation in rabbits

·         Skin sensitization in mice - LLNA (read across)

·         Bacterial reverse mutation test

·         In vitro chromosome aberration test inChinese hamster cells

·         In vitromammalian cell gene mutation test with L5178Y mouse lymphoma cells(read across)

·         Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity  Screening Test in the Rat.

·         Subchronic oral toxicity in rats (read across)

·         Prenatal Developmental Toxicity Study (read across)

 

Physico-chemical properties

The substance is a substituted phenol organic monoconstituent liquid with a molecular weight of 178.27 g/mol. It is characterized by a low water solubility of 120 mg/l at 20 °C and a medium range partition coefficient (log Pow 3.64). 

Toxicological Profile:

The group was tested for acute oral toxicity in male and female rats in two separate studies. Death of animals was seen at and above dose levels of 1024 mg/kg in male, and 819 mg/kg in female.  In dead animals, hydrothorax, black patch in the thymus, black patch and retention of brownish urine in the urinary bladder, white patch in the forestomach, black patch in the glandular stomach, white, brown or black contents in the small intestine, white patch in the small intestine and white contents in the large intestine were observed. In the animals surviving until the end of observation period, yellow patch in the liver, adhesion with the liver and the spleen, diaphragm, or retro-peritoneum, atrophy of the spleen, adhesion with the spleen and the kidney, thickening of the forestomach, and adhesion with the forestomach and the liver or the diaphragm were observed. In the surviving male animals at the end of the observation period, severe ulceration with granulomatous inflammation, and hyperplasia of the stratified squamous epithelium with granulomatous inflammation in the forestomach, and granulomatous inflammation in the liver were observed. This implies that the substance is readily absorbed following dosing, and has adverse effects following dosing.

 

Dermal toxicity was evaluated in 4 separate studies; two on rats and two on rabbits. Within the rabbit, severe effects were noted following dosing. Common signs of systemic toxicity noted were hunched posture, lethargy, decreased respiratory rate, laboured respiration and increased salivation with additional signs or isolated incidents of ataxia, dehydration, pallor of the extremities, hypothermia, ptosis, gasping, increased or noisy respiration, loss of righting reflex and red/brown staining around the snout. Surviving animals showed an expected gain in bodyweight during the study. Haemorrhagic lungs were noted at the necropsy of all animals that died during the study with additional abnormalities of dark liver, pale spleen, haemorrhage or slight haemorrhage of the gastric mucosa, epithelial sloughing of the gastric mucosa, haemorrhage or slight haemorrhage and epithelial sloughing of the non-glandular region of the stomach. It appears that the substance is readily absorbed through rabbit skin, where it causes haemorrhagic effects in the rabbit. Such effects were also seen in the skin irritation study with the rabbit. A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits resulted in the death of one animal one day after treatment. It also became necessary to kill one animal for humane reasons later the same day owing to the development of severe respiratory problems. The surviving animal showed very slight to well-defined erythema and slight oedema at the treatment site during the study. One rabbit in this group was also found dead two days after treatment. The two surviving animals showed very slight to well-defined erythema and very slight to moderate oedema at the treatment sites during the study. Incidents of crust formation were also noted. It was concluded that the deaths seen during this study were due to the toxicity of the test material. Based on the results of the surviving animals the test material produced a primary irritation index of 3.8 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme. Similar effects were noted in the eye; a single instillation of the test material to the non-irrigated eye of one rabbit produced areas of diffuse corneal opacity, iridial inflammation and moderate conjunctival irritation. Other adverse ocular effects noted were a dulling of the normal lustre of the corneal surface and petechial haemorrhage of the nictitating membrane. Two days after treatment the animal showed signs of respiratory problems which were thought to be treatment-related. The animal was therefore killed for humane reasons in accordance with current U.K. Home Office guideline and no further animals were treated. The test material produced a maximum total score of 39.0 and was considered to be at least a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system (based on one rabbit only).

  

However, such effects were not noted in the rat. Two additional dermal toxicity studies in the rat are available; one on neat test substance and one on a 55% formulation containing the substance. Following 24-hour exposure in both studies,very slight to well-defined erythema were observed. However, this was clear at the 14-day observation, and gross pathological examinations at terminal necropsy revealed no test article-dependent findings. As a result of this, it is clear that the substance does not penetrate the rat dermis to any significant extent, and appears to not be readily absorbed.

 

Skin sensitisation was assessed via read across to a structural analogue, CAS 85-60-9 using a LLNA study. No sensitisation effects were noted as a result of this read across, and minimal irritation was observed on the ears. The substance is therefore not considered to be a skin sensitiser, and effects are more closely correlated with toxicity than sensitisation.

 

Given the large distinction between the results noted in the rat and the rabbit, it was considered appropriate to assess why rats are largely unaffected by the substance, whereas rabbits demonstrate a toxic response to the substance when applied dermally.  The studies themselves carry no definitive explanation of this difference of toxicity observed, and it is concluded that this is a species specific effect. An assessment of the literature data was undertaken, specifically:

 

Skin PermeabilityIn Vivo: Comparison in Rat, Rabbit, Pig and Man;Journal of Investigative Dermatology(1972)58, 114–123; doi:10.1111/1523-1747.ep12538909 Accepted 26 October 1971.

 

This paper details that in a comparative percutaneous absorption study undertaken in rats, rabbits, miniature swine and man, the results obtained indicated that skin permeability decreases in the following order: rabbit, rat, pig and man. It may be therefore that the permeability of rabbit skin is much greater for the substance than it is in the rat, and the toxic response is proliferated by rabbit skin to a greater extent.For the purposes of hazard assessment however, it is considered appropriate to consider that the substance may be absorbed through the human skin and hence dermal absorption is considered to be 100% on a worst case basis.

 

The substance was not mutagenic in a reverse mutation test on bacteriainSalmonella typhimurium (TA 98, TA 100, TA1535, TA1537) and E. coli WP2 uvr A. The substance was also non-clastogenic in an in vitro chromosome aberration test withcultured Chinese hamster cells (CHL/IU). Finally, evaluation of the mutagenic activity in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells was conducted utilising read across to the structural analogue, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol. The substance, CAS85-60-9, is analogous to the substance to be registered, but is approximately twice the size of CAS 1879-09-0 – this is essentially a second “duplicate” molecule of the substance to be registered. In the presenceandabsence of S9-mix, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol did not induce a significant increase in the mutation frequency.It was concluded that 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report. As such, it is considered inappropriate to conduct a further assessment ofin vitrogenotoxicity when available mutagenicity studies on the substance and other group members clearly confirm that the substance is not genotoxic.

 

There are two sets of data included for assessment of the repeated dose effects for the substance as follows:

 

OECD Guideline 422 – 28 days – Substance itself.

NOEL (male): 6 mg/kg bw/day

NOEL (female): 30 mg/kg bw/day

 

90-Day study – Read across to85-60-9

NOAEL (male/female): 10 mg/kg/day

 

In the 28-day study, hypertrophy of centrilobular hepatocytes, degeneration of hepatocytes, necrosis of centrilobular hepatocytes and single cell necrosis in liver were seen in 150 mg/kg females at necrosis on day 4 of lactation. Parakeratosis in tongue and esophagus, hypertrophy of centrilobular hepatocytes as well as various degeneration, single cell necrosis and increase in mitotic figures in liver were observed in dead animals and the animals whose pups all died. In addition, degeneration of proximal convoluted tubules, proteinaceous casts and deposition of PAS positive granules at the papilla in kidney were observed in the female of this group. In the histopathology examination, the following findings were considered to be due to the test substance administration. Hypertrophy of centrilobular hepatocytes was observed in the male group treated with 150 mg/kg. Parakeratosis in tongue and esophagus, hypertrophy of centrilobular hepatocytes as well as various degeneration, single cell necrosis and increase in mitotic figures in liver were observed in dead animals and the animals whose pups all died. In addition, degeneration of proximal convoluted tubules. Similar effects were seen in the CAS 85-60-9 90-day repeated dose study. Macrophages with distended cytoplasm were present in sinusoids and portal areas of livers from both sexes at the highest dose level. The only notable changes in organ weights were slight increases in absolute liver weights for males at the highest dose level, and for females at the two highest dose levels. Liver to body weight ratios were slightly increased for both sexes at the highest dose level. Other increases in organ to body weight ratios were attributed to low terminal body weights, rather than a direct treatment effect on the organs.

 

On the basis of the results in the repeated dose studies, it is clear that the substance is absorbed, and effects on the liver and kidneys can be anticipated.

 

Assessment of fertility was conducted as part of the OECD Guideline 422 study listed above. No effect due to the test substance administration was observed in the copulation capability, fertility or estrus cycle observation. In the observation at parturition, one female of 150 mg/kg group died during delivery. In addition, there were 3 females whose pups all died in this group. The viability index on day 4 was inclined to show low value, and it is suggested that the test substance has the possibility to induce disorder in parturition or lactation function. However, no effect due to the test substance administration was seen during gestation period or delivery duration. In the external examination of neonates, no external anomalies were observed, and the body weight of neonates increased normally until day 4 of lactation. Abnormal findings considered to be due to the test substance administration were not seen in the rate of stillbirth and death rate of pups, or on the necropsy on day 4 of lactation.

From the above results, no effect on male fertility were observed by the administration of 150 mg/kg/day. Therefore, no observed effective level is estimated to be 150 mg/kg/day. The effect on fertility of female and development and growth of neonates were observed by the administration of 150 mg/kg/day. Therefore, no observed effective level is estimated to be 30 mg/kg/day.

 

On the basis of the evaluation of the developmental endpoints in the OECD 422 study, further assessment of developmental toxicity was conducted via read across tothe structural analogue CAS125643-61-0. No indication for developmental toxicity was seen at dose levels of 20 and 40 mg/kg bw/day. At 80 mg/kg body weight/day, the mean fetal weights were statistically significantly reduced. There were more visceral abnormalities in the fetuses than in the control group. There was reduced cranial ossification, as well as reduced digit/limb ossification. The findings from the skeletal and cartilage examinations suggest a possible minor disturbance in the development of the axial skeletons of the fetuses. Nevertheless, since the fetal findings were seen at the highest tested dose of 80 mg/kg bw/day which clearly was toxic to the dams, they were considered to be attributable to maternal toxicity. In conclusion, some development toxicity related to the test substance was seen, however at a dose level which clearly was toxic to the dams. Based on the results of this study, the maternal NOAEL was set at 40 mg/kg body weight/day. The NOAEL for developmental toxicity also was set at 40 mg/kg body weight/day.

On the basis of the available data, the substance is not deemed to pose a hazard to reproductive or developmental endpoints. Toxicity is associated with repeated dose effects and not reproductive effects.

Absorption, distribution, metabolism and excretion of substituted phenols

The substance is a substituted phenol monoconstituent substance, used as alubricant additives in the production of lubricants, functional fluids and greases. Based on the available data, the substance does appear to contribute significantly to toxicokinetic behaviour.

 

The World Health Organisation has assessed hindered phenols as part of its review on “Food Additives Series 46:Phenol And Phenol Derivatives”, available at:

 

http://www.inchem.org/documents/jecfa/jecmono/v46je09.htm#_46091300

 

The WHO discusses the potential metabolism of phenol derivatives, including a range of xylenol substances that have been evaluated for food additive usage. Whilst the substance subject to registration is not a direct food additive, toxicokinetic parameters evaluated as part of this study can be applied in the review of the likely mechanism of action of the substance when ingested.

Data taken from the WHO assessment indicates that when phenol and its derivatives are ingested, they are rapidly absorbed from the gastrointestinal tract and participate in common pathways of metabolism (Hughes & Hall, 1995).Alkyl-, alkenyl-, and aryl-substituted phenols and their corresponding esters known to be conjugated with sulfate and glucuronic acid after hydrolysis of the esters and excreted primarily in the urine. Other metabolic pathways, observed mainly at high doses, include ring hydroxylation and side-chain oxidation.A diagrammatical representaton of the proposed metabolic pathways is detailed below.

Monoalkyl-substituted phenols are conjugated mainly with sulfate or glucuronic acid in a manner similar to phenol. They undergo a small degree of side-chain oxidation or ring hydroxylation, which results in a second hydroxyl grouporthoorparato the existing hydroxyl group (Williams, 1959). In rabbits and dogs, a single oral dose of 200 mg/kg bw ofortho- ormeta-cresol was hydroxylated to a very minor extent (< 1 %), while up to 10% ofpara-cresol was oxidized on the side-chain and excreted aspara-hydroxybenzoic acid (Bray et al., 1950a). In rats, < 0.2% of an oral dose of para-cresol of 100 mg/kg bw was ring hydroxylated andortho-methylated to yield 4- and 5-methylguaiacol (2-methoxy-4 or 5-methylphenol) (Bakke, 1970; Bakke & Scheline, 1970).

Incorporation of a second alkyl substituent does not significantly alter the sulfate and glucuronic acid conjugation pathway. Doses of 1 g of 2,5-xylenol, 2,6-xylenol, or 3,4-xylenol (2;5-methylphenol, 2,6-methylphenol, and 3,4-methylphenol) given to rabbits were excreted as xylenyl sulfates (8-16%) and glucuronides (50-72%); only 1-3% of the dose was excreted unconjugated. Ring hydroxylation was evident in only a small portion of the metabolites of the 2,6-xylenol isomer (Bray et al., 1950).

Discussion of toxicokinetics

 

The data of the acute dermal toxicity and dermal irritation test indicate a variable potential dermal permeability, which is species dependant, When applied to rabbit skin, significant absorption can be seen resulting in toxic effects; however when applies to rat skin, only minor effects are noted owing to the fact that only minor systemic and irritating effects are observed. These were observed to be fully reversible within the respective test periods, implying that significant absorption through the dermal route is not applicable.

 

Taking the results of the sub-acute oral toxicity and repro / developmental toxicity study into account, the substance appears to be absorbed from the gastrointestinal tract as evidenced by the liver and kidney effects in the higher dose groups. This effect is anticipated, on the basis of the fact that substituted phenols are known to be rapidly absorbed and metabolised for excretion mainly in the urine. The effects of metabolic activities can be seen in the high dose group kidney effects. Following ingestion, the substance is assumed to be conjugated mainly with sulfate or glucuronic acid to the corresponding metabolite for subsequent excretion. The substance is therefore proposed to be readily metabolized and excreted and that accumulation seems to be unlikely. There are no data on dermal route of administration or exposure by inhalation for long term exposure. An absorption rate of 100% for the oral route is proposed to be taken for risk characterisation purposes, whereas dermal and inhalation absorption is assumed to be 100% (defaults). The assumption of a default dermal absorption value of 100% is supported by the effects noted in the irritation and dermal toxicity studies as a worst case. Due to the potential for absorption and the lack of experimental data, a default absorption value of 100% is also assumed for inhalative uptake.

The substance has been demonstrated to have a partition coefficient value which indicates the potential to accumulate biologically. However, the data from an actual bioaccumulation study does not support this presumption. The substance demonstrated only a low level bioconcentration factor in fish (BCFfish of 263). In conclusion, the substance is proposed not to fall into a BCF level where classification is applicable. These factors indicate that accumulation and significant distribution within body tissues is unlikely to occur. 

Conclusion

The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to the toxicokinetics of the substance. The data indicate that whilst there is the possibility of potential dermal absorption, there are only species specific predicted effects noted from this route. Systemic effects associated with absorption have been observed following oral ingestion, with the liver and kidneys demonstrating systemic effects associated with metabolic activities. Bioaccumulation and storage of the material in fatty tissue (adipose cells) of the substance can most probably be excluded due to the bioaccumulation behaviour. Based on the results of all mutagenicity assays and assessment of relevant literature, a metabolisation towards genotoxic structures can be ruled out. It is proposed that following ingestion, the substance will be conjugated to more soluble forms, followed by further metabolism using standard metabolic mechanisms for subsequent elimination.  Toxicity associated with this metabolic pathway can be predicted for higher doses, but at lower doses, no significant effects are proposed.

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to the toxicokinetics of the substance. The data indicate that whilst there is the possibility of potential dermal absorption, there are only species specific predicted effects noted from this route. Systemic effects associated with absorption have been observed following oral ingestion, with the liver and kidneys demonstrating systemic effects associated with metabolic activities. Bioaccumulation and storage of the material in fatty tissue (adipose cells) of the substance can most probably be excluded due to the bioaccumulation behaviour. Based on the results of all mutagenicity assays and assessment of relevant literature, a metabolisation towards genotoxic structures can be ruled out. It is proposed that following ingestion, the substance will be conjugated to more soluble forms, followed by further metabolism using standard metabolic mechanisms for subsequent elimination. Toxicity associated with this metabolic pathway can be predicted for higher doses, but at lower doses, no significant effects are proposed.

Executive summary:

The data of the acute dermal toxicity and dermal irritation test indicate a variable potential dermal permeability, which is species dependant, When applied to rabbit skin, significant absorption can be seen resulting in toxic effects; however when applies to rat skin, only minor effects are noted owing to the fact that only minor systemic and irritating effects are observed. These were observed to be fully reversible within the respective test periods, implying that significant absorption through the dermal route is not applicable.

 

Taking the results of the sub-acute oral toxicity and repro / developmental toxicity study into account, the substance appears to be absorbed from the gastrointestinal tract as evidenced by the liver and kidney effects in the higher dose groups. This effect is anticipated, on the basis of the fact that substituted phenols are known to be rapidly absorbed and metabolised for excretion mainly in the urine. The effects of metabolic activities can be seen in the high dose group kidney effects. Following ingestion, the substance is assumed to be conjugated mainly with sulfate or glucuronic acid to the corresponding metabolite for subsequent excretion. The substance is therefore proposed to be readily metabolized and excreted and that accumulation seems to be unlikely. There are no data on dermal route of administration or exposure by inhalation for long term exposure. An absorption rate of 100% for the oral route is proposed to be taken for risk characterisation purposes, whereas dermal and inhalation absorption is assumed to be 100% (defaults). The assumption of a default dermal absorption value of 100% is supported by the effects noted in the irritation and dermal toxicity studies as a worst case. Due to the potential for absorption and the lack of experimental data, a default absorption value of 100% is also assumed for inhalative uptake.

The substance has been demonstrated to have a partition coefficient value which indicates the potential to accumulate biologically. However, the data from an actual bioaccumulation study does not support this presumption. The substance demonstrated only a low level bioconcentration factor in fish (BCFfish of 263). In conclusion, the substance is proposed not to fall into a BCF level where classification is applicable. These factors indicate that accumulation and significant distribution within body tissues is unlikely to occur.