2,2'-(2-methylpropylidene)bis[4,6-xylenol]

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Remarks:

Written assessment

Type of information:

other: Written assessment

Adequacy of study:

other information

Study period:

October 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Written assessment

Justification for type of information:

At the current level of registration, a written assessment is suitable.

Data source

Reference

Reference Type:

other: Written assessment

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Objective of study:

other: Written assessment of ADME

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

Written assessment based on available study data.

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]

EC Number:

251-394-8

EC Name:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]

Cas Number:

33145-10-7

Molecular formula:

C20H26O2

IUPAC Name:

2-[1-(2-hydroxy-3,5-dimethylphenyl)-2-methylpropyl]-4,6-dimethylphenol

Test material form:

solid: particulate/powder

Details on test material:

Identification: Lowinox® 22IB46
Appearance: White to cream coloured powder
Batch: WB44L0016
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 18 November 2018 (retest date)

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

See overall remarks below

Details on distribution in tissues:

See overall remarks below

Details on excretion:

See overall remarks below

Metabolite characterisation studies

Details on metabolites:

See overall remarks below

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

See overall remarks below

Any other information on results incl. tables

Summary and discussion of toxicokinetics

Introduction

This assessment of the toxicokinetic properties of 2,2’-(2-methylpropylidene)bis[4,6-xylenol] 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

-         Skin irritation

-         Eye irritation

-         Skin sensitisation

-         Subacute (28 day) oral toxicity incorporating an Reproduction/Developmental Toxicity Screening test 

-         Bacterial reverse mutation test

-         In vitro chromosome aberration test

-         In vitro mammalian gene mutation test

Toxicological Profile

 2,2’-(2-methylpropylidene)bis[4,6-xylenol] is amono constituent substance (origin: organic). The substance has a molecular weight of298.46, awater solubility of 0.203mg/L at 20 °Cand an estimated log Kow of 5.6

The substance was tested for acute oral toxicity in female rats (Wistar strain).After a single oral administration, female rats administered with 2000 mg/kg were all sacrificed for humane reasons on Day 2. Clinical signs included: lethargy, tremor, hunched posture, uncoordinated movements, slow breathing, shallow respiration, piloerection, watery discharge from eyes, lean appearance and/or hypersensitivity to touch were noted for the animals on Days 1 and/or 2. At 2000 mg/kg, the animals showed body weight loss during the first two days of the study. No mortality occurred in any of the female rats that were received a single dose of 300 mg/kg. There were however clinical signs including lethargy, tremor, hunched posture, uncoordinated movements, rales and/or piloerection which was noted for the animals between Days 1 and 5. The body weight of the rats administered 300mg/kg were comparable to animals of the same age and strain. No abnormalities were found at macroscopic post mortem examination of the animals.  The oral LD50 value of the substance in Wistar rats was established to be within the range of 300-2000 mg/kg body weight. According to the test guideline, the LD50 cut-off value was considered to be 500 mg/kg body weight..

Similarly, single dermal application of >2000 mg/kg body weight of the substance, ontomale and female rats (Wistar strain)produced no deaths. Symptoms of systemic toxicity included lethargy, tremors, flat- and hunched posture, lateral recumbency, rales, uncoordinated movements, shallow respiration, piloerection, chromodacryorrhoea (snout), hypersensitivity to touch and/or ptosis were noted for the animals of both sexes. The male animals had recovered from the symptoms by Day 4 and the females by Day 6. These local effects were considered not to have affected the conclusion of the study, and were likely to be associated with grooming and associated oral exposure rather than dermal effects. No abnormalities were found at macroscopic post mortem examination of the animals.

Skin irritation tests utilising in vitro assessment techniques indicated that the substance was non-irritant and non-corrosive. Use of in vitro eye irritation assessment methodologies did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.9 after 240 minutes of treatment. The administration of the substance into the conjunctival sac of rabbit eyes had negligible effects on the cornea, iris orconjunctiva. Instillation of the test item resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 24 hours in all animals. Consequently, the test substance is not irritating to the skin or the eyes.

A Local Lymph Node Assay was performed to assess Skin Sensitisation. Application of three concentrations (2%, 5% and 10%).  No irritation was observed in any of the animals. The scaliness as shown by the control animals and some animals treated at 2 and 5% was considered not to have a toxicologically significant effect on the activity of the nodes. All auricular lymph nodes of the animals control group and animals treated at 2% were considered normal in size. The nodes of some animals treated at 5 and 10% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 928, 748 and 1029 DPM, respectively. The mean DPM/animal value for the vehicle control group was 690 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 1.3, 1.5 and 1.5, respectively. Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, the substance was not considered to be a skin sensitizer.

A 28 day repeated dose toxicity with reproduction developmental screening study by the oral route was conducted on the substanceat dose levels of 3, 10 and 30 mg/kg/day. Uncoordinated movements were observed for a major part of the study period in both high dose males and females. In some of these females,temporary behavioural changes, including lethargy, restlessness or fearfulness,were occasionally observed. The functional observation test performed towards the end of the treatment period showed decreased fore limb grip strength in both high dose males and females and an accelerated decrease in movements and ambulations in the motor activity test in high dose females only in contrast to a more gradual decrease in activity in a normal habituation pattern. There might be a relation between the clinical signs and behavioural changes which might be interpreted as signs of neurotoxicity caused by treatmentat 30 mg/kg/day. In the absence of changes in gait/motility and effects on motor activity in the mid dose animals, the minimal decreases in fore limb grip strength observed at this dose level were considered to be non-adverse and of no toxicological significance.
Small increases in liver weights were observed in high dose males. Histopathology of the liver revealedminimal hepatocellular hypertrophy, but in the absence of any degenerative findings this was considered to be a non-adverse finding.
Furthermore, histopathology also revealed follicular cell hypertrophy of the thyroid gland in high dose males. The minor increase in incidence and/or severity (up to slight degree) and the fact that it is usually an adaptive response to induction of hepatic enzymes, this finding was regarded to be an adaptive change and considered to be non-adverse.

No treatment-related and/or toxicologically significant changes were noted in any of the other parental parameters investigated in this study (i.e. body weight, food consumption, clinical laboratory investigations and macroscopic examination).

 

No reproduction toxicity was observed up to the highest dose level tested (30 mg/kg/day). Lower body weights in male and female pups of high dose dams treated at 30 mg/kg/day were observed during the lactation phase.The median for the absolute anogenital distance in female pups from high dose dams was reduced. Since the normalized anogenital distance in female pups was similar to that in the other groups, the change in anogenital distance was considered to be related to the lower body weights observed in the female pups. Therefore, no toxicological significance was attached to the changes in anogenital distance itself, but this finding must be evaluated in combination with the lower body weights and might indicate a retarded development of the high dose pups in comparison with control pups. It was striking that less or no milk in the stomach was observed among pups from high dose females only early in the lactation phase, whereas this phenomenon was not observed in pups from females in the other dose groups. The reduced amount of milk in the stomach in nine (out of sixteen) the pups that were found dead (at first litter check) or went missing in the high dose group, might have been related to its death.It could not be established from the results obtained in this study if the reduced amount of milk was the result of effects in the females or of effects in the pups itself. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, lactation indices, duration of gestation, parturition, sex ratio, maternal care, clinical signs, areola/nipple retention (PND 13 males) and T4 thyroid hormone levels (PND 4 and 13-15).

In the Ames test, with use of four strains of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E. coli WP2 uvr A, the substance produced neither a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any one of the test points and according to these results it is considered non mutagenic in the system. 

In a chromosome aberration test using human lymphocytes, the substance did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of two independently performed experiments. It was concluded that the substance is not clastogenic in human lymphocytes under the experimental conditions.

Evaluation of the mutagenic activity of the substance in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells was undertaken. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). In the both the presence and absence of S9-mix, the substance did not induce a significant increase in the mutation frequency. It was concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described. 

 

Toxicokinetic parameters

Specific toxicokinetic information is not available. Therefore, an evaluation of the data indicates the following:

Absorption

Oral

In the acute oral toxicity study and the repeated dose toxicity study there were treatment-related effects noted that resulted in classification for exposure via this route. This implies that the substance is absorbed following oral ingestion, and hence oral availability of the substances is relatively high. The test substance has water solubility of 0.203 mg/L at 20 °C and a log Kow value 5.60. The absorption of highly lipophilic substances (log Kow ≥ 4) may be limited by the inability of such substances to dissolve in gastrointestinal fluids and therefore make contact with the mucosal surface. However, the absorption of such substances will be increased if they undergo micellular solubilisation by bile salts. As a worst case, for risk assessment purposes the oral absorption of the test substance is set at 100%.

Dermal

The results of the acute dermal toxicity study with the test substance and the skin irritation and skin sensitisation studies with provide no evidence to support significant skin absorption or subsequent retention.

The log Kow value of the test substance 5.60 therefore the dermal absorption of the substance is expected to be limited based on the high log Kow value. 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 will 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 an estimated water solubility of 0.203 mg/L at 20 °C therefore dermal uptake is likely to be low. In conclusion, dermal absorption of the substance is expected to be low. In addition, the relatively large molecular weight is approaching the value where absorption in precluded. In the absence of significant absorption, for risk assessment purposes the dermal absorption of the test substance is set at 10%.

Inhalation

The substance has a low vapour pressure (2.5 x 10 E-6 Pa at 20°C) therefore a significant inhalation exposure to vapours is not expected. The substance has a relatively high percentage of small particulates (62.13% < 10.00 μm) and hence is potentially respirable; however exposure to such dusts is strictly controlled at the site of manufacture. 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 high log Kow value (5.60) therefore it may be taken up by micellular solubilisation particularly as the substance is poorly soluble in water (0.203 mg/L at 20 °C). As a worst case, for risk assessment purposes the inhalation absorption of the test substance is set at 100%.

Distribution

In a 28 day oral repeated dose toxicity study effects were observed in the rat liver and thyroid gland, suggesting distribution to these organs. The test substance is lipophilic therefore it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues. Metabolites (presumably phenolics) released following hydrolysis of the test substance in the gastrointestinal tract are likely to be widely distributed in the body. Substances with high log Kow values tend to have longer half-lives unless their large volume of distribution is counterbalanced by a high clearance. There is the potential for highly lipophilic substances to accumulate in individuals that are frequently exposed to that substance. Once exposure stops, the concentration within the body will decline at a rate determined by the half-life of the substance. However, as detailed below, the test substance is likely to undergo conjugation following absorption so only low and transient exposure to the parent compound is expected.

Metabolism and excretion

The phenol groups in the test substance are likely to undergo conjugation with glucuronic acid and sulfate. Quantitatively, the most important sites of phenolic conjugation are the liver, lung and gastrointestinal mucosa. The relative roles played by these tissues depend on the route of administration and the dose. The liver is an important site of phenolic metabolism. Other tissues, such as lungs, intestines and kidneys, also play an important role in phenolics metabolism. Phenolic sulfotransferases, which catalyse phenolic sulfation, occur in a variety of human tissues (intestinal wall, lungs, platelets, adrenal glands, brain, placenta, etc.). On the basis of the mutagenicity assays it appears that the substance is not metabolised toward genotoxic structures.

Urinary excretion is the major route of phenol elimination in animals and humans. Only minor amounts of unchanged phenolics are known to be excreted in exhaled air or in faeces. Phenolic conjugates may also be excreted in the bile of rats and it has been suggested that biliary excretion of phenolic substances plays an important role when urinary excretion is impeded.

Conclusion

In conclusion, there is no evidence thatthe substanceis significantly absorbed via the dermal route or the inhalation route. The results of an oral repeated dose toxicity study indicate thatthe substancemay be absorbed orally. It is likely that the following ingestion, the substanceundergoes conjugation with glucuronic acid and sulfate within the gastrointestinal tract. Following absorption, the fatty acids are transported to the tissues of the body including the liver where they undergo oxidation in the cells. These solubilised metabolites are then most probably excreted in the urine in conjugated form. Consequently, the substance is considered to have low bioaccumulation potential.

 

Applicant's summary and conclusion

Conclusions:

In conclusion, there is no evidence that the substance is significantly absorbed via the dermal route or the inhalation route. The results of an oral repeated dose toxicity study indicate that the substance may be absorbed orally. It is likely that the following ingestion, the substance undergoes conjugation with glucuronic acid and sulfate within the gastrointestinal tract. Following absorption, the fatty acids are transported to the tissues of the body including the liver where they undergo oxidation in the cells. These solubilised metabolites are then most probably excreted in the urine in conjugated form. Consequently, the substance is considered to have low bioaccumulation potential.

Executive summary:

In conclusion, there is no evidence that the substanceis significantly absorbed via the dermal route or the inhalation route. The results of an oral repeated dose toxicity study indicate thatthe substancemay be absorbed orally. It is likely that the following ingestion, the substanceundergoes conjugation with glucuronic acid and sulfate within the gastrointestinal tract. Following absorption, the fatty acids are transported to the tissues of the body including the liver where they undergo oxidation in the cells. These solubilised metabolites are then most probably excreted in the urine in conjugated form. Consequently, the substance is considered to have low bioaccumulation potential.