4-methylstyrene

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Objective of study:

metabolism

Principles of method if other than guideline:

Metabolism of the test substance in rats was studied by investigating urinary metabolites after injection of different doses. The urinary metabolites analysed were: thioesters, p-methylmandelic acid, p-methylphenylglyoxylic acid, the glycine conjugates of p-methylbenzoic acid, p-methylphenylacetic acid and p-vinylbenzoic acid. The role of cytochrome P-450 in the formation of the metabolites was studied by inhibiting its catalytic activity.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 350-400 g
- Diet (e.g. ad libitum): commercial pellet diet from Astra-Ewos
- Water (e.g. ad libitum): tap water

Route of administration:

intraperitoneal

Vehicle:

olive oil

Duration and frequency of treatment / exposure:

Single intraperitoneal dose

Dose / conc.:

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

Remarks:

First experiment

Dose / conc.:

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

Remarks:

First experiment

Dose / conc.:

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

Remarks:

First experiment

Dose / conc.:

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

Remarks:

First experiment

Dose / conc.:

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

Remarks:

Second and third experiments

Control animals:

yes, concurrent vehicle

Metabolites identified:

yes

Details on metabolites:

After rats received a single intraperitoneal injection of 50 mg/kg, 55% of the dose was found as urinary metabolites, mainly in the first 6 h; at higher doses, slightly smaller percentages were found (Heinonen, 1984). The principal urinary metabolites were thioethers (25%), p-methylmandelic acid (5.7%), p-methylphenylglyoxylic acid (11.9%), p-methylbenzoyl glycine (9.3%), p-methylphenylacetyl glycine (2.5%), and p-vinylbenzoyl glycine (1%). The excretion of these metabolites was prevented by pretreatment with an inhibitor of the cytochrome P450 monoxygenases. Further, the test substance was found to bind to hepatic cytochrome P450, and the reduced glutathione content of the liver and kidney was decreased in rats after a single intraperitoneal injection (Heinonen and Vainio, 1980). These findings suggest that metabolism of the test substance is catalyzed by cytochrome P450, producing vinyl toluene-7,8-oxide as the main reactive intermediate, with subsequent conjugation to glutathione or hydration to diols (Heinonen, 1984).

Conclusions:

Under the study conditions, following intraperitoneal injection to the rat, metabolism is catalysed by cytochrome P450 producing vinyl toluene-7,8-oxide as the main reactive intermediate, with subsequent conjugation to glutathione or hydration to diols. The main route of excretion of metabolites was via the urine (55% within first 6 h).

Executive summary:

A study was conducted to determine the metabolism of the read-across substance, vinyltoluene, in rats by investigating urinary metabolites after injection of different doses. Male Wistar rats received the test substance by single intraperitoneal injection in three different experiments. In the first, the test substance dissolved in olive oil was administered at 50, 250, 500 and 1000 mg/kg bw. Four rats were sacrificed after 12 h at each dose level and three rats at the dose levels of 50, 250 or 500 mg/kg bw after 23 h. In the second experiment, rats were given the test substance (500 mg/kg bw in olive oil), 1-phenylimidazole (50 mg/kg bw in DMSO) or both. The control rats received olive oil or DMSO alone. 1-Phenylimidazole and DMSO were administered 1.5 h before the test substance. Four rats of each group were sacrificed 12 h after injection of the test substance and 3 in each group after 23 h. In the third experiment, rats were exposed to PCBs (500 mg/kg bw in olive oil), test substance (500 mg/kg bw in olive oil) or both Controls received olive oil only. The dose of PCB was given 5 d before the test substance. Three animals in each group were sacrificed 23 h after injection of the test substance. Urine was collected during the exposure from all rats in all groups. The urinary metabolites analysed were: thioesters, p-methylmandelic acid, p-methylphenylglyoxylic acid, the glycine conjugates of p-methylbenzoic acid, p-methylphenylacetic acid and p-vinylbenzoic acid. The role of cytochrome P-450 in the formation of the metabolites was studied by inhibiting its catalytic activity.The highest excretion rate was obtained with doses of 50, 250 and 500 mg/kg bw already within the first 6 h. However, the dose of 500 mg/kg bw did not increase the excretion rates of these metabolites compared to the dose of 250 mg/kg bw, suggesting that the metabolic pathways begin to be saturated with the amount of 250 mg/kg bw. At 50 mg/kg bw, 55% of the dose was detected as urinary metabolites within 23 hr, mainly within the first 6 h. The amounts of the excreted metabolites expressed as % of injected dose (250 or 500 mg/kg bw) were lower than that caused by 50 mg/kg bw, and a noticeable amount of the total sums were excreted within 11–23 h, suggesting that the excretion was still continued with the doses of 250 and 500 mg/kg bw 23 h after injection. The excretion of all analyzed metabolites was prevented by the pre-treatment of the rats with 1-phenylimidazole, an inhibitor of cytochrome P-450 monooxygenases. This indicates that these metabolites were formed as catalyzed by cytochrome P-450. The structures of the analyzed metabolites suggest that the main reactive intermediate of the test substance is vinyltoluene-7,8-oxide. Furthermore, the amounts of the excreted metabolites showed that the main detoxification pathways of vinyltoluene-7,8-oxide were the conjugation with reduced glutathione and hydration to diols. Pre-treatment of the rats with PCBs increased the excretion rates of the metabolites. However, the PCB pre-treated rats excreted less thioethers (62%) compared to the rats treated only with the same amount of test substance, whereas the total sum of the other metabolites was about the same in these both groups. This result suggests that PCBs change the metabolism of the test substance to some other pathway which could be glucuronide conjugation because PCBs increased the activity of UDP-glucuronosyltransferase in a dose-dependent manner (Heinonen, 1984).

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

No studies are available on the toxicokinetics of the test substance. However, ADME information can be obtained from the physico-chemical properties and the results of toxicity testing, as described in Chapter R.7.12.2.1 of REACH Guidance R7c (June 2017).

Absorption

Oral

The test substance has a molecular weight below 500 (118.179), moderate water solubility (89 mg/L) and a log Pow between -1 and 4 (3.44). These characteristics favour oral absorption via the gastro-intestinal tract, possibly via passive diffusion. Uptake though this route is confirmed in oral toxicity testing where clinical signs indicative of systemic effects were noted.

Dermal

Dermal absorption of the test substance is likely to be limited due to its volatility (the liquid can evaporate off the skin surface). However, as for the oral route, the molecular weight, water solubility and log Pow are in a range that favours dermal absorption of the test substance fraction that does not evaporate. Moderate dermal absorption is therefore assumed.

Inhalation

The test substance is volatile, so that exposure via inhalation is likely. As for the oral route, the molecular weight, water solubility and log Pow are in a range that favours direct absorption through the respiratory tract epithelium via passive diffusion. Also, clinical signs indicative of systemic effects were noted in inhalation toxicity testing in rat.

Based on the above information, oral, dermal and inhalation uptake rates of 100, 50 and 100% are assumed for risk assessment purposes.

Distribution

No information could be identified on the distribution of the test substance in the organism once taken up systemically.

Metabolism and excretion

A study was conducted to determine the metabolism of the read-across substance, vinyltoluene, in rats by investigating urinary metabolites after injection of different doses. Male Wistar rats received the test substance by single intraperitoneal injection in three different experiments. In the first, the test substance dissolved in olive oil was administered at 50, 250, 500 and 1000 mg/kg bw. Four rats were sacrificed after 12 h at each dose level and three rats at the dose levels of 50, 250 or 500 mg/kg bw after 23 h. In the second experiment, rats were given the test substance (500 mg/kg bw in olive oil), 1-phenylimidazole (50 mg/kg bw in DMSO) or both. The control rats received olive oil or DMSO alone. 1-Phenylimidazole and DMSO were administered 1.5 h before the test substance. Four rats of each group were sacrificed 12 h after injection of the test substance and 3 in each group after 23 h. In the third experiment, rats were exposed to PCBs (500 mg/kg bw in olive oil), test substance (500 mg/kg bw in olive oil) or both Controls received olive oil only. The dose of PCB was given 5 d before the test substance. Three animals in each group were sacrificed 23 h after injection of the test substance. Urine was collected during the exposure from all rats in all groups. The urinary metabolites analysed were: thioesters, p-methylmandelic acid, p-methylphenylglyoxylic acid, the glycine conjugates of p-methylbenzoic acid, p-methylphenylacetic acid and p-vinylbenzoic acid. The role of cytochrome P-450 in the formation of the metabolites was studied by inhibiting its catalytic activity.The highest excretion rate was obtained with doses of 50, 250 and 500 mg/kg bw already within the first 6 h. However, the dose of 500 mg/kg bw did not increase the excretion rates of these metabolites compared to the dose of 250 mg/kg bw, suggesting that the metabolic pathways begin to be saturated with the amount of 250 mg/kg bw. At 50 mg/kg bw, 55% of the dose was detected as urinary metabolites within 23 h, mainly within the first 6 h. The amounts of the excreted metabolites expressed as% of injected dose (250 or 500 mg/kg bw) were lower than that caused by 50 mg/kg bw, and a noticeable amount of the total sums were excreted within 11–23 h, suggesting that the excretion was still continued with the doses of 250 and 500 mg/kg bw 23 h after injection. The excretion of all analyzed metabolites was prevented by the pre-treatment of the rats with 1-phenylimidazole, an inhibitor of cytochrome P-450 monooxygenases. This indicates that these metabolites were formed as catalyzed by cytochrome P-450. The structures of the analyzed metabolites suggest that the main reactive intermediate of the test substance is vinyltoluene-7,8-oxide. Furthermore, the amounts of the excreted metabolites showed that the main detoxification pathways of vinyltoluene-7,8-oxide were the conjugation with reduced glutathione and hydration to diols. Pre-treatment of the rats with PCBs increased the excretion rates of the metabolites. However, the PCB pre-treated rats excreted less thioethers (62%) compared to the rats treated only with the same amount of test substance, whereas the total sum of the other metabolites was about the same in these both groups. This result suggests that PCBs change the metabolism of the test substance to some other pathway which could be glucuronide conjugation because PCBs increased the activity of UDP-glucuronosyltransferase in a dose-dependent manner (Heinonen, 1984).