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Description of key information

There are no repeat dose toxicity data on this intermediate. However, there are data on the repeated dose toxicity of major and minor components present i.e. n-hexane, toluene, ethylbenzene, styrene and xylenes which demonstrate significant target organ toxicity. 

Key value for chemical safety assessment

Additional information

There are no repeat dose toxicity data on the intermediate. However, there are data on the repeated dose toxicity of all the components present.

For cyclohexane, there are no oral data but slight liver effects were induced after 14 or 90 day inhalation exposure. Increases in absolute and relative liver weight and centrolobular hypertrophy were noted in both rats and mice at dose levels between 6000 and 7000 ppm. The NOAEC for hepatic effect is estimated to be 2000 ppm (6,880 mg/m3) (Malley et al, 2000). 

Human exposure data on cyclohexane is reviewed in the EU RAR (2004). It has been reported that cyclohexane exposure at the concentrations up to 274 ppm (959 mg/m3) did not induce induced any significant increase in the prevalence of subjective symptoms or measured biochemical parameters of liver and kidney function (Yasugi et al., 1994). A 1991 UK HSE review pointed out that cyclohexane, with other chemicals, was implicated by some authors in the peripheral neuropathy reported among in workers in Italian shoe factories, printing plants and in paint workers; cyclohexane frequently being a major component of the solvents and adhesives used (De Rosa et al., 1985; Mutti et al., 1982; Franco et al., 1979). However, such workers received mixed exposure to various solvents, including n-hexane, and HSE concluded from the experimental evidence that the neuropathy was most likely caused by the n-hexane component of the solvents and adhesives used in these industries (as cited in the EU RAR, 2004). The neurotoxicity of cyclohexane during occupational exposure in a luggage factory of 18 women (aged from 18 to 56) who were exposed for eight hours a day to glue containing 75.6% cyclohexane, 12% toluene and 0.9% n-hexane was assessed in a study was carried out by Yasugi (1994). Overall, there was no evidence of neurotoxicity during a relatively short period of exposure at low concentrations (below the time weighted average threshold limit values of the US (150 ppm)).

Repeat dose toxicity data exist for 4-vinylcyclohexene in rats and mice up to 2 years in duration. NTP (1986) administered 4-vinylcyclohexene by oral gavage to groups of rats (50-800 mg/kg bw/d) and mice (75-1200mg/kg bw/d ) for up to 13 weeks. In rats there was decreased body weight gain in males at >400 mg/kg body weight/day and females at 800 mg/kg/day, a minimal increased severity of hyaline droplet degeneration of the renal proximal convoluted tubule in males at 800 mg/kg/d and some inflammatory changes in non-glandular stomach of high dose males and females. Overall the findings indicate a sub-chronic oral NOAEL of 200-400 mg/kg body weight/day for male and female rats, respectively. In mice, there was a high level of early mortality in high dose (1200 mg/kg/d) animals of both sexes, with lower mortality in females at 300 mg/kg bw/d, however this is of doubtful relevance due to evidence of mis-dosing at necropsy. Occasional mild acute inflammation of the stomach was recorded following histopathological examination of high dose males and females. Histological re-evaluation of ovaries from females dosed at 1200 mg/kg/d (post completion of the two year mouse study; lower dose groups not examined) revealed a reduction in the number of primary follicles and mature graafian follicles. There were no other microscopic changes. A NOAEL of 600 mg/kg body weight/day was identified, based on early mortality and stomach lesions. No no-effect level is available for the ovarian effects due to the absence of data from the lower treatment groups. Whilst there was some mortality in female mice dosed at 300 mg/kg/day, no conclusions can be drawn due to the high incidence of mis-dosing reported across the study as a whole.

In a chronic gavage investigation (NTP, 1986), rats and mice were administered 4-vinylcyclohexene for 103 weeks at doses of 200 and 400 mg/kg. Survival in rats was significantly decreased by week 103 in males at both dose levels and in females dosed at 400 mg/kg. In both sexes there was also an increased incidence of epithelial hyperplasia of the forestomach (more pronounced in males); this was statistically significant in males surviving beyond week 93. With respect to chronic toxicity, no NOAEL was determined; a chronic LOAEL of 200 mg/kg body weight per day is proposed based on decreased survival in male rats, and the occurrence of histological abnormalities in the stomach of rats (both sexes, but more pronounced in males). In mice, survival was decreased in the high-dose animals of both sexes, with stomach abnormalities (which included ulcers, inflammation, and epithelial hyperplasia of the forestomach) and lung congestion at necropsy of mice that survived to termination. Histopathology revealed a significant increase in the incidence of hepatic centrilobular congestion and atrophy of splenic red pulp in high dose males only, with adrenal gland congestion and cortex alterations and ovarian changes in females from both treatment groups. No NOAEL for chronic toxicity was obtained. A chronic LOAEL of 200 mg/kg body weight per day is based on reduced survival and histological abnormalities in the stomach of mice (males and females), the liver and spleen of male mice, and the adrenal gland and ovary of female mice.

Bevan et al. (1996) exposed groups rats (up to 1500 ppm) and mice (up to 1000 ppm) to 4-vinylcyclohexene for 13 weeks. In rats statistically significant lethargy was reported in males at 250 ppm and both sexes at 1500 ppm. There was reduced body weight and/or weight gain in both sexes exposed at 1000 ppm and 1500 ppm. Liver weights were significantly increased in male and female rats exposed to >1000 ppm, and kidney weights in males exposed to >1000 ppm and females at 1500 ppm. No histopathological anomalies were attributed to 4-vinylcyclohexene and haematological, clinical chemistry and urinalysis parameters were unaffected by treatment. The authors report a NOAEC for rats of 1000 ppm based on lethargy and lowered body weights at 1500 ppm (4424 mg/m3). However, based on effects on bodyweight and liver weights at 1000ppm, the NOAEC is proposed as 250ppm (equivalent to conclusions reached by the HPV Challenge Program). All high-dose males and most female mice died on or before day 12. There were increased incidences of mortality, lethargy, tremors and ovarian atrophy (as diagnosed by histopathology) at 1000 ppm. There were no effects on haematological, clinical chemistry and urinalysis parameters at any dose level. These findings indicate a sub-chronic NOAEC of 250 ppm in mice (1106 mg/m3).

Other specific components present in this intermediate are n-hexane, toluene and also minor components ethylbenzene, styrene and xylenes. All of these are identified as producing serious target organ toxicity following repeated oral, dermal or inhalation exposures in animals and man:

n-Hexane (Classification: EU -Harmful Xn, R48/20; GHS/CLP - STOT-RE Category 2, H373): In animals following oral or inhalation exposure n-hexane can produce neurologic dysfunction of motor or sensorimotor nerves resulting in weight loss and paralysis (Spencer and Schaumberg, 1985). Epidemiological studies on n-hexane have shown an association between inhalation exposure to n-hexane and neurological symptoms in occupationally exposed individuals. However, the extent of exposure to n-hexane in many, if not all, of the occupational studies is imprecise, and subjects were likely exposed concurrently to other solvents. The NOAEC for peripheral neuropathy is reported to be 20 ppm (70 mg/m3).

Toluene (Classification: EU - Harmful Xn, R48/20; GHS/CLP - STOT-RE Category 2, H373): Toluene exposure can produce central nervous system pathology in animals after high oral doses. Repeated inhalation exposure can produce ototoxicity in the rat and high concentrations are associated with local toxicity (nasal erosion). In humans neuropsychological effects and disturbances of auditory function and colour vision have been reported, particularly when exposures are not well controlled and/or associated with noisy environments. The NOAEC for subchronic oral toxicity in rats is 625 mg/kg/day based on neuropathology (NTP, 1990). The NOAEC for inhalation toxicity in the rat is 300 ppm (1131 mg/m3) based on effects on body weight, mortality and adverse local effects (nasal erosion) (Gibson and Hardisty, 1983). The NOAEC for neuropsychological effects, auditory dysfunction and disturbances of colour vision in humans is 26 ppm (98 mg/m3) (Seeber et al, 2004); Schaper et al, 2003, 2004).

Minor components impacting classification:

Ethylbenzene: No repeated dose toxicity studies in humans have been identified. The EU transitional RAR (EU, 2008a) concluded "Repeat-dose or prolonged exposure to ethylbenzene specifically affected the nervous system, but did not induce overt toxicity of any other organ system." The auditory system is the most sensitive to the toxic effects of ethylbenzene after inhalation exposure (Gagnaire et al, 2007) while the liver is the most sensitive following oral exposure (Mellert et al, 2006). The LOAEL for ototoxicity was 200 ppm (868 mg/m3) and the NOAEL for hepatotoxicity was 75 mg/kg/day in a 13 week oral gavage study in rats.

Styrene: Inhalation studies in animals have reported damage to the nasal olfactory epithelium in rats and mice, liver damage in mice, eye irritation in rats and guinea pigs, ototoxicity in rats and impaired nerve conduction velocity. The EU transitional RAR (EU, 2008b) has identified a NOAEC of 50 ppm in humans based on colour vision discrimination effects in occupationally exposed workers.


Additional References

EU RAR (2004).European Union Risk Assessment Report: Cyclohexane. EC Joint Research Centre

EU (2008a). Draft Risk Assessment Report for Ethylbenzene.

EU (2008b). Annex XV Transitional Dossier: Styrene. echa. europa. eu/doc/trd_substances/styrene/RAR/trd_rar_uk_styrene. Rtf

Justification for classification or non-classification

A mandatory classification is triggered by ≥10% n-hexane and toluene, therefore the intermediate should be classified as Xn, labelled R48/20 according to Dir 1999/45/EC and Cat 2, H373 according to Reg (EC) 1272/2008.