Reproductive and developmental toxicity

11.Effects on Humans

Eye irritation of l-limonene was measured using goggles instrumentation and a relatively short exposure time of 2 minutes. The threshold level for irritation in 12 volunteers was 1700-3400 mg/m³ (300-600 ppm approximately) (Mølhave et al., 2000).

Oxidised Histo-clear solvent, identified elsewhere as limonene-based (Langman, 1995), produced respiratory irritation requiring medical treatment in a histology technician (Zitko, 2001). It was considered that the autoxidation products might be the cause of the irritation. An increase in nasal or throat irritation was also reported by 4/12 workers in a facility where a limonene-based cleaner was stored in an uncovered tank (Kiefer et al., 1994).

In a study in which the sensitivity of four patch testing systems (Finn chamber, Hill Top patch, Van der Bend chamber, and Webril patch) was evaluated in volunteers, d-limonene (perfumegrade) reacted strongly in all types of patches within 1015 minutes of exposure *(York et al., 1995). Skin irritation was assessed before application, as well as immediately and 1, 24, 48, and 72 hours after removal of the patch, using a scoring system based broadly on that used for rabbit irritation studies *(OECD, 1981c), but modified to account for the nature of reactions on human skin. There was evidence of sensory effects and urticarial responses on removal of the patches. Significant irritation persisted for 24 hours, and these reactions persisted for 48 and 72 hours in many volunteers *(York et al., 1995). Dermal exposure to dlimonene (98%) for 2 hours in one subject caused burning, itching, aching, and a longlasting purpuric rash *(Falk et al., 1991).

dLimonene infused directly into the bile system of human volunteers to dissolve gallstones caused pain in the upper abdomen, nausea, vomiting, and diarrhoea, as well as increases in serum aminotransferases and alkaline phosphatase *(Igimi et al., 1976, *Igimi et al., 1991). The oral administration of 20 g dlimonene to volunteers resulted in diarrhoea, painful constrictions, and proteinuria, but no biochemical changes (total protein, bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase) in the liver *(Igimi et al., 1976). Dose related gastrointestinal side effects such as nausea and diarrhoea were observed following oral administration of d-limonene to patients with advanced cancer in a Phase I clinical trial. Dosages were 0.5 to 12 g/m² body surface area (BSA) per day and the maximum tolerated dose was 8 g/m² BSA (Vigushin et al., 1998). The authors reported that d-limonene had low toxicity after single and repeated dosing for up to 1 y.

There are no studies involving inhalation exposure in which effects on the liver have been investigated.

Reports of contact allergy to dipentene have appeared *(Calnan, 1979; *Rycroft, 1980). In one investigation, 15 of 22 people with an allergy to oil of turpentine also reacted to dipentene *(Cachao et al., 1986). Patch testing in consecutive dermatitis patients from Sweden and Belgium revealed positive reactions in 1.52% of the subjects tested with oxidized dlimonene, a finding similar to that observed with other common sensitisers, such as formaldehyde (A.T. Karlberg, personal communication, 1996; Karlberg & Dooms-Goossens, 1997). The oxidation products d-cis- and d-trans-limonene-1,2-oxide, carvone, and cis- and trans-limonene-2-hydroperoxide were found to be sensitisers. Some patients not sensitive to the oxidised limonene were found to be sensitive to the hydroperoxide fraction, and vice versa. It is believed that limonene itself has a very low sensitising capacity and that sensitisation is due to the oxidation products formed on exposure to air.

No sensitising effect was observed when 25 volunteers were exposed to dlimonene in a Human Maximization Test *(Grief, 1967). Skin patch testing carried out at an Australian dermatology practice in 1999 indicated that 2/470 patients reacted to limonene, and a larger number reacted to other terpenes. The oxidation status of the limonene was not tested (Fewings, 2001).

Prolonged or repeated exposure to limonene in histology laboratories is reported to cause respiratory effects such as difficulty in breathing and tightness in the chest (Dapson & Dapson, 1995). Bronchial hyper-responsiveness was related to indoor concentrations of limonene in dwellings (Norbäck et al., 1995).

Health effects reported in the NICNAS phone survey of formulators and users included skin drying and irritation, dermatitis, eye irritation, headache and “lightheadedness” from the strong odour, and respiratory symptoms when product is atomised (see Appendix 3).

dLimonene reduced nonimmunological contact urticaria caused by cinnamic aldehyde, with competitive receptor inhibition suggested as the mechanism of suppression *(Guin et al., 1984). Quenching (inhibition of the sensitising capacity of another substance) of the effects of citral by limonene has been reported in humans (European Union, 2000). However the EU SCCNFP noted conflicting evidence and concluded on the balance of evidence currently available that the quenching of fragrance allergens by other specific fragrance components should be regarded as a hypothesis only.