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Description of key information

Chronic oral toxicity (similar to OECD TG 453, according to GLP; NTP 2003c-d)
rat: NOAEL 100 mg/kg bw/d
mouse: LOAEL 60 mg/kg bw/d for females
Subchronic inhalation toxicity (Weight of evidence: Gaworski 1992,1993)
rat: NOAEC 34 ppm = 215 mg/m3

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
LOAEL
60 mg/kg bw/day

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEC
215 mg/m³

Additional information

Oral exposure

Groups of male and female F344/N rats or B6C3F1 mice were exposed to diets containing a microencapsulated preparation with a load of 31.3% citral to minimize loss by volatilisation (Kuhn 1991). The test protocol of the 14-week toxicity study used for dose selection of the chronic toxicity study was similar to OECD Guideline 408 (except for partly different investigated organ weights and clinical chemistry parameters). The protocol of the combined chronic toxicity and carcinogenicity study was similar to OECD Guideline 453 except that a complete histopathology was performed for animals from all groups. Clinical chemistry, haematological or urinalyses parameters were not analysed in the chronic study, however, from the 14-week study there are no indications that there are direct effects related to citral exposure (Key studies: NTP 2003 a-d).

   In the subchronic toxicity study with rats (similar to OECD TG 408, according to GLP), diet concentrations were 0 (vehicle control), 3900, 7800, 15600 and 31300 ppm citral yielding daily dosages of 0, 345, 820, 1785, and 1585 mg citral/kg bw/d in males and 335, 675, 1330, and 2125 mg/kg bw/d in females, based on the actual food consumption (NTP 2003a). At 31300 ppm, all animals were killed moribund in the second study week. The LOAEL was 3900 ppm or 345 mg/kg bw/d in male rats and 335 mg/kg bw/d in female rats with significant decreases of final body weight and of body weight change compared to controls as adverse effects. Further effects observed were transient changes of hematological (erythrocytes, platelets) and clinical chemistry parameters (from 7800 and 3900 ppm onward, respectively), which appeared to be secondary to the decreased food consumption. Histopathological findings in bone marrow (athrophy and hemorrhage from 15600 ppm onward), minimal to mild nephropathy not mediated by alpha2µ globulin (significant from 7800 ppm onward) and forestomach (epithelial hyperplasia and hyperkeratosis only at 31300 ppm) seemed to be associated with treatment. A NOAEL could not be derived due to dose selection. 

In the study in mice (similar to OECD TG 408, according to GLP), subchronic feeding of diets with 0, 3900, 7800, 15600 and 31300 ppm citral resulted in daily dosages of 0, 745, 1840, 3915, and 8110 mg citral/kg bw/d in males and 790, 1820, 3870, and 7550 mg/kg bw/d in females, based on the actual food consumption (NTP 2003b). The LOAEL corresponded to the lowest doses of 745 mg/kg bw/d in male mice and 790 mg/kg bw/d in female mice with significantly reduced body weight gain and final body weights below controls (body weight loss during study in the 31300 ppm dose group) as adverse findings. Changes of hematological parameters (lymphopenia) as well as the histopathological finding of hypoplasia of ovaries (from 15600 ppm onward) appeared to be secondary to an overt general toxicity, i.e. decreased body weights and poor general condition of the test animals at high dose levels, and not a organ specific toxicity of citral. A NOAEL could not be derived.

Data on the long-term toxicity of citral are available from a combined toxicity and carcinogenicity study in Fischer 344 rats and B6C3F1 mice (NTP 2003). Diet concentrations were 0, 1000, 2000, and 4000 ppm yielding daily dosages of 0, 50, 100, and 210 mg citral/kg bw/d for rats. Mice were exposed to diets with 0, 500, 1000, and 2000 ppm citral yielding daily dosages of 0, 60, 120, and 260 mg citral/kg bw/d.

In the study in rats (similar to OECD TG 453, according to GLP), application of citral for 2 years

resulted in no treatment-related non-neoplastic or neoplastic effects (NTP 2003c). In high-dosed rats, mean body weights were generally reduced compared to vehicle controls from week 49 onward in males and from week 25 onward in females while food consumption was comparable in all groups. Based on this decrease of mean body weight the LOAEL in male and female rats was 4000 ppm or 210 mg/kg bw/d. The NOAEL was 2000 ppm or 100 mg/kg bw/d.

In the study in mice (similar to OECD TG 453, according to GLP), application of citral for 2 years resulted in

reduced body weights compared to controls in males from 1000 ppm onward and in females in all dosed groups, while food consumption was not affected (NTP 2003d). No treatment-related non-neoplastic lesions with clear toxicological significance were observed. The increased incidence of malignant lymphoma in females at 260 mg/kg bw/d was evaluated to be of equivocal evidence (for detailed discussion of neoplastic lesions see Section "Carcinogenicity"). The LOAEL, based on a persistent reduction of body weight, was 500 ppm or 60 mg/kg bw/d in female mice and 1000 ppm or 120 mg/kg bw/d in male mice.

In a 14-day study with F344 rats and B6C3F1 mice (Supporting study: Dieter 1993), effects of twelve gavage applications (5 d/w, vehicle corn oil) were compared with continuous uptake via the microencapsulated preparation in feed.

In the rat study with dietary doses ranging from 142 to 2280 mg/kg bw/d, body weight parameters were affected at 1140 and 2280 mg/kg bw/d reflecting an initially decreased food consumption. At 2280 mg/kg, absolute weights of liver, spleen and kidneys were decreased. Morphological changes in the respiratory epithelium of the nose, with minimal to mild hyperplasia, indicated local irritation starting at a dose of 1140 mg/kg bw/d. The overall NOAEL was 570 mg/kg bw/d, both for male and female rats. After gavage exposure to doses of 570, 1140, or 2280 mg citral/kg bw/d, the LOAEL in male rats was 2280 mg/kg bw/d based on local irritation of the forestomach with minimal hyperplasia of the squamous epithelium as only effect. Accordingly, der NOAEL for males was 1140 mg/kg bw/d and the NOEL for female rats was set at 2280 mg/kg bw/d, due to the absence of effects. After feeding to mice at doses ranging from 534 to 8550 mg/kg bw/d, the only observable effects were initial weight loss and initially slightly reduced food consumption, and a reduction of final body weights by about 10% at 8550 mg/kg bw/d. The NOAEL was 4275 mg/kg bw/d for both sexes. Administration to mice via gavage with dosages of 534, 1068, or 2137 mg citral/kg bw/d, resulted in liver weight changes starting from 534 mg/kg bw/d in females (with histopathological correlate starting from 1068 mg/kg bw/d) and from 1068 mg/kg bw/d in males. In histopathology cytoplasmic vacuolization of hepatocytes has been observed. The high dose bolus applications resulted in direct irritant effects on the forestomach mucosa indicated by inflammation, necrosis and hyperplasia (both sexes at 1068 and 2137 mg/kg), as well as mortality (males at 1068 and 2137 mg/kg) and moribundity with premature sacrifice (both sexes at 2137 mg/kg).

Inhalation exposure

The repeated dose inhalation toxicity of citral can be evaluated based on a weight of evidence approach with data from 21-day and 13-week inhalation toxicity studies (Gaworski 1993) and maternal toxicity data from a developmental toxicity study (Gaworski 1992). In both studies, exposure atmospheres contained citral concentrations of 1, 3, 10 and 34 ppm (both as vapour), or 68 ppm (aerosol/vapour mixture) corresponding to 6, 19, 63, 215, 430 mg/m3.The exposure condition at 68 ppm comprised an aerosol/vapour mixture with the intention to produce signs of toxicity. However, an exposure of this type would not be expected to occur under normal conditions of use and probably far exceeds any realistic human consumer exposure (Gaworski 1992).

Groups of F344 rats were exposed for 6 hrs/day for either 21 consecutive days at concentration of 10 and 34 ppm (both as vapour), or 68 ppm (aerosol/vapour mixture), or for 13 w, 5 days per week, at concentrations of 1, 3, or 10 ppm (all vapour atmospheres). During the 21 day study, no mortalities occurred. Rats in the 68 ppm-dose group displayed signs of severe ocular, oral and nasal irritation, and significantly reduced body weights. In the nasal respiratory epithelium treatment-related lesions consisted of a dose-related chronic-active inflammation, hyperplasia, squamous metaplasia and goblet cell atrophy of the nasal respiratory epithelium. High-dose rats developed changes indicative of irration in the tracheas and lungs, as well as corneal inflammation and ulceration. In the subchronic inhalation toxicity study there was no mortality and no treatment related signs of toxicity. Body weight gains, clinical pathology indices and organ weights were not adversely affected by exposure. Rats exposed to 10 ppm citral developed minimal hyperplasia and squamous metaplasia of the laryngeal epithelium; however, these changes were completely reversed during a 5-week recovery period. No significant lesions were observed in rats exposed to 1 or 3 ppm.

Taken together, there was no change for the worse as exposure duration increased from 21 days to 13 weeks (Gaworski 1993).

During a developmental toxicity study, maternal toxicity was indicated at 68 ppm (430 mg/m3) by decreased body weights and by clinical signs as ocular opacity, breathing difficulty, nasal discharge and salivation. These signs of maternal toxicity were secondary to the stress produced by severe respiratory tract irritation, as recovery of body weight and clinical signs of toxicity occurred after completion of the exposure period. At 10 and 34 ppm, findings were incidental and not siginficantly different from control animals (Gaworski 1992).

Overall, the NOAEC has been set to 34 ppm or 215 mg/m3 based on evident local irritation and systemic effects, i.e. body weight changes, observed at 68 ppm or 430 mg/m3.

Dermal exposure

A sequence of studies investigated the induction of different types of prostratic hyperplasia (atypic prostrate hyperplasia APH and benign prostrate hyperplasia BPH) in adolescent male rats after topical application of citral with the intention to find a rat model for the development of benign prostratic hyperplasia in man.

Increased prostrate weight and histological changes characteristic of prostratic hyperplasia appeared in Wistar rats from day 10 of dermal treatment (150 mg/kg bw/d, 5 d/w) and progressed with duration of treatment up to 90 days (Servadio 1986).

A comparison between a daily topical application regimen and administration adjusted to the circadian minimum of testerone concentration in rat plasma (4-day intervals) with citral (185 mg/kg bw/d) was performed in Wistar rats (Abramovici 1987). Daily application resulted in BPH with increased prostrate weights, whereas 4 – day interval treatment promoted APH without increase of prostrate weight, beeing identical to the findings of APH in human prostrates.

Hyperplasia of glandular epithelium and interglandular stroma of the ventral prostrate without increase in prostrate weight were visible inrats after 5 weeks and increased in severity during a 4 month period with topical treatment with 62 mg citral per rat, thrice a week. These authors suggested the prostratic hyperplasia-inducing capacity of citral to be due to the estrogenic activity proven in assays for cellular proliferation of vaginal epithelium and in an in vitro estrogen receptor binding assay (for details see Chapter “ Toxicity to reproduction”) (Geldof 1992).

 

Among different rat strains the histological pattern of untreated rat ventral prostrate varied at adolescence which might be explained by different endocrine homeostasis. Normal morphology was found in untreated Wistar, Sprague-Dawley and F344 rats, whereas untreated ACI/Ztm rats showed benign hyperplastic changes. Citral (150 mg/kg bw applied topically for 30 days, intermittently every 3 to 4 days) induced BPH in Wistar and Sprague-Dawley rats, and APH in ACI/Ztm rats (no data for F344 rats). Additional treatment with testosterone, promoted APH in Wistar and ACI/Ztm rats and induced BPH in F344 and SD rats. Experiments with castrated rats demonstrated that citral seems to be effective in inducing prostrate hyperplasia only in the presence of endogenous or exogenous testosterone. Principally, the F344 strain was the most resistant to prostrate hyperplasia, possibly due to decreased androgen sensitivity of the prostrate. In rats with APH, the simultaneous presence of APH and BPH lesions in the histological picture is suggestive of a correlative and progressive evolution of BPH lesions toward APH lesions in rats. In contrast, in the human condition BPH is considered an autonomous lesion. However, the APH changes found in the Wistar and ACI/Ztm rats, were found to be analogous to human APH lesions (Scolnik 1994).

In summary, citral was shown to induce benign and atypic prostrate hyperplasia (BPH and APH), in adolescent male rats after topical administration. Possible interactions with testosterone levels or estrogen-like effects of citral were discussed in chapter “Toxicity to reproduction”.

Justification for classification or non-classification

The present data on repeated dose toxicity do not fulfill the criteria laid down in 67/548/EEC and CLP, and therefore, a non-classification is warranted.