(Z)-3,7-dimethylocta-2,6-dienal

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Additional information

Reproductive toxicity studies

In the chosen key study, a reproduction toxicity screening test in rats was performed according to OECD Guideline 421 and GLP (Yoshimura, 2002).Male and female Sprague Dawley rats were exposed to citral by gavage at dosages of 0, 40, 200, and 1000 mg/kg bw/d in corn oil as vehicle. Male rats were treated for 14 days before mating, throughout the mating period, and up to day 46. Females were dosed from 14 days before mating, throughout the gestation period up to lactation day 3.

In the dose group, receiving 1000 mg/kg bw/day, parental toxicity was found in terms of decreased body weights (significant for body weight changes), temporarily decreased food consumption and histological changes in the forestomach, indicating a irritative potential of the test substance in the GI tract. No test substance related effects were detected in terms of reproductive performance, parental organ weights or histopathology of the reproductive organs. Test substance related developmental toxicity was found at 1000 mg/kg bw/d in terms of reduced pup body weights on postnatal days 0 to 4, whereas no other adverse effects were observed. The NOAELs for developmental toxicity and parental toxicity is set at 200 mg/kg bw/day.The NOAEL for reproductive toxicity in rats is set at 1000 mg/kg bw/day.

Supporting information from repeated dose toxicity studies

Several repeated dose toxicity studies are available, providing supportive information concerning fertility. In subchronic and chronic studies of Fischer 344 rats or B6C3F1 mice, exposed to diets containing a microencapsulated preparation with citral for 14 weeks, histopathological assessment on adrenal gland, clitoral gland, mammary gland, ovary, parathyroid gland, pituitary gland, preputial gland, prostate gland, testis with epididymis and seminal vesicles, thyroid gland and uterus was performed ( see Chapter “repeated dose toxicity” and “carcinogenicity”; NTP, 2003). No adverse effects on these organs were noted that were attributable to a substance-specific effect.

Administration of citral (210 mg citral/kg bw/d)to female rats for 2 years resulted in significantly decreased incidences of clitoral gland adenoma or carcinoma and of mammary gland fibroadenoma. NTP (2003) discussed these to be putatively related to an antiestrogenic effect of citral. However specific studies on endocrine effects of citral (see Discussion for “Toxicity to reproduction other studies” below) and the findings of the fertility screening study according to OECD 421 (see above) do not support this assumption.

Supporting information from in vivo and in vitro studies on endocrine activity

The biological significance of in vitro binding of citral to the estrogen receptor at high concentrations is uncertain as an estrogenic activity was not confirmed by uterotrophic assays at doses up to 1000 mg/kg bw/d (For further details see: Discussion for “Toxicity to reproduction other studies” below). Consequently, there is no indication that fertility might be affected by an action as endocrine disruptor.

Short description of key information:
Reproduction toxicity screening test (OECD Guideline 421, GLP), rat, gavage:
reproductive toxicity NOAEL = 1000 mg/kg bw/d; parental toxicity NOAEL = 200 mg/kg bw/d (Yoshimura et al., 2002)

Effects on developmental toxicity

Description of key information

Developmental toxicity study, rat, gavage, exposure GD 6-15: developmental toxicity LOAEL = 60 mg/kg bw/d, no NOAEL identified; maternal toxicity LOAEL = 60 mg/kg bw/d, no NOAEL identified (Nogueira 1995)
Reproduction toxicity screening test according to OECD TG 421 and GLP,  rat, gavage, exposure 14 d premating up to lactation day 3: developmental toxicity  NOAEL = 200 mg/kg bw/d; parental toxicity  NOAEL = 200 mg/kg bw/d (Yoshimura 2002)
Developmental toxicity study, rat, inhalation, 6h/day, exposure GD 6-20: developmental toxicity NOAEC = 68 ppm (429 mg/m3);  NOAEC= 34 ppm (215 mg/m3) (Gaworski 1992)

Effect on developmental toxicity: via oral route

Dose descriptor:

LOAEL

60 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Dose descriptor:

NOAEC

430 mg/m³

Additional information

Developmental toxicity

In a developmental toxicity study, comparable to OECD Guideline 414, citral was orally administered via gavage to Wistar rats (0, 60, 125, 250, 500, 1000 mg/kg bw/day) from day 6 to day 15 of pregnancy (Nogueira 1995). A decrease in the corrected body weight gain (- uterus weigths) revealed maternal toxicity at 500 and 1000 mg/kg bw/d. Furthermore, significant reduction in body weight gains during gestation days 6 -11 at 60 and 125 mg/kg bw/d are considered to represent maternal toxicity since embryo weights are insignificant during this gestational phase. Thus, citral was found to be maternally toxic over the dose range tested, and severity of effects correlated with the dose applied.

A slight but statistically significant increase in the ratio of resorptions per implantations was observed in the 60 and 125 mg/kg bw/d dose group, beeing indicative for post implantation losses. Doses higher than 125 mg/kg led to a dose-dependent reduction of the ratio of pregnant per mated female indicating pre- or peri-implantation losses. Citral seemed to have induced whole-litter rather than intra-litter individual losses. The dose dependent differences in the effects observed, indicated that citral-induced gestational losses occurred earlier as the dose increased. Further developmental effects were observed from 125 mg/kg bw/day onwards, i.e. fetal growth retardation, increased incidences of minor skeletal abnormalities and increases in fetal spleen weights.The overlapping with overt maternal toxicity substantiate, that substance-induced developmental effects were secondary to maternal adverse effects.

In conclusion, developmental effects were observed starting at a dose of 125 mg/kg bw/d. Additionally, citral increased the ratio of resorptions per implantations at 60 and 125 mg/kg bw/d, and impaired implantation in doses higher than 125 mg/kg bw/d. Maternal toxicity, i.e. decreased body weight parameters were observed in all dose groups. Consequently the LOAEL for maternal toxicity and developmental toxicity is set at 60 mg/kg bw/d and no NOAEL is established from this study. The adverse effects on implantations and resorptions could not be confirmed in a guideline reproduction screening study (see below).

In a reproduction toxicity screening test in rats performed according to OECD Guideline 421, male and female Sprague Dawley rats were exposed to citral by gavage at dosages of 0, 40, 200, and 1000 mg/kg bw/d in corn oil as vehicle (Yoshimura et al., 2002).

Male rats were treated for 14 days before mating, throughout the mating period, and up to day 46. Females were dosed from 14 days before mating, throughout the gestation period up to lactation day 3.

In the dose group, receiving 1000 mg/kg bw/day, parental toxicity was found in terms of decreased body weights (significant for body weight changes), temporarily decreased food consumption and histological changes in the forestomach, indicating a irritative potential of the test substance in the GI tract.No test substance related effects were detected in terms of reproductive performance, parental organ weights or histopathology of the reproductive organs.Test substance related developmental toxicity was found at 1000 mg/kg bw/d in terms of reduced pup body weights on postnatal days 0 to 4, whereasno other adverse effects were observed. The NOAELs for developmental toxicity and parental toxicity is set at 200 mg/kg bw/day.The NOAEL for reproductive toxicity in rats is set at 1000 mg/kg bw/day

In a developmental toxicity study comparable to OECD Guideline 414, Sprague-Dawley rats were exposed to citral by inhalation (0, 10, 34, 68 ppm or 63, 215, 430 mg/m3) for 6 hours per day on gestation days 6 -15 (Gaworski et al. 1992). Maternal toxicity was observed at 68 ppm by maternal body weight loss during exposure period and by clinical signs, such as ocular opacity, breathing difficulties, nasal discharge and salivation. The clinical signs were considered to be secondary to the stress produced by severe respiratory tract irritation, and recovery occurred after completion of the exposure period.The number of corpora lutea, implantations, resorptions, foetal viability, litter size, and sex ratio were not adversely affected at any dose level. Although, there was a slight increase in preimplantantion loss in the test substance exposed groups, no biological relevance can be attributed to this finding since no siginficant reduction in litter size was observed. A slight non significant reduction in mean fetal body weights and a slight increase in the incidence of hypoplastic bones was observed in the 68 ppm dose group, beeing considered as secondary to the maternal toxic effects observed. No exposure-related malformations were observed. A NOAEC for maternal toxicity is set at 34 ppm (215 mg/m3) and a NOAEC for developmental toxicity is set at 68 ppm (430 mg/m3), i.e. the highest concentration tested.

In summary, signs of developmental toxicity have been observed after oral or inhalative exposure with citral in the presence of maternally toxic doses. No teratogenic effects, leading to specific malformations were found. Consequently, the observed effects on developmental toxicity are considered to be secondary to maternal toxicity. In a weight of evidence, citral is not considered to be a developmental toxicant.

Toxicity to reproduction: other studies

Additional information

Endocrine activity

A sequence of studies in adolescent male rats after topical application of citral identified the induction of different types of prostratic hyperplasia (atypic prostrate hyperplasia APH and benign prostrate hyperplasia BPH) (For details see Chapter “repeated dose toxicity”).

Assessment of prostratic hyperplasia by citral treatment of male Copenhagen rats via the transdermal route (62 mg per rat, thrice weekly) showed no significant increase in postrate weights and proliferation rate (BrdUrd incorporation) up to 2 weeks of treatment despite observed cellular hyperplasia (Geldof 1992). A putative estrogenic activity was suggested to be causative, since in another study from the same authors, direct application of citral to the vagina of female, ovariectomized rats (27 mg/rat/day for 4 days) resulted in increased cellular proliferation (BrdUrd incorporation) in vaginal epithelial cells. No treatment-related alterations in serum testosterone and estradiol concentrations in males or females were observed in these studies. In an in vitro estrogen receptor binding assay using cytosolic uterus or prostate fractions, citral inhibited estrogen binding to the estrogen receptor, while no such inhibition was observed with testosterone for androgen receptors. A putative estrogen-like effect of citral on the female reproductive tract was observed in an unphysiological condition of ovariectomy resulting in decreased endogenous estrogen levels combined with a high local citral dose by direct application to the target tissue.

Further in vitro assays demonstrated the ability of citral to bind to the estrogen receptor at high concentrations (about 1.000.000-fold that of ß-estradiol) and to inhibit binding of estradiol (Geldof 1992; Howes 2002). In contrast, citral showed no estrogenic or anti-estrogenic acitvity in the estrogen-responsive human endometrial cell line Ishikawa Var I, and no activity in a yeast screen for androgenic and anti-androgenic activity (Howes 2002).

The putative estrogenic activity of citral could not be confirmed in in vivo assays. Citral was inactive in the uterotrophic assay (no increase of uterus weights as an indicator of estrogen-like activity), both after oral exposure (300 or 1000 mg/kg bw/d for 3 days) of rats (BASF07R0155/98090) or dermal exposure of mice (950 mg/kg bw/d for 3 days) (Howes 2002). Also, there was no indication of an estrogenic activity of citral in an acute vascular permeability assay of the uterus after dermal application of a single dose of 950 mg/kg bw (Howes 2002).

In line, a reproduction toxicity screening test in rats according to OECD Guideline 421 and GLP revealed no test substance related effects on reproductive ability, organ weights or histopathology of the reproductive organs, on delivery or on maternal behavior (Yoshimura et al., 2002; see above).

Justification for classification or non-classification

The present data on reproductive toxicity do not fulfill the criteria laid down in 67/548/EEC and 272/2008/EEC, and therefore, a non-classification is warranted.