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Key value for chemical safety assessment

Effects on fertility

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 2002)

Effect on fertility: via oral route
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Additional information

Reproductive toxicity studies

In 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 an 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 further 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.

 

Study proposal for an extended one-generation toxicity study

The present data for Citral do not contain concerns to be addressed by the DIT and DNT cohorts of an EOGRTS. Repeated administration of Citral up to the limit dose (1000 mg/kg bw/d) via feed in the key subchronic/chronic repeated dose toxicity studies (rat, mouse) did neither result in clinical signs indicative for neurotoxicity nor histopathological changes of neuronal tissues such as the brain. Likewise, oral administration of Citral in the reproduction toxicity screening test and the Uterotrophic assay in rats did not lead to any relevant clinical signs up to 1000 mg/kg bw/d. Clinical signs seen in developmental toxicity studies stem from general systemic toxicity and irritation of the respiratory tract after inhalation, however, they do not represent evident neurotoxic effects. Specific neurological investigations in rats and mice are available in literature. Neurological modulation (specific sedation of the CNS, hypnotic/neuroleptic/anticonvulsant effects) of Citral has been investigated after oral and intraperitoneal administration in rats and mice (Carlini 1986). In contrast to intraperitoneal administration, oral administration of Citral up to 200 mg/kg bw did not affect open-field behavior, spontaneous motor activity, intestinal transit or the Barbiturate induced sleeping-time. Application of Citral (intraperitoneal and/or oral) did not significantly affect performance on the Rota-rod or tests for neuroleptic effects (catatonic reaction, palpebral ptosis, blockade of stereotyped behavior induced by apomorphine) and electroshock/pentylenetetrazol induced convulsions were not modulated. Given the absence of any neurological effects after oral administration, representing the route of exposure in question for the proposed EORGTS, the inclusion of the DNT cohort is not warranted. 

On the basis of the subchronic/chronic repeated dose toxicity study in rats, no indication of an immunotoxic potential of Citral could be identified. There were no changes in leucocyte and differential blood cell counts. No evident and test substance related histopathological changes in immuno-competent organs such as bone marrow, lymph nodes, spleen or thymus were observed up to limit dose. Excessive dosing above the limit dose resulted in atrophies of the thymus and the bone marrow, which can be seen as secondary effects to decreased body weights and severe general toxicity. In mice, decreased leucocyte and lymphocyte counts were observed together with a marked suppression in mean body weights, reflecting a physiological response consistent with a stress-related and/or corticosteroid-induced lymphopenia. No histopathological changes in immuno-competent organs were reported. In literature, no indication of immunotoxicity in an antibody plaque-forming cell assay and a host-resistance assay in Citral-treated mice was observed (Gaworski 1994). In this study, Citral was orally applied daily for 5 days at 200, 400 and 800 mg/kg bw/d to CD1 mice, and no effects on mortality, average survival time, spleen and thymus weights were observed after bacterial challenge with L. monocytogenes. Furthermore, spleen cellularity and IgM antibody plaque forming cell response to SRBCs was not altered, providing evidence for the absence of an adverse modulation of the immune reponse by Citral.

 

Indications of an estrogenic activity of Citral could not be confirmed in several in vivo studies, since Citral was inactive in an 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). In line, the present 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). Overall, a putative estrogenic activity of Citral cannot be used as an argument for justifying the inclusion of cohorts for DNT and DIT.

 

Therefore, no triggers for the inclusion of Cohorts 2A and 2B (developmental neurotoxicity) and Cohort 3 (developmental immunotoxicity) were identified.

Generally, a Cohort 1 B including the mating of the F1 animals to produce the F2 generation is not mandatory. This can be based on sound scientific evidence:

  • Piersma et al. (1) investigated the necessity of producing a second generation to assess the potential for human health risks. The analysis included 498 rat multi-generation studies representing 438 different test substances. Detailed assessment of study reports revealed no critical differences in sensitivities between the generations on the basis of a consideration of all endpoints evaluated (1). This analysis indicated that the second generation mating and offspring will very rarely provide critical information (1).
  • Martin et al. (2) conducted an analysis on 329 multi-generation studies on 316 chemicals using the ToxRefDB dataset of USEPA. This analysis supports the hypothesis that the second, F2 generation in these 329 studies would rarely impact either the qualitative or quantitative evaluations of these studies.
  • Janer et al. (3) evaluated 176 multi-generation studies to assess potential differences between the first and the second generation and came to the conclusion that the second generation in the two-generation studies considered affected neither the overall NOAEL nor the critical effect, and had no impact on the ensuing risk assessment, nor on classification and labelling. Therefore, if the second generation had not been included in the reproductive toxicity test, the ensuing risk assessment and classification and labelling would have basically been the same for the substances that were considered (3).
  • Beekhuijzen et al. (4) reviewed nine two-generation studies in rats and recommended that further breeding should not be conducted when the effects on parental fertility and fecundity are clear after first pairing. However, mating of the F1 generation is warranted if the effects on parental reproduction/breeding parameters are ambiguous (4). Likewise, if development and/or viability of the F1 generation are adversely affected, a second mating deserves considerations as potential exists for compromised reproductive integrity of the F1 animals (4).
  • Rorije et al. (5) analysed the possible impact on classification and labelling decisions of effects observed in second generation parental (P1) and offspring (F2) parameters in multi-generation studies. This was done for 50 substances classified as reproductive toxicants in Europe, for which a multi-generation study was available (5). The analysis shows that, except for a single case, effects observed in second generation mating and offspring did not impact the decision on classification and labelling for reproductive toxicity (5). Moreover, the single case where second generation mating and offspring effects appeared to be instrumental for classification would be identified without any doubt as a reproductive toxicant in an extended one-generation reproductive toxicity study without second generation mating and offspring (5).


Therefore, the extended one-generation reproductive toxicity study in rats, oral route (test method: OECD 443) with Cohort 1A would be fully sufficient in order to allow for a proper risk assessment including valid classification and labelling. There is no scientific and regulatory reason to include Cohort 1B to mate the F1 animals to produce the F2 generation and Cohort 2 A/B and 3 at this stage. Also for animal welfare reasons it is generally proposed to make the second generation optional based on the results observed during the study and to leave this decision to the registrant.

Effects on developmental toxicity

Description of key information

Developmental toxicity study, rabbit, gavage, exposure GD 6-28 (OECD 414, GLP): developmental toxicity NOAEL = 60 mg/kg bw/d; maternal toxicity NOAEL = 60 mg/kg bw/d (BASF 2016; 40R0410/07R055)

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, 6 h/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 (rabbit)

In the chosen key study for developmental toxicity according to OECD 414 and GLP, citral was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28) at dose levels of 20, 60 and 200 mg/kg bw/day.

Generally, no toxicologically relevant signs of maternal toxicity were observed in any of the control and test groups receiving 20 or 60 mg/kg bw/d. Neither clinical examinations nor determination of food consumption and body weights/body weight gain and necropsy revealed any relevant effect on the animals of these dose groups. However, signs of distinct maternal toxicity were noted in the high-dose group (200 mg/kg bw/d).

 

·        Two high-dose females were found dead during the last third of the administration period (on GD 23 and GD 28). Gross pathological examination revealed a severe confluent reddening of the stomach mucosa in one of these animals.

·        One high-dose female was sacrificed after abortion ahead of schedule (GD 26). As multiple ulcerations were found in the stomach of this doe at necropsy, a relationship of this abortion to the treatment is assumed.

·        One high-dose doe had 4 dead fetuses at term. Alike abortions, this is considered an expression of maternal toxicity in rabbits, in particular as this animal also suffered from markedly reduced food consumption (5.5 g/animal /d vs. 101.8 g/animal /d mean of the respective dose group at GD 28-29) and body weight loss (-150g at GD 28-29) near term.

·        One high dose doe with a litter including 5 cases of malrotated limbs was particularly affected by maternal toxicity in terms of constantly and markedly reduced food consumption until it almost stopped eating. Accordingly, this doe lost more (net) body weight during the treatment period than the average (net) weight loss in the high-dose group.

 

The food consumption of the high-dose group (200 mg/kg bw/d) was constantly below control during the treatment period, but the difference gained statistical significance only on GD 7-8. However, throughout treatment period (GD 6-28), the average food consumption of the high-dose does was almost 10% below the control group. Average carcass weights as well as gross and corrected (net) body weight change were also not significantly different from control in all treatment groups, although some high-dose individuals suffered from more pronounced reductions of food consumption and (net) body weight losses than the majority of animals.

Generally, these findings are indicative of a local irritating potential of the test item in the gastrointestinal tract which because of the peculiarity of rabbit digestive system subsequently led to reduced food consumption, distinct body weight loss, doe mortality, abortion and fetal mortality in the most sensitive individuals exposed to the top dose of 200 mg/kg bw/d. It is notable that all casualties occurred towards the end of pregnancy, when the rapid growth of the offspring makes it a particularly demanding pregnancy phase to the mothers.

 

There were no differences of biological relevance between the control and the substance-treated groups (20, 60 and 200 mg/kg body weight/day) in conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio or in the values calculated for the pre- and the post-implantation losses.

 

No test substance-related differences were recorded for placental and fetal body weights, or for fetal sex ratio.

 

With one exception the external, soft tissue and skeletal examinations of the fetuses revealed no differences between the controls and the test substance-treated groups, which might be related to the test substance. Number and type of fetal external, soft tissue and skeletal findings, which were classified as malformations and/or variations, did not show any differences of toxicological relevance between the groups.

 

The exception are five cases of malrotated limbs which were clustered in one litter of the high-dose group. These findings were accompanied by pale discolored placentae in some offspring of this litter. Malrotation of the limbs is occasionally also observed in untreated control fetuses. Generally it is seen as a subtle inward rotation of the limb and in many cases is a result of reduced amnion levels and/or compression of the uterus caused by maternal toxicity, combined with some influence of positioning of the foetus in the uterus. This doe was particularly affected by maternal toxicity. It constantly and markedly reduced its food consumption from GD 18 (61% of the average in the high-dose group) onwards until it almost stopped eating from GD 27 onwards. Accordingly, this doe lost 766.5 g (net) body weight during the treatment period while the average (net) weight loss in the high-dose group was 192.9 g. As proven by skeletal examination the limb malrotations were not caused by any abnormalities of the underlying skeleton. Considering all this, there is sufficient evidence that these fetal findings are a direct consequence of the severe maternal toxicity. No similar or less severe findings of related nature were noted in any doe of the high-dose group less affected by maternal toxicity.

Altogether there is no evidence for selective developmental toxicity of the test substance. The test substance is not teratogenic in rabbits at the tested dose levels.

 

Under the conditions of this prenatal developmental toxicity study, the oral administration of citral to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of maternal toxicity at the high dose of 200 mg/kg bw/d, such as reduced food consumption, distinct body weight loss, doe mortality, and abortion in the most sensitive individuals. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 60 mg/kg bw/d.

Adverse fetal findings at 200 mg/kg bw/d such as mortality or limb malrotations noted in one individual litter, respectively, were a direct consequence of the maternal toxicity. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg bw/d.

There is no evidence for selective developmental toxicity of citral. The test substance is not teratogenic in rabbits at the tested dose levels.

 

Developmental toxicity (rat)

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 weights) 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, which indicates 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 onward, 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 the guideline developmental toxicity study in rabbits (see above) and the reproduction screening study in rats (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, 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.

 

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, fetal viability, litter size, and sex ratio were not adversely affected at any dose level. Although, there was a slight increase in pre-implantation loss in the test substance exposed groups, no biological relevance can be attributed to this finding since no significant 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, which are 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. Overall, 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 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.