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Key value for chemical safety assessment

Effects on fertility

Description of key information

Extended One Generation Reproductive Toxicity Study, EOGRTS (OECD Guideline 443, GLP), rat, gavage (BASF 2021; BASF 2021; 90R0410/07R076):

NOAEL (general, systemic toxicity): 25 mg/kg bw/d (based on pathological findings in the gastrointestinal tract at the LOAEL of 80 mg/kg bw/d)

NOAEL (fertility and reproductive performance): 250 mg/kg bw/d (highest tested dose)

NOAEL (developmental toxicity F1/F2): 250 mg/kg bw/d (highest tested dose)

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

Key study on reproductive toxicity

In the key study for reproductive toxicity, i.e. an Extended One Generation Reproductive Toxicity Study (EOGRTS) according to OECD 443 and GLP, Citral N was administered to groups of 25 male and 25 female healthy young Wistar rats asan aqueous preparation by stomach tube at dosages of 0, 25, 80 and 250 mg/kg body weight/day(BASF 2021; 90R0410/07R076). F0 animals were treated at least for 2 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations. Cohort 1B (=F1 generation parental animals) were selected to produce F2 pups. F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were offered an aqueous preparation by stomach tube at different dosages (0, 25, 80 and 250 mg/kg bw/d) of the test substance post weaning, and the breeding program was repeated to produce a F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 parental animals. Control animals were dosed daily with the vehicle(0.5% Sodium carboxymethyl cellulose (CMC) suspension in drinking water (with 5 mg/ 100mL Tween 80).

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Water, food consumption and body weights of the F0 and F1 parents and F1 rearing animals was determined regularly and a detailed clinical observation (DCO) was performed in all F0 and F1 parents and F1 animals in cohorts 1A.

Estrous cycle data were evaluated for F0 and F1B parental females. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically. Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 parental and rearing animals was recorded.

All surviving F1 and F2 pups were examined for the presence or absence of nipple/areola anlagen on PND 13. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one PND 20. The number of nipple/areola anlagen were counted. Anogenital distance measurements were conducted on all live F1 and F2 male and female pups on PND 1.

Urine and blood samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Further blood samples were taken from a maximun of 10 surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group.

Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males and cohort 1A males at scheduled sacrifice or after appropriate staining.

All F0 and F1 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females cohort 1A. All F1 rearing animals were assessed by different pathological and histopathological examinations.

No test substance-related mortalities or adverse clinical observations were observed in any of the groups, indicating systemic toxicity. In particular, detailed clinical observations revealed no test substance-related adverse systemic effects.

Transient salivation during a short time-period after gavage dosing was noted for nearly all high-dose and some mid-dose male and female animals (F0 and F1 animals across all cohorts) during all sections of the study. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is not considered to be an adverse finding of systemic toxicity but may, however, had subsequent consequences, namely increases of food and water consumption. In the high-dose group (F0 and F1B animals) intermittent increases of food and water consumption were noted during all study segments. Similar changes, though to a much lesser extent, were also observed in the mid-dose group. Concurrent with food and water consumption the mean body weights and body weight gain of the high-dose group (F0 and F1B animals in particular) showed intermittent increases during several study segments. All these food/water consumption and body weight increases in the high-dose group were rather mild, in the lower dose groups they were even limited to short episodes in individual animals. Supposedly, the attempt of the animals to attenuate an unpleasant taste and/or smell after gavage dosing of the test material led to those phases of increased food/water consumption and their consequences. However, as an increase of food/water consumption was observed, the described changes were neither considered as adverse findings nor as signs of systemic toxicity.

Concerning clinical pathology, no treatment-related, adverse effects were observed in the parental F0 generation as well as in the offspring F1A rats up to a dose of the compound of 250 mg/kg bw/d.

Regarding pathology, the forestomach was the target organ in the F0 generation parental animals. The macroscopically observed thickening of the forestomach wall in males and thickening of the margo plicatus in females corresponded to hyperplasia (diffuse or focal) with hyperkeratosis (diffuse or focal) by light microscopy. Single animals revealed erosion/ulcer in addition which - in combination with the other findings - represented an aggravation of lesions. Mid and high dose male and female animals (80 and 250 mg/kg bw/d) were affected, with increasing number and severity in the high dose group (250 mg/kg bw/d).

The absolute liver weight in mid and high dose males (80 and 250 mg/kg bw/d) and kidney weight in high dose males (250 mg/kg bw/d) were above historical control data. These weight changes were assumed to be treatment-related but not adverse for the following reasons: no histopathologic correlate, no findings in clinical pathology, relative liver and kidney weights were within historical control data.

Male and female F1 generation rearing animals of cohort 1A revealed hyperplasia (diffuse or focal) with hyperkeratosis (diffuse or focal) in the forestomach. Single animals revealed erosion/ulcer in addition which - in combination with the other findings - represented an aggravation of lesions. Mid and high dose male and female animals (80 and 250 mg/kg bw/d) were affected with increasing number and severity in the high dose group (250 mg/kg bw/d).

High dose F1 males (250 mg/kg bw/d) revealed a significant increase of absolute and relative weight of cauda epididymis, kidney and liver; high dose F1 females (250 mg/kg bw/d) revealed a significant increase in absolute and relative weight of the liver. The altered weights were above historical control values. Nevertheless, these findings were regarded as treatment-related but not as adverse, because neither in histopathology nor in clinical pathology findings were observed which could explain the increased organ weights.

All other findings in the investigated internal organs of the F0 and F1A animals occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and without any relation to treatment.

In the F1-generation cohort 1B males and females of the high dose group (250 mg/kg bw/d), the liver weights were significantly increased (males: absolute and relative, females: absolute only). In concordance with the discussion for the F0 and F1 cohort 1A animals these changes were regarded to be treatment-related but not as adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered incidental or spontaneous in origin and without any relation to treatment.

In the surplus F1-generation pups on PND 22 (F1 weanlings not selected for cohorts) neither treatment-related organ weight changes nor gross lesions were detected. Histopathology was not performed.

There were no indications from clinical examinations, that Citral N adversely affected the fertility or reproductive performance of the F0 and F1B parental animals up to and including the administered high-dose of 250 mg/kg bw/d. Estrous cycle data, sperm quality of males, mating behavior, conception, gestation, parturition, lactation and weaning were comparable between the rats of all groups including control and ranged within the historical control data of the test facility. The same is true for sexual organ weights and gross and histopathological findings of these organs in F0 and F1A females of all dose groups. Specifically, the results of the differential ovarian follicle count (DOFC) in F1A females – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – showed no significant differences between the control and the high dose group.

For all liveborn male and female pups of the F0 and F1B parents, no test substance-induced signs of developmental toxicity were noted at dose levels as high as 250 mg/kg bw/d. Postnatal survival, pup body weight gain as well as post-weaning development of the offspring until puberty remained unaffected by the test substance. Furthermore, clinical and/or gross necropsy examinations of the F1 and F2 pups revealed no adverse findings.

Measurement of thyroid hormones revealed no effect caused by the test substance, neither in the F0 parental animals nor in the liveborn F1 offspring.

Neither the anogenital distance/index nor the check for the presence of nipples/areolas, both very sensitive marker of potential endocrine-mediated imbalances, revealed any test substance-related effects.

Vaginal opening and preputial separation are commonly used developmental markers for onset of puberty in laboratory rats. No delays beyond a normal range of biological variation in rat (multi)generation studies which might be attributable to the treatment were noted in any of the test substance-treated groups.

Thus,under the conditions ofthe present extended one-generation reproduction toxicity studythe NOAEL (no observed adverse effect level) for general, systemic toxicity is 25 mg/kg bw/d, based on pathological findings in the gastrointestinal tract at the LOAEL of 80 mg/kg bw/d. The NOAEL for fertility and reproductive performance for the parental rats is250 mg/kg bw/d, the highest tested dose. The NOAEL for developmental toxicity in the F1 and F2 progeny is250 mg/kg bw/d, the highest tested dose.

Dose selection for the key study was based on a preceding range-finding study (i.e. a reproduction/ developmental screening study acc. to OECD 421; BASF project 80R0410/07R074), showing signs of toxicity at dose levels of 100, 300, 500 and 1000 mg/kg bw/d. In this study, Citral N was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). As severe signs of intoxication were observed at 1000 mg/kg bw/d (particularly females, details see below) during the first days of administration (premating phase), the dose was lowered to 500 mg/kg bw/d from premating day 4 until the end of the study. Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 5 mg/ 100mL Tween 80)). The duration of treatment covered about 4 weeks in-life period (males) including 2 weeks mating (mating pairs were from the same test group) as well as a 2 weeks premating period (females), 2 weeks mating period, the entire gestation, about 3 weeks of lactation period and about 5 weeks postmating period in females up to one day prior to the day of scheduled sacrifice of the animals.

The 1000 mg/kg bw/d dose caused mortality (3 females) and severe clinical signs of intoxication (i.e. unsteady gait, severely reduced muscle tonicity and prone-position with abducted limbs) immediately after the start of treatment. After the high-dose was lowered to 500 mg/kg bw/d (premating day 4), still adverse clinical observations (hypothermia, unsteady gait, apathy, abdominal position, salivation, increased water consumption, decreased food consumption) were recorded, along with slight anemia, as well as a macroscopically visible thickening of the forestomach at necropsy. Histopathological examination of the forestomach in the 1000/500 mg/kg bw/d animals revealed a full thickness necrosis of the forestomach epithelium in the 3 female decedents, focal and transmural inflammation in 9 males and all females (most likely caused by erosion/ulceration that was not always visible on the slide), erosion/ulcer in 6 males and 6 females, as well as hyperplasia of the squamous epithelium and hyperkeratosis in 10 males and 7 females.

The 300 mg/kg bw/d dose caused salivation and marginally decreased food consumption at the beginning of the study. Histopathological examination of the forestomach revealed inflammation in 8 males and 9 females, erosion/ulcer in 2 males and 2 females, hyperplasia of the squamous epithelium and hyperkeratosis in 10 males and 9 females, as well as focal degeneration of the squamous epithelium in1 female.

In the 100 mg/kg bw/d dose group histopathological examination of the forestomach revealed inflammation in 1 male, hyperplasia of the squamous epithelium and hyperkeratosis in 1 male, as well as diffuse degeneration of the squamous epithelium in1 male.

Thus, dose levels of 25, 80 and 250 mg/kg body weight/day were selected as dose levels for the present study. The high dose was intended to cause a similar adverse pathology as in the rangefinder while the low dose was intended to be a NOAEL.

 

Further 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 EOGRTS and the reproductive toxicity screening studies 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 of Citral as an endocrine disruptor.

Effects on developmental toxicity

Description of key information

Developmental toxicity study, rabbit, gavage, exposure GD 6-28 (OECD 414, GLP; BASF 2016; 40R0410/07R055):

NOAEL (maternal toxicity): 60 mg/kg bw/d

NOAEL (developmental toxicity): 60 mg/kg bw/d

 

Developmental toxicity study, rat, gavage, exposure GD 6-15 (similar to OECD 414; Nogueira 1995):

LOAEL (maternal toxicity): 60 mg/kg bw/d, no NOAEL identified

LOAEL (developmental toxicity): 60 mg/kg bw/d, no NOAEL identified

 

Developmental toxicity study, rat, inhalation, 6 h/day, exposure GD 6-20 (similar to OECD 414; Gaworski 1992):

NOAEC (maternal toxicity): 34 ppm (215 mg/m3)

NOAEC (developmental toxicity): 68 ppm (429 mg/m3)

 

Extended One Generation Reproductive Toxicity Study, EOGRTS (OECD Guideline 443, GLP), rat, gavage (BASF 2021; BASF 2021; 90R0410/07R076):

NOAEL (general, systemic toxicity): 25 mg/kg bw/d

NOAEL (fertility and reproductive performance): 250 mg/kg bw/d

NOAEL (developmental toxicity F1/F2): 250 mg/kg bw/d

Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
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 (BASF 2016; 40R0410/07R055).

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 these aspects, 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). Furthermore, in the Extended One Generation Reproduction Toxicity Study (EOGRTS), implantation was not affected and postimplantation losses were not observed in F0 and F1 generation treated animals up to 250 mg/kg bw/d Citral, as described in the Chapter “Effects to fertility”.

  

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 assays and the Extended One Generation Reproductive Toxicity Study (EOGRTS) in rats did not confirm ED related effects of Citral after exposure duration of 2 generation (see Chapter “Effects to fertility”).

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.

Additional information