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Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Study duration:
subacute
Additional information

Studies specifically designed to assess the reproductive toxicity potential of tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran are currently not available.

However, reproductive organs and fertility parameters of adult rats in two repeated dose toxicity studies provide data to assess potential effects on male and female fertility (see also IUCLID chapter 7.5.1).

In a subacute study according to OECD TG 407 and GLP, tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran (Rose oxide 90) was administered in corn oil to Wistar rats by gavage (groups of 5 male and female rats) at concentrations of 0, 100, 300 and 1000 mg/kg bw/day over a period of 4 weeks (BASF 30C0624/07S043, 2012).

In mid and high dose males (300 and 1000 mg/kg bw/d), sperm function and morphology in the cauda epididymidis were adversely affected by the test substance. A significant increase of the epididymides weight (relative +26%, absolute +33%) of high dose males was observed. Histopathological assessment of the left epididymis revealed the presence of immature ducts in the distal corpus and/or cauda epididymis in all males of the two upper dose groups. Immature ducts increased in severity, ranging from minimal to slight (300 mg/kg bw/d) up to severe in high dose males (1000 mg/kg bw/d). With increasing severity, the immature ducts were accompanied by increasing interstitial edema, which correlated with the significant epididymis weight increase. In only two of five high dose males (1000 mg/kg bw/d), additional intraductal granulocytic infiltration was observed in single distended ducts of the cauda with apparent sperm stasis. All these findings were attributed to treatment and were regarded as adverse. No test substance-related effects on estrous cycle length and the number of cycles were obtained in treated females. No treatment related changes were observed in organ weight parameters and histopathology of other primary and secondary reproductive organs. No treatment-related effects on parameters addressing reproductive toxicity were seen in low dose males (100 mg/kg bw/d) and in females of all dose groups. Therefore, the no observed adverse effect level (NOAEL) for reproductive toxicity was considered to be 100 mg/kg bw/d for male and 1000 mg/kg bw/d for female Wistar rats.

In the key study for repeated dose toxicity, i.e. an oral subchronic toxicity study in rats according to OECD TG 408 and GLP, tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran, encapsulated in alginate microcapsules (termed as Rose oxide 90 (encapsulated)) was administered via the diet over a period of 90 days (BASF 50C0087/18S026). Rose oxide 90 (encapsulated) was administered at concentrations of 0 ppm (pure maintenance diet), 0 ppm (Placebo Alginate – without Rose oxide 90), 7500 ppm, 25000 ppm and 75000 ppm to groups of 10 male and 10 female Wistar rats over a period of 3 months. Based on a content of about 16% pure Rose oxide 90 in the alginate capsule material, the effective concentrations of Rose Oxide 90 (a.i.) were 0 ppm, 0 ppm, 1200 ppm, 4000 ppm and 12000 ppm, respectively.

In the sperm analysis performed, no treatment-related effects were observed concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis. Further, sperm head counts in the testis and in the cauda epididymidis were not affected. Organ weight analysis did show a significant increase in relative weights of testis (+9.2% vs. placebo ctrl.) and epididymis (+8.9% vs. placebo ctrl) in high dose males (12000 ppm a.i.), but these were not reflected in absolute organ weights, as the final body weights in this dose group was significantly decreased. No histopathological findings were observed in epididymides in any animal of the low, mid and high dose groups. Histopathology in testes revealed minimal tubular degeneration in 1/10 high dose and 1/10 placebo control group males. Overall, no relevant treatment related changes were observed in organ weight parameters and histopathology of all primary and secondary reproductive organs investigated. Estrous cycle data revealed regular cycles of all test groups including the controls. In all test substance treated male and female animals, no treatment related alterations of T3, T4 and TSH levels were observed after the administration period.

Considering the outcome of the subchronic toxicity study, the spermatotoxic and adverse effects on the epididymis (as observed in the subacute repeated dose toxicity study after administration of tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran via gavage for 28 days) could not be confirmed in the subchronic study after continuous administration of encapsulated tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran in feed over a longer period of test substance application (90 days). Further experimental reproductive toxicity studies are planned to further elucidate the potential of tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran for reproductive toxicity.

Effects on developmental toxicity

Additional information

In the key study for developmental toxicity (acc. to OECD 414 and GLP), tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran was administered as an oily preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 50, 250 and 800 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19 (BASF 2019; 30R0624/07R071). The control group was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 24-25 females per group had implantation sites. Water consumption, food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including sampling of thyroid glands (with parathyroid glands) and weight determinations of the kidneys, liver, spleen, thyroid glands (with parathyroid glands), unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

 

All females of the high- and mid-dose groups (800 and 250 mg/kg bw/d) and 8 females of the low-dose group (50 mg/kg bw/d) showed transient salivation during the treatment period. Furthermore, all females of the high- and mid-dose groups and 12 females of the low-dose group ploughed nose-first into bedding. Both findings occurred in most of the respective animals only within the 2-hour examination interval immediately after treatment, however in some mid- and high-dose dams it continued beyond 2 hours for a maximum of 5-hours. They are considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. They are not considered to be signs of systemic toxicity.

For 19/25 high-dose females (800 mg/kg bw/d) unsteady gait shortly after treatment (i.e. 0-2h) was recorded on GD 7-14, the highest number of animals being affected on GD 9 and 10. Furthermore, all females of the high-dose group had piloerection during the treatment period. Both findings are considered as treatment-related signs of systemic maternal toxicity.

Generally, clinical observations revealed no effects of treatment-related systemic maternal toxicity in the animals receiving 50 mg/kg bw/d of the test substance.

The mean food consumption of the high-dose dams (800 mg/kg bw/d) was decreased statistically significantly on GD 6-13 (up to -44% vs. ctrls.). Afterwards it recovered, but during the treatment period (GD 6-19), the high-dose dams consumed 14% less food in comparison to the concurrent control group. The high-dose of tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran consistently affected the gross and corrected (net) body weight gain (body weight loss GD6-8) of the dams, gaining overall about 16% (gross) or 33% (net) less weight than the concurrent control during the treatment period (GD 6-19). The carcass weight was about 4% below the concurrent control group. These effects were regarded to be treatment-related and adverse.

Regarding clinical pathology, in high dose dams (800 mg/kg bw/d) a regenerative hypochromic, macrocytic anemia was observed because of decreased red blood cell (RBC) counts and mean corpuscular hemoglobin concentration (MCHC) as well as increased absolute reticulocyte counts, mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH). Lower inorganic phosphate and calcium levels in these individuals was found, but the reason for these findings cannot be elucidated. In mid dose dams (250 mg/kg bw/d), RBC counts and MCHC were lower and MCV and MCH higher compared to controls. However, all values in this test group were within historical control ranges. Thus, these alterations are likely precursory to a more distinct anemia, but still adaptive changes which are not yet adverse.

In mid and high dose dams (250 and 800 mg/kg bw/d) decreased T3 and T4 values and increased TSH values indicated a hypothyroidism.

Histopathology revealed a minimal hypertrophy/hyperplasia of the follicular cells in 2/24 high-dose dams (800 mg/kg bw/d) in the thyroid glands. The minimal incidence and grading of these findings were assumed as possibly treatment-related and in combination with the results of hormonal measurements (increased TSH and decreased T3 and T4) as potentially adverse. However, no treatment related alterations of T3, T4 and TSH levels were observed in treated male and female rats after a 90 day continuous administration of tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran at higher doses via feed according to the key study for repeated dose toxicity (BASF 2019; 50C0087/18S026).

A metabolome analysis in the 4 week subacute toxicity study in rats (assessing a plasma profile with 272 endogenous metabolites) revealed matches for liver toxicity, paracetamol-induced toxicity, liver enzyme induction and indirect effects on the thyroid in female animals, when compared to the metabolite patterns for different toxicological modes of action available in the MetaMap®Tox database (BASF 97Z0624/07K080). Although a comparable database for pregnant animals has not been fully established yet, the data of the subacute study give indications for an induction of liver enzymes by treatment with tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran (Rose oxide 90) in females, which has the potential for an increased metabolism of thyroid hormones. Further examinations are planned to assess the role of liver enzyme induction in pregnant rats after treatment with tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran.

Significant absolute and relative weight increase of the liver in the mid and high dose animals (250 and 800 mg/kg bw/d) was consistent with an increased hepatocytic accumulation of, most likely, glycogen. Histopathologically, this accumulation was similarly present in both test groups and was regarded as treatment-related but not as adverse since neither signs of cytotoxicity nor changes in clinical chemistry parameters indicative of hepatic alteration were noted.

In high dose dams, the absolute and relative kidneys and spleen weights were significantly increased above the historical control ranges. In a previous study with a 28-day exposure to the same test substance, significant absolute and relative kidneys weight increases occurred in females at 300 and 1000 mg/kg bw/d without a histopathological correlate, whereas significant relative spleen weight increases at 1000 mg/kg bw/d were consistent with extramedullary hematopoiesis and hematological signs of regenerative anemia (BASF 2012; 30C0624/07S043). The data of the 28-day study and the absence of altered clinical chemical markers of kidney toxicity in the present study indicate that the kidney weight increases were treatment-related but not adverse. Although no histopathological investigation of the spleen was performed in the present study, the significant weight increases of the organ in the high dose group were assumed to be a treatment-related adaptive response, relating to the evident adverse hematological signs of regenerative anemia.

No differences of toxicological relevance between the control and the treated groups (50, 250 or 800 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and post-implantation loss. Similarly, no toxicologically relevant influence of tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran on sex distribution and anogenital distance/index of the fetuses was noted at any dose.

The mean fetal weights of the mid- and high-dose groups (250 and 800 mg/kg bw/d) were statistically significantly reduced (about 5% and 11% below control). This correlated with mild delays in ossification of a few skeletal elements (skull, sternebrae, sacral arch). These delays of ossification do not constitute structural alterations of the skeleton, as the underlying cartilage template was completely intact in all these cases. Further, the delayed ossification noted in the mid- and high-dose group was associated with beginning (250 mg/kg bw/d) or distinct (800 mg/kg bw/d) maternal toxicity. As can be seen from the historical background data, increased incidences of such incomplete or non-ossifications of skeletal elements are routinely quantified and are among the most frequently noted skeletal variants in control populations of this Crl:WI(Han) rat strain. This indicates that these findings reflect species-specific anatomic variation at the time around birth without any detrimental effects on further development. Thus, their toxicological relevance is considered to be rather low.

Under the conditions of this study tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran is not teratogenic.

 

In conclusion, the oral administration of tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of distinct maternal toxicity (such as adverse clinical findings, reductions of food consumption and body weight/body weight gain, anemia, thyroid perturbation at 800 mg/kg bw/d) under the conditions of this prenatal developmental toxicity study. Beginning maternal toxicity was detectable already at 250 mg/kg bw/d where clinical findings, precursory signs of anemia and thyroid perturbation were noted. Each of these effects was not strong enough to constitute adverse events when looked at them individually, but as a whole provide evidence for disturbance of maternal homeostasis. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg bw/d.

Further, tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran caused a slight delay of embryofetal development (lower fetal weights, mild delays in ossification) at dose levels of 250 and 800 mg/kg bw/d. These dose levels also caused maternal toxicity. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 50 mg/kg bw/d. However, tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran did not show any teratogenic potential under the conditions of this study.

Toxicity to reproduction: other studies

Additional information

No data available

Justification for classification or non-classification

The present data on reproductive toxicity are sufficient to fulfill the criteria laid down in regulation (EU) 1272/2008 and a classification as Repr. Cat3; R62 (67/548/EEC) and reproductive toxicant; Cat2f (regulation (EU) 1272/2008) is warranted.