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Effects on fertility

Description of key information

In a combined repeated dose toxicity and reproduction/developmental toxicity screening study according to OECD TG 422 and GLP, tetrahydro-2-isobutyl-4-methyl-2H-pyran was administered daily to groups of 10 male and 10 female Wistar rats by gavage at doses of 50, 150 and 500 mg/kg bw/d in corn oil (BASF 2018; 85R0080/13R165). The NOAEL for fertility and reproductive performance was 500 mg/kg bw/d tetrahydro-2-isobutyl-4-methyl-2H-pyran, i.e. the highest dose tested.

Additional information

In the chosen key study for the toxicity to reproduction; i.e. a combined repeated dose toxicity and reproduction/developmental toxicity screening study according to OECD TG 422 and GLP, tetrahydro-2-isobutyl-4-methyl-2H-pyran was administered daily to groups of 10 male and 10 female Wistar rats by gavage at doses of 50, 150 and 500 mg/kg bw/d in corn oil (BASF 2018; 85R0080/13R165).

The dose was selected based on signs of toxicity noted at a dose level of 1000 mg/kg bw/d in a previously conducted dose range-finding study in Wistar rats, which preceded this definitive OECD 422 study. At 1000 mg/kg bw/d, significantly increased water consumption (up to 30.5%), significantly reduced food consumption (up to 15.6%) and significantly reduced body weight gain (up to 43.5%) were noted in males, while females showed moderately increased water consumption (up to 11.8%), significantly reduced food consumption (up to 17.6%) and they significantly lost body weight (weight change -139.7%) during the course of the study. Also, a massive increase of liver weights was noted in both sexes (rel. 42%/48%) and kidney weights were moderately increased (rel. 14%/18%). In addition, both sexes frequently exhibited salivation, piloerection and unsteady gait. In this range-finding study a dose level of 300 mg/kg bw/d produced similar effects on food consumption, body weight gain and liver weight, but less severe.

In the main study, the duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), 7 days postmating in males as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear.

A detailed clinical observation, food consumption and body weights of the F0 parents was determined. Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females. Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed, their viability was recorded and at necropsy on PND 13, all pups were examined macroscopically for external and visceral findings. Anogenital distance measurements were conducted on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13.

Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4).

The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature and correct concentrations of the test substance preparations were verified.

No adverse findings related to tetrahydro-2-isobutyl-4-methyl-2H-pyran were observed concerning clinical examinations, clincal-/pathology and reproductive performance in the F0 parental animals and concerning clinical examinations and gross findings in the F1 pups at any dose level tested.

In the clinical examinations of the F0 parental animals, signs of a bad taste of tetrahydro-2-isobutyl-4-methyl-2H-pyran and/or local affection of the upper respiratory and/or digestive tract such as temporary salivation and animal “ploughed nose first into bedding” were noted in a dose-dependent fashion. As they were transient and appeared only shortly after dosing they were not considered as signs of systemic toxicity.

Observed organ weight changes comprised of decreased mean thymus weights in males and females of all treated groups, increased mean liver and thyroid gland weights in males and females treated with 500 mg/kg bw/d, and increased mean adrenal gland weights in females of the 500 mg/kg bw/d treated group. Because the weight changes of the thymus, thyroid glands and adrenal glands were not supported by relevant histopathologic changes they were considered to be of negligible toxicologic relevance. The increase in mean liver weights in males and females of the 500 mg/kg bw/d treated group was considered to be related to minimal centrilobular hepatocellular hypertrophy, encountered microscopically. This hepatocellular hypertrophy is believed to represent the morphologic hallmark of a low level of enzyme induction and is as such considered an adaptive non-adverse response.

Regarding fertility and reproductive performance, as well as pre-postnatal development no signs of toxicity were observed in male or female parental animals or their offspring of all test groups during the entire study. F0 parental animals across all dose groups proved to be fertile and those control individuals failing to generate offspring did not show any specific gross or histopathological findings. Mating behavior, conception, implantation and parturition were not influenced.

A small decrease in pup body weights of the high-dose group was observed, but not noteworthy enough to be considered adverse, as it showed a tendency to recover during the course of lactation until PND 13. Neither a test substance-related influence on body weight change nor any corroborative effects on pup well-being or survival were noted. The determination of anogenital distance/index and the count of nipple/areola anlagen revealed no treatment-related changes up to and including a dose of 500 mg/kg bw/d tetrahydro-2-isobutyl-4-methyl-2H-pyran. In male and female pups at PND13, no treatment-related alterations of T4 levels were observed.

Accordingly, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity in the offspring was 500 mg/kg bw/d, the highest dose tested.

In a supporting screening study on testes toxicity in male Wistar rats, initial information on the effect of tetrahydro-2-isobutyl-4-methyl-2H-pyran on male sexual organs after repeated oral administration was investigated (BASF 00R0804/11N025; see endpoint 7.5.1 "repeated dose toxicity: oral"). The test substance was administered once per day to groups of 5 Wistar rats via gavage at 1000 mg/kg bw/d in corn oil for a maximum of 14 days. During the study, all animals were observed for any clinically abnormal signs. One day after the last administration the animals were sacrificed and assessed by gross pathology, special attention was given to the reproductive organs for which a histopathological assessment and sperm analyses were performed. Besides salivation, no adverse effects on male reproductive organs and no signs of general systemic toxicity were observed.

Effects on developmental toxicity

Description of key information

In a combined repeated dose toxicity and reproduction/developmental toxicity screening study according to OECD TG 422 and GLP, tetrahydro-2-isobutyl-4-methyl-2H-pyran was administered daily to groups of 10 male and 10 female Wistar rats by gavage at doses of 50, 150 and 500 mg/kg bw/d in corn oil (BASF 2018; 85R0080/13R165). The NOAEL for developmental toxicity was 500 mg/kg bw/d tetrahydro-2-isobutyl-4-methyl-2H-pyran, i.e. the highest dose tested.

Additional information

In the chosen key study for the toxicity to reproduction; i.e. a combined repeated dose toxicity and reproduction/developmental toxicity screening study according to OECD TG 422 and GLP, tetrahydro-2-isobutyl-4-methyl-2H-pyran was administered daily to groups of 10 male and 10 female Wistar rats by gavage at doses of 50, 150 and 500 mg/kg bw/d in corn oil (BASF 2018; 85R0080/13R165).

The dose was selected based on signs of toxicity noted at a dose level of 1000 mg/kg bw/d in a previously conducted dose range-finding study in Wistar rats, which preceded this definitive OECD 422 study. At 1000 mg/kg bw/d, significantly increased water consumption (up to 30.5%), significantly reduced food consumption (up to 15.6%) and significantly reduced body weight gain (up to 43.5%) were noted in males, while females showed moderately increased water consumption (up to 11.8%), significantly reduced food consumption (up to 17.6%) and they significantly lost body weight (weight change -139.7%) during the course of the study. Also, a massive increase of liver weights was noted in both sexes (rel. 42%/48%) and kidney weights were moderately increased (rel. 14%/18%). In addition, both sexes frequently exhibited salivation, piloerection and unsteady gait. In this range-finding study a dose level of 300 mg/kg bw/d produced similar effects on food consumption, body weight gain and liver weight, but less severe.

In the main study, the duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), 7 days postmating in males as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear.

A detailed clinical observation, food consumption and body weights of the F0 parents was determined. Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females. Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed, their viability was recorded and at necropsy on PND 13, all pups were examined macroscopically for external and visceral findings. Anogenital distance measurements were conducted on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13.

Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4).

The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature and correct concentrations of the test substance preparations were verified.

No adverse findings related to tetrahydro-2-isobutyl-4-methyl-2H-pyran were observed concerning clinical examinations, clincal-/pathology and reproductive performance in the F0 parental animals and concerning clinical examinations and gross findings in the F1 pups at any dose level tested.

In the clinical examinations of the F0 parental animals, signs of a bad taste of tetrahydro-2-isobutyl-4-methyl-2H-pyran and/or local affection of the upper respiratory and/or digestive tract such as temporary salivation and animal “ploughed nose first into bedding” were noted in a dose-dependent fashion. As they were transient and appeared only shortly after dosing they were not considered as signs of systemic toxicity.

Observed organ weight changes comprised of decreased mean thymus weights in males and females of all treated groups, increased mean liver and thyroid gland weights in males and females treated with 500 mg/kg bw/d, and increased mean adrenal gland weights in females of the 500 mg/kg bw/d treated group. Because the weight changes of the thymus, thyroid glands and adrenal glands were not supported by relevant histopathologic changes they were considered to be of negligible toxicologic relevance. The increase in mean liver weights in males and females of the 500 mg/kg bw/d treated group was considered to be related to minimal centrilobular hepatocellular hypertrophy, encountered microscopically. This hepatocellular hypertrophy is believed to represent the morphologic hallmark of a low level of enzyme induction and is as such considered an adaptive non-adverse response.

Regarding fertility and reproductive performance, as well as pre-postnatal development no signs of toxicity were observed in male or female parental animals or their offspring of all test groups during the entire study. F0 parental animals across all dose groups proved to be fertile and those control individuals failing to generate offspring did not show any specific gross or histopathological findings. Mating behavior, conception, implantation and parturition were not influenced.

A small decrease in pup body weights of the high-dose group was observed, but not noteworthy enough to be considered adverse, as it showed a tendency to recover during the course of lactation until PND 13. Neither a test substance-related influence on body weight change nor any corroborative effects on pup well-being or survival were noted. The determination of anogenital distance/index and the count of nipple/areola anlagen revealed no treatment-related changes up to and including a dose of 500 mg/kg bw/d tetrahydro-2-isobutyl-4-methyl-2H-pyran. In male and female pups at PND13, no treatment-related alterations of T4 levels were observed.

Accordingly, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity in the offspring was 500 mg/kg bw/d, the highest dose tested.

Toxicity to reproduction: other studies

Additional information

No data available.

Justification for classification or non-classification

The present data on reproductive toxicity do not fulfill the criteria laid down in regulation (EU) 1272/2008, and therefore, a non-classification is warranted.

Additional information