Tetrahydro-4-methyl-2H-pyran

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-20 to 207-06-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(2016)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Justification for the selection of the test system
This study provides information concerning the effects of a test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This screening test gives information at an early stage of assessing the toxicological properties of chemicals, or on chemicals of concern.

The test item was administered orally via gavage, daily, i.e. 7 days per week in graduated doses to several groups of test animals (male and female), one dose level per group with a maximum exposure of 63 days in total in females (at least 14 days pre-mating, up to 14 days mating, approximately 22 days of gestation and up to post-natal day 12).
Males were dosed for a minimum of four weeks until up to one day before the scheduled sacrifice (this includes a minimum of two weeks prior to mating, during the mating period and approximately two weeks post-mating).

During the administration period the animals were observed closely each day for signs of toxicity. An animal which died during the test period was necropsied and, at the conclusion of the test, surviving animals were sacrificed and necropsied.

- Justification for the selection of dose levels
Based on information supplied by the sponsor, 4-methyloxane (CAS no. 4717-96-8) was dosed orally via gavage (suspended in 0.1% Tween 80 + 1% CMC) in a previous study at dose levels of 0, 30, 120 and 500 mg/kg bw/day for 28 days to Sprague-Dawley rats. An additional 2 week withdrawal period was included after the administration to examine reversibility of any toxicological changes. The maximum dose selected was based on preliminary study where 3 animals/sex/group were dosed at 15, 60, 250 and 1000 mg/kg bw/day for 14 days. At 1000 mg/kg bw/day decreased body weight, anesthetic effects in males and emaciation immediately before death and anesthetic effects in females were observed. Therefore 1000 mg/kg bw/day was deemed to exceed the maximum tolerated dose (MTD), with 500 mg/kg bw/day set as the MTD.

Treatment related effects included decreased motor activity at week 4, early onset during the recording period in high dose group males. No effects were observed following withdrawal of administration. Functional examinations and grip strength were unaffected in all treatment groups.
Increased water intake accompanied by low urine osmolality values were recorded in high dose females. These effects were no longer present following withdrawal of the test article.

Haematological parameters were unaffected at the end of treatment. However, high dose group males and females in the withdrawal group showed a significantly lower mean haemoglobin and white blood cell count, respectively.

Blood clinical chemistry, food consumption and body weight parameters remained unaffected.

Increased relative liver weights were observed in high dose group males (+14%), with accompanying histological examination confirming centrilobullar hypertrophy of hepatocytes in both high dose group males and females. Relative kidney weights were increased in high dose group males (+12%), without any accompanying histopathology. These changes were no longer observed after withdrawal of administration.

The NOEL was deemed to be 120 mg/kg bw/day for both males and females following dosing via oral gavage over 28 days. The NOEL was based on increased relative liver weights with accompanying histopathology (centrilobullar hypertrophy) in both males and females, with increased relative kidney weight (without any adverse histopathology) in males only. The effects observed during the administration period were all reversible.

- Route of administration
The route of administration was orally, via gavage

- Justification for the selection of the test method
No valid in vitro method is available for screening the reproductive / developmental toxicity.

Specific details on test material used for the study:

- Test Material: 4-methyltetrahydropyran (MTHP)
- Lot/Batch No.: MTHP-72715
- Purity: 99.9%
- CAS No.: 4717-96-8
- Stability of test compound: Confirmed stable for the duration of the study.
Concentration analysis of formulation samples were determined at three concentrations, 3 mg/mL, 12 mg/mL and 50 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The mean recoveries observed for the LD dose group was between 75.9% and 100.6% of the nominal value, between 84.9% and 99.3% for the MD dose group and between 90.0% and 101.6% of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 93.7%, 93.1%, and 96.2% of the nominal concentration, respectively.
- Storage condition of test material: Room temperature, protected from light
- Vehicle and/or positive control: 0.1% (v/v) Tween 80-supplemented 1% (w/v) carboxymethylcellulose sodium solution (CMC, [0.1% Tween 80 + 1% CMC]) / none

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Test animals:
Species: Rat
Strain: Crl: WI(Han)
Age: 14-15 wks
Weight at dosing: M: 330-420g; F: 227-299g
Source: Charles River, France

Sex:

male/female

Details on test animals or test system and environmental conditions:

Acclimation period: 8 days
Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
Water: Municipal tap water, ad libitum
Housing: Groups of 2 animals / sex / cage during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to their original cage

Route of administration:

oral: gavage

Vehicle:

other: 0.1% Tween 80 + 1% CMC

Details on exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day (PND) 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Animals were dosed orally via gavage, employing a dose volume of 10 mL/kg bw.

Details on mating procedure:

- Mating:
Mating was performed using a ratio of 1:1 (male to female). Vaginal smears were checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Dose Formulation Analysis
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the chosen vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich study no. 171449). Further stability testing was performed during the treatment period due to change of procedure (use of glass ware). Prestart homogeneity investigation includes samples collected from various levels (top, middle and bottom) of a high dose and low dose formulation preparation.

As the test item was not shown to be homogenous after 60 minutes without stirring, during the study samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups. Based on low recovery of measured study samples from week 1 beyond acceptance criteria of 15%, new formulations were prepared and measured again in week 1.

Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data. The B-samples were retained at -15 to -35°C at BSL Munich (test facility) and discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day (PND) 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

Once daily, orally via gavage

Details on study schedule:

Refer above

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

120 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/gp

Control animals:

yes, concurrent vehicle

Details on study design:

- Animal assignment:
Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software). After randomisation before the first administration, due to irregularity of estrous cycles one already randomised female was manually replaced by a regularly cycling female taking its body weight into account.

- Dose selection rationale:
Refer above to Justification for study design

Positive control:

not applicable

Parental animals: Observations and examinations:

Once daily.

Clinical observations included check of spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Any changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded if present.

Oestrous cyclicity (parental animals):

Oestrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Testes, epididymides, prostate, seminal vesicles and coagulating gland organ (wet) weights were undertaken.

Litter observations:

The duration of gestation was recorded and calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded. The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Postmortem examinations (parental animals):

All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Special attention was paid to the organs of the reproductive system. The following tissues from all male and female animals were preserved in 4% neutral-buffered formaldehyde except for testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol:
- Preserved and examined tissues: all gross lesions, epididymides, ovaries, prostate and seminal vesicles with coagulating glands as a whole, testes, uterus with cervix, vagina, thyroid/parathyroid glands

- Intercurrent deaths: organs preserved and examined: adrenal glands, all gross lesions, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum
colon, duodenum, epididymides, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary), ovaries, oviducts, prostate and seminal vesicles with coagulating glands as a whole, rectum, spleen, stomach, testes, thymus, thyroid/parathyroid glands, trachea, urinary bladder, uterus with cervix, vagina

Organ weights: epididymides, testes, ovaries, uterus with cervix, prostate, seminal vesicles and coagulating gland, thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females), was weighed after fixation

Postmortem examinations (offspring):

Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Reproductive indices:

- Oestrus cycle: Vaginal smears were taken prior to mating.
- Mating index: Copulation was confirmed by sperm in vaginal smears. Mating indices were calculated for each sex of animals.
- Fertility index: (number of pregnant females/number of copulated females x 100) pregnancy was confirmed by the occurrence of parturition or by the presence of implantation sites in the uterine horns.
- Conception index: (no. of animals copulated/no. of pairs x 100) determined by the occurrence of pregnant females vs. the number of females mated.
- Gestation index: parturitions resulting in birth of one or more live pups were considered to be normal.
- Duration of gestation: represented as the days from detection of copulation to completion of parturition.
- Number of implantation sites: the number of implantation sites in the uterus was counted for each female.

Offspring viability indices:

Refer to litter observations above

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Remarkable clinical signs related to the treatment with the test item were observed primarily in the high dose group. Predominant clinical signs were slightly to severely reduced spontaneous activity and apathy. Reduced spontaneous activity was observed in all males and all females of the high dose group. Males were affected during the whole treatment period of 28 days whereas females were affected during the whole premating period but only transiently during mating and gestation period and not during lactation period. Additionally, apathy was transiently noticed in one male of the high dose group and three females of the high dose group.

In the mid dose group, three males were seen with reduced spontaneous activity on one single day of treatment (premating day 2) and one male on 7 days of treatment (premating day 2 to 8). Females of the mid dose group were not affected.

Moving the bedding was observed transiently in all males and all females of the high dose group on most days of treatment and in 4/10 females of the mid dose group on single days of treatment. Furthermore, salivation was noted transiently in all males and 9/10 females of the high dose group, in 1/10 females of the mid dose group and in 1/10 males and 1/10 females of the low dose group. Moving the bedding and salivation were observed in short timely relation to dose application and thus were considered to be a sign of discomfort and/or a local reaction to the test item. These slight signs were not considered as adverse systemic effects.

One female of the mid dose group was seen regurgitating test item formulation directly after dosing and showed abnormal breathing afterwards but recovered the day thereafter, surviving to the scheduled sacrifice.

None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

n/a

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female no. 77 of the high dose group was found dead directly after dose administration on premating day 4. All remaining animals survived their scheduled study period.

It is considered that the animal died due to a technical gavage error. Clinical signs reported immediately after dosing on pre-matig day (PMD) 4 (fell dead immediately after dosing, with blood on gavage tube) and macroscopic findings seen at necropsy (clear fluid in the thoracic cavity and discoloured dark lung) support the conclusion of a dosing error. In addition, this is supported by the histopathological evaluation.

Description (incidence and severity):

In males and females, the mean body weight increased in the control, the low and mid dose groups during the study.

After initiation of treatment, males of the high dose group showed a moderate and statistically significant loss of body weight (mean body weight change -7.10g compared to +4.19g in controls). Although body weight increased subsequently, mean body weight gain of the male high dose group was moderately and statistically significantly lower when related to the whole treatment period (47% of controls). However, there was no statistically significant effect on mean body weight in the high dose group. Mean body weight was only slightly (up to 6%) below controls within the study period.

For females, a marginal and not statistically significant loss of body weight occurred in the first week of treatment in the high dose group (-1.11g compared to +0.60g in controls). Body weight increased subsequently and without statistically significant difference to controls. In the third week of gestation a slightly but statistically significantly higher body weight gain was observed in the low dose group (133% of controls). This transient, statistically significant difference to controls without dose-dependency was considered as incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In males and females, in correlation to the body weight change, mean food consumption per animal in the high dose group was marginally but not statistically significantly lower in the first week of the premating period when compared to controls (males 86% of controls, females 83% of controls). There was no biologically or statistically significant difference to controls during the remaining study period or in the male and female LD and MD groups.

Food efficiency:

not examined

Description (incidence and severity):

n/a

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

n/a

Ophthalmological findings:

not examined

Description (incidence and severity):

n/a

Haematological findings:

not examined

Description (incidence and severity):

n/a

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no statistically or biologically significant differences for measured thyroxine (T4) serum hormone levels of adult males and pups on post natal day (PND)13 between the dose groups and controls. This was also reflected in thyroid/parathyroid gland mena weights in pups on PND 13, where no changes were observed.

Urinalysis findings:

not examined

Description (incidence and severity):

n/a

Behaviour (functional findings):

not examined

Description (incidence and severity):

n/a

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

n/a

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Under the conditions of this study, one female at 500 mg/kg bw/day died during the course of the study. It is deemed that this animal died due to a gavage error.

There were no gross lesions or histological findings that could be attributed to treatment with the test item. Sperm staging did not reveal any indication of toxicity.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

n/a

Other effects:

not examined

Description (incidence and severity):

n/a

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The test item had no biologically significant effect on the estrous cyclicity analyzed during the 2 week premating period after the first administration

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The test item had no biologically significant effect on the estrous cyclicity analyzed during the 2 weeks premating period after the first administration. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group. There were no statistically significant differences for the number of abnormal and normal cycles in the dose groups compared to the control group.

Reproductive performance:

no effects observed

Description (incidence and severity):

Length of the pre-coital interval and mean duration of gestation were within the normal range of variation for animals of this age and strain in the dose groups and the control group. There were no toxicologically relevant differences. One single female of the high dose group (no. 73) was seen with a slightly prolonged gestation length of 24 days which was considered as biological variation without relation to the treatment with the test item.

The copulation index (no. of animals copulated/no. of pairs x 100) was 100% in all groups including control.

The fertility index (number of pregnant females/number of copulated females x 100) was 90% in the low dose group and each 100% in the mid and high dose group when compared to 90% in the control group and therefore the reduced fertility index in the low dose group was considered to be incidental in nature and not treatment related.

Histologically there was no morphological reason for the non-mating of female no. 49 of the control group and no. 54 of the low dosegroup with their male pairing partners (no. 9 and 14, respectively). Therefore, the slightly reduced fertility index in the control and the low dose group was considered as incidental in nature.

The delivery index (no. of dams with live pups born / no. of pregnant dams x 100) was 100% in the dose groups but moderately reduced in the control group (77.78%). This was related to two dams of the control group (no. 41 and no. 42) which had implantation sites at necropsy but were not seen littering which was considered as incidental.

Dose descriptor:

NOAEL

Remarks:

(parental)

Effect level:

ca. 120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

(reproductive)

Effect level:

ca. 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects up to the maxiumum tolerated dose

Critical effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no remarkable, toxicological relevant external abnormalities.

One pup of the low dose group (dam no. 52) which was missing on PND 1 was noted as small and without showing indication of suckling on the day of its birth. One pup of the mid dose group (dam no. 62) which was found dead on PND 1 showed no preceding external findings.

One single pup of the mid dose group (dam no. 62) was seen cold and pale and with a flat abdomen and limp lower body part on the day of birth but it recovered and survived until scheduled sacrifice.

Two pups of the high dose group (dam no. 71 and dam no. 78) showed no indication of suckling on the day of birth but no further findings on the following days. Furthermore, one pup of dam no. 75 of the HD group showed a discolored (red) head on PND 0.

These single transient findings in few pups were not considered as toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

n/a

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

All pups of the high dose group and the control group survived from PND 0 to PND 4.

One single pup of the low dose group (dam no. 52) was missing on PND 1 which was attributed to cannibalism by the dam. One single pup of the mid dose group (dam no. 62) was found dead on PND 1. This led to a marginally increased mortality of 1.01% in the low dose group and 0.74% in the mid dose group when compared to 0.00% in the control group on PND 1 but without achieving statistical significance. Without following a dose-dependent pattern these single deaths of pups were considered as biological variation without relation to the treatment with the test item.

All live pups on PND 4 in the dose groups and the control group survived until terminal sacrifice on PND 13.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects of toxicological relevance on litter weight data.

Mean weight of pups and total litter weight on PND 0, PND 4 and PND 13 of the dose groups and control group was in the normal range of variation without dose-dependent differences.

However, marginally but statistically significantly lower mean weight of male pups on PND 0 was noted in the mid dose group (6.28g) when compared to the respective controls (6.78g) (93% of controls) and also of female pups in the MD (6.04 g) and the LD group (6.04 g) when compared to the respective controls (each 93% of controls). Without following a dose-dependent pattern this was not considered test item-related.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

n/a

Food efficiency:

not examined

Description (incidence and severity):

n/a

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

n/a

Ophthalmological findings:

not examined

Description (incidence and severity):

n/a

Haematological findings:

not examined

Description (incidence and severity):

n/a

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no statistically or biologically significant differences for measured thyroxine (T4) serum hormone levels in pups on PND 13 between the dose groups and controls.

Urinalysis findings:

not examined

Description (incidence and severity):

n/a

Sexual maturation:

not examined

Description (incidence and severity):

n/a

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There was no test item-related effect on mean weight of thyroid/parathyroid glands of pups on PND 13.

Gross pathological findings:

not examined

Histopathological findings:

not examined

Description (incidence and severity):

n/a

Other effects:

not examined

Description (incidence and severity):

n/a

Behaviour (functional findings):

not examined

Description (incidence and severity):

n/a

Developmental immunotoxicity:

not examined

Description (incidence and severity):

n/a

- Anogenital Distance and Nipple Retention
The mean absolute and relative anogenital distance of male pups was marginally but statistically significantly higher in the high dose group (2.68 mm and 1.42, respectively) when compared to the control group (2.47 mm and 1.30, respectively) (109% of controls). Statistical significance was also achieved for the relative (to cube root of pup weight) anogenital distance of male pups in the mid dose group. Mean anogenital distance of male pups of the mid and low dose groups were comparable to controls. No dose response was therefore seen for absolute and relative anogenital distance. Mid dose group absolute value was lower when compared to control and low dose group, the mean relative mid dose group value was also lower when compared to the low dose group. Additionally, no dose response was found for mean absolute and relative values of female pups. Mean absolute and relative anogenital distance and cube root of pup weight of the both control groups and all dose groups were in the normal range of variation. The effects observed in males are therefore concluded to be incidental and not test article related.

No statistically or biologically significant effects were noted on anogenital distance of female pups in the high dose group. However, marginally but statistically significantly lower absolute mean value was noted in the mid dose group (0.83 mm) when compared to the control group (0.94 mm) (88% of controls) but achieved no statistical significance when related to pup weight. Furthermore, the relative but not absolute anogenital distance of female pups of the low dose group was marginally but statistically significantly higher (115% of controls). These marginal differences in female pups to controls without dose-dependency were considered as incidental.

No effect of toxicological relevance was observed for nipple retention in male pups

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on no adverse effects when tested up to the MTD for the paternal generation

Critical effects observed:

no

Reproductive effects observed:

no

Table CA 7.8.1/01-1:
Overview of F0 parental generation treated orally via gavage with MTHP: bodyweight and food consumption

Parameter		M (mg/kg bw/d)				F (mg/kg bw/d)
	Day	0	30	120	500	0	30	120	500
Body weight (g)
Pre mating	1 7 14	389.5 394.4 402.3	380.7 386.8 392.1	380.9 371.8 380.3	378.9 371.8 380.3	256.6 257.2 257.3	259.1 260.2 262.4	264.2 266.7 269.3	260.0 258.8 263.8
Mating and post mating	7 14	407.1 414.1	398.9 400.9	395.7 396.5	385.5 390.5	-
Gestation	0 7 14 20	-				257.1 280.6 305.7 359.8	261.1 282.7 311.2 383.3	270.0 289.8 316.4 386.0	262.0 285.6 308.7 372.4
Lactation	0 4 13					286.6 305.0 321.3	289.7 313.9 323.7	304.6 315.0 327.4	295.6 310.4 319.6
Terminal bwt (g)		414.2	402.4	398.1	394.1	318.6	323.3	327.7	318.9
Bwt gain (g)
Pre-mating 1 – 7		4.90	6.10	2.90	-7.10***	0.60	1.10	2.50	-1.11
Pre-mating day 1 – 14		7.90	5.30	5.70	8.50	0.10	2.20	2.60	5.00
Gestation day 0 – 20		-				102.7	122.2	116.0	110.4
Lactation day 0 - 4						18.4	24.2	10.4	14.9
Lactation day 4 - 13						16.3	9.8	12.4	9.1
Food consumption (g/animal)
Day 1 – 7 pre-mating		124.9	120.4	117.0	108.0	90.30-	90.8	92.6	75.0*
Day 7 - 14 pre-mating		143.8	139.2	137.1	133.7	109.5	109.3	109.7	100.8
Gestation day 0 – 7		-				134.2	126.4	130.9	122.0
Gestation day 7 - 14						146.4	148.3	127.6	144.3
Gestation day 14 - 20						140.6	141.2	137.4	135.8
Lactation day 0 - 4						124.7	119.3	114.7	129.8
Lactation day 4 - 9						201.9	160.2	207.7	232.8
Lactation day 9 - 13						189.3	208.2	206.8	196.3
* p=0.05; **p=0.01

Table CA 7.8.1/01-2:
Overview of F0 parental generation treated orally via gavage with MTHP: reproductive performance

Parameter	F (mg/kg bw/d)
	0	30	120	500
Pairs mated	10	10	10	10
Oestrous cycle (mean, days)	4.00	4.00	4.00	4.26
Pre-coital interval (days)	1.70	2.40	2.56	2.78
Evidence of copulation (N)	10	10	10	9
Females achieving pregnancy (n)	9	9	10	9
Conceiving days 1 - 5 (N)	10	10	9	9
Conceiving days 6 - . . .(1) (N) and more	0	0	0	0
Duration of gestation (days)	22.71	22.56	22. 22	22.78
Dams with live young born (N)	7	9	10	9
Corpora lutea	11.44	13.89	13.20	13.11
Implantation sites	10.33	13.56	12.90	12.89
Pre Implantation Loss (%)	12.71	2.28	2.23	1.48
Post Implantation Loss (%)	27.76	3.60	3.81	8.04
Delivery index (%)	100	100	100	100
Copulation index (%)	90	90	100	100
Fertility index (%)	77.78	100	100	100
Mean no. of live pups at birth/litter	8.89	13.11	12.56	11.89
Mean no. of live pups on PND 4 (before interim sacrifice)	8.89	13.00	12.44	11.89
Mean no. of live pups on PND 4 (after interim sacrifice)	7.56	11.22	10.67	10.11
Mean no. of live pups on PND 13	7.56	11.22	10.67	10.11
* p=0.05; **p=0.01

Table CA 7.8.1/01-3:
Overview of F0 parental generation treated orally via gavage with MTHP: organ weight and clinical chemistry

Parameter	F (mg/kg bw/d)				F (mg/kg bw/d)
	0	30	120	500	0	30	120	500
Bwt at sacrifice (g)	414.2	402.4	398.1	394.1	318.6	323.3	327.7	318.9
Testes            Abs                          Rel:	3.7027 0.9003	3.561 0.08955	3.5675 0.0576	3.5416 0.901	-
Epidid.           Abs                          Rel	1.3310 0.3232	1.2990 0.3240	1.3125 0.3298	1.2823 0.3258
Prostate          Abs                          Rel	2.5564 0.6203	2.4880 0.6193	2.5880 0.6477	2.4161 0.6143
Ovaries          Abs                          Rel	-				0.1179 0.0373	0.1011 0.0317	0.1017 0.0312	0.1134 0.0358
Uterus + Cer.Abs                          Rel					0.6265 0.1994	0.4870 0.1511	0.4705 0.1439	0.4288 0.1359
Thyroid/para Abs                          Rel:	0.0318 0.0077	0.00300 0.0074	0.0264 0.0066	0.0309 0.0079	0.0249 0.0078	0.0239 0.0074	0.0217 0.0067	0.0242 0.0076
T4 (mmol/L)	73.07	73.83	73.99	76.46	-
Bwt: bodyweight Abs.: absolute (g) Rel.: relative Epidid.: epididymides Uterus + cer.: uterus with cervix Thyroid/para.: thyroid/parathyroid

 

Table CA 7.8.1/01-4:
Overview of F1 offspring generation: survival

Parameter	M (mg/kg bw/d)				F (mg/kg bw/d)
	0	30	120	500	0	30	120	500
Survival (mean no. of live pups)
PND 0 PND 1 PND 2 PND 3 PND 4 (prior to interim sacrifice)	5.00 5.00 5.00 5.00 5.00	7.44 7.33 7.33 7.33 7.33	5.44 5.33 5.33 5.33 5.33	6.33 6.33 6.33 6.33 6.33	3.89 3.89 3.89 3.89 3.89	5.67 5.67 5.67 5.67 5.67	7.11 7.11 7.11 7.11 7.11	5.56 5.56 5.56 5.56 5.56
Parameter	0		30		120		500
Total mortality (PND 0 - 4) [%]	0.00		1.01		0.74		0.00
Parameter	0	30	120	500	0	30	120	500
PND 4 (post interim sacrifice) PND 13	6.43 6.43	7.00 7.00	5.33 5.33	6.33 6.33	3.29 3.29	4.22 4.22	5.33 5.33	3.78 3.78
Parameter	0		30		120		500
Total mortality (PND 0 - 13) [%]	0.00		0.00		0.00		0.00

Table CA 7.8.1/01-5:
Overview of F1 offspring generation: anogenital distance and nipple retention

Parameter	M (mg/kg bw/d)				F (mg/kg bw/d)
	0	30	120	500	0	30	120	500
Bwt at sacrifice (g)	6.78	6.59	6.28*	6.78	6.52	6.04**	6.04**	6.36
Cube root of bwt	1.89	1.87	1.84***	1.89	1.87	1.82***	1.82***	1.85
Anogenital distance
Absolute (mm)	2.47	2.48	2.41	2.68*	0.94	1.05	0.83*	0.97
Relative	1.30	1.33	1.31	1.42*	0.50	0.58*	0.45	0.52
Pup nipple retention (n)	0.01	0.25	0.17	0.19	-
* p<0.05, ** p<0.01, *** p<0.001

Table CA 7.8.1/01-6:
Overview of F1 offspring generation: thyroid/parathyroid glands weight and clinical chemistry

Parameter	M (mg/kg bw/d)				F (mg/kg bw/d)
	0	30	120	500	0	30	120	500
Bwt at sacrifice (g)	6.78	6.59	6.28*	6.78	6.52	6.04**	6.04**	6.36
Cube root of bwt	1.89	1.87	1.84***	1.89	1.87	1.82***	1.82***	1.85
Abs (g)	0.00071	0.0052	0.0057	0.0060	0.0067	0.0057	0.0060	0.0063
T4 (mmol/L)	87.44	83.66	80.80	79.07	-
** p<0.01, *** p<0.001 Bwt: bodyweight

Conclusions:

Under the conditions of this study NOAEL of 4-Methyltetrahydropyran for parental toxicity is deemed to be 120 mg/kg bw/day; the NOAEL for reproductive toxicity is deemed to be 500 mg/kg bw/day (considered to be a maximum tolerated dose), the highest dose tested, based on no adverse effects. The NOAEL for parental toxicity is based on remarkable clinical signs (slightly to severely reduced spontaneous activity, apathy) primarily in the male and female high dose group (persisting in male animals) and moderately and statistically significantly lower mean body weight gain in male high dose group when related to the whole treatment period.

The NOAEL for offspring toxicity is deemed to be 500 mg/kg bw/day (the parental MTD) based on no adverse effects.

Executive summary:

The aim of this study was to assess the possible effects of 4-Methyltetrahydropyranon male and female fertility and embryofetal development after repeated dose administrationin Wistar rats following OECD 421 (2016) test guideline.

 

Four groups of 10 male and 10 female Han Wistar rats received the test article via oral gavage at concentrations of 0 (0.1% Tween 80 + 1% CMC), 30, 120 or 500 mg/kg bw/day employing a dose volume of 10 mL/kg bw for a treatment period of up to 63 days (i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females). Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

 

The effects on dams and the F1 offspring were examined. During pregnancy and lactation clinical signs, body weights and food consumption were recorded. The dams were allowed to deliver and the gestation length was calculated. After birth the F1 offspring were examined for the numbers of live born and stillborn pups, gross abnormalities and pup viability.The anogenital distance of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels of the thyroid hormone thyroxine (T4).

 

Males were sacrificed after completion of the mating period on day 29 and the females along with their pups were sacrificed on PND 13. Non-pregnant females or females without delivery were sacrificed on day 26 after mating. The number of implantation sites and corpora lutea were recorded for each parental female at necropsy.

 

Pups sacrificed on PND 4 for interim sacrifice for blood sampling, those found dead and all surviving pups before terminal sacrifice were carefully examined for gross external abnormalities.

 

A set of organs/tissues was preserved and the weight of a subset of organs/tissues was taken.

 

A full histopathological evaluation of the preserved reproductive organs was performed on high dose and control animals and in a non-pregnant female and its male mating partner of the low dose and mid dose group. These examinations were not extended to animals of all other dosage groups for treatment-related changes that are observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined.Any gross lesion macroscopically identified in any animal was examined microscopically.

 

During the treatment period a single female from the high dose was found dead directly after dose administration on premating day 4. Although there were no histological findings that could be related to the cause of death, it is considered that this animal died due to a technical gavage error and therefore not deemed to be treatment related. All remaining animals survived their scheduled study period.

 

Remarkable clinical signs related to the treatment with the test item were observed primarily in the high dose group. Predominant clinical signs were slightly to severely reduced spontaneous activity and apathy. Reduced spontaneous activity was observed in all males of the high dose group during the whole treatment period of 28 days and all females of the high dose group during the whole premating period but only transiently during mating and gestation period and not during lactation period. Additionally, apathy was transiently noticed in one male of the high dose group and three females of the high dose group. In the mid dose group, three males were seen with reduced spontaneous activity on one single day of treatment (premating day 2) and one male on 7 days of treatment (premating day 2 to 8). Females of the mid dose group were not affected.

 

Clinical signs of moving bedding/increased salivation, which were seen in animals of the high dose group, with mid and low group animals also displaying the same effectsshortly post dosing and therefore were considered to be a general sign of discomfort and/or local reaction to the test item although there are no effects in stomach. These slight signs were not considered as adverse systemic effects.

 

None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.

 

Although body weight increased subsequently, mean body weight gain of the male high dose group was moderately and statistically significantly lower when related to the whole treatment period (47% of controls). However, there was no statistically significant effect on mean body weight in the high dose group. In the third week of gestation a slightly but statistically significantly higher body weight gain was observed in the female low dose group (133% of controls). This transient, statistically significant difference to controls without dose-dependency was considered as incidental. No further statistical significant differences between female dose and control group were observed. The mean body weight increased in the control, the low dose and the mid dose group with the progress of the study for the male and females.

 

In males and females, in correlation to the body weight change, mean food consumption per animal in the high dose group was marginally but not statistically significantly lower in the first week of the premating period when compared to controls (males 86% of controls, females 83% of controls). There was no biologically or statistically significant difference to controls during the remaining study period or in the male and female low dose and mid dose groups.

 

The test item had no biologically significant effect on the oestrous cyclicity analyzed during the 2 week premating period after the first administration.

 

Length of the pre-coital interval and mean duration of gestation were within the normal range of variation for animals of this age and strain in the dose groups and the control group.

 

There were no effects of toxicological relevance on the number of corpora lutea, number of implantation sites and pre- and post-implantation loss in the dose groups when compared to the control group.

 

There were no test item related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group. A slightly reduced fertility index of 90% was observed in the low dose group compared to the mid dose and high dose groups, however the reduced fertility index was comparable to the control group (90%) and therefore considered as incidental in nature and not treatment related.

 

There were no statistically or biologically significant effects on the mean number of total pups delivered (live and dead), mean number of male and female pups and sex ratio of the dose groups when compared to the control group. There were no dose-dependent differences. No stillbirths or runts were observed in any of the dose groups or control group.

 

There were no test item related effects on litter weight data.

 

All pups of the high dose group and the control group survived from PND 0 to PND 4. Without following a dose-dependent pattern the single deaths of pups in the low dose group and mid dose group were considered as biological variation without relation to the treatment with the test item. All live pups on PND 4 in the dose groups and the control group survived until terminal sacrifice on PND 13.

 

The mean absolute and relative anogenital distance of male pups were marginally, but statistically significantly higher in the high dose group when compared to the control group. Mean anogenital distance (absolute and relative) of male pups of the mid dose and the low dose group were comparable to controls. No dose response was therefore seen for absolute and relative anogenital distance. mid dose group absolute value was lower when compared to control and low dose group, the mean relative mid dose value was also lower when compared to low dose group. Additionally, no dose response was found for mean absolute and relative values of female pubs. Mean absolute and relative anogenital distance and cube root of pup weight of the both control groups and all dose groups were in the normal range of variation. The effects observed in males are therefore concluded to be incidental and not test article related.

 

No statistically or biologically significant effects were noted on anogenital distance of female pups in the high dose group. Marginally but statistically significantly lower absolute mean value was noted in the mid dose group when compared to the control group without statistical significance when related to pup weight. Furthermore, the relative anogenital distance of female pups of the low dose group was marginally but statistically significantly higher. These marginal differences in female pups to controls without dose-dependency were considered as incidental. No effect of toxicological relevance was observed for nipple retention in male pups.

 

There were no statistically or biologically significant differences for measured serum T4 hormone levels of adult males and pups on PND 13 between the dose groups and controls.

 

Furthermore, there was no test article related effects on mean weight of thyroid/parathyroid glands of pups on PND 13.No remarkable, toxicologically relevant gross external abnormalities on PND 0 until terminal sacrifice were observed in the pups in any of the groups.

 

Dose formulation analysis for nominal concentrations were confirmed throughout the study period for all dose groups except for samples of the LD dose group in week 1. Samples were prepared fresh and measured once again. The nominal concentrations were confirmed. All concentrations were within acceptance criterion of 15%.

 

Under the conditions of this study NOAEL of 4-Methyltetrahydropyran for parental toxicity is deemed to be 120 mg/kg bw/day; the NOAEL for reproductive toxicity is deemed to be 500 mg/kg bw/day (considered to be a maximum tolerated dose), the highest dose tested, based on no adverse effects. The NOAEL for parental toxicity is based on remarkable clinical signs (slightly to severely reduced spontaneous activity, apathy) primarily in the male and female high dose group (persisting in male animals) and moderately and statistically significantly lower mean body weight gain in male high dose group when related to the whole treatment period.

 

The NOAEL for offspring toxicity is deemed to be 500 mg/kg bw/day (the parental MTD) based on no adverse effects.

Materials and methods

Test animals

Species:

rat