Tetrahydro-4-methyl-2H-pyran

Endpoint summary

Description of key information

In the GLP, test guideline compliant 28-day repeat dose oral study, rats produced liver enlargement as the main toxicological effect. High dose males (500 mg/kg bw/day) also showed increased relative kidney weight, without any associated histopathology and reduced motor activity (without associated histopathology), with high dose group females showing a significant increase in water intake, with significantly reduced urine osmolality. These effects were all reversible following withdrawal of administration. Histopathological liver changes were in all cases associated with liver enlargement and liver enzyme induction (hypertrophy of centrilobular hepatocyte).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-22 to 2010-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 4-methyltetrahydropyran (MTHP)
- CAS no.: 4717-96-8
- EC-no.: not assigned
- Source and lot/batch No.of test material: Kuraray / MTHP213536
- Expiration date of the lot/batch: not stated
- Molecular weight: 100.16 g/mol
- Purity: 100%

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

refer to "Details on test animals and environmental conditions"

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age: 6 weeks at dosing
Weight at dosing: M: 188-214g; F: 151-174g
Source: Charles River Laboratories Japan, Inc.
Acclimation period: 8 days
Diet: Pelleted diet CR-1 (Oriental Yeast Co., Ltd), ad libitum
Water: Municipal water supply, ad libitum
Housing: individually

Environmental conditions
Temperature: 20-24°C
Humidity: 30-70%
Air changes: 10-15 times/hr
Photoperiod: 12 hours light/dark

Route of administration:

oral: gavage

Details on route of administration:

Refer to "Details on study design"

Vehicle:

other: 0.1% v/v Tween-80 supplemented with 1%w/v Na CMC

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test doses were prepared weekly. Refer to "Details on analytical verification of dose or concentrations"

VEHICLE: 0.1% v/v Tween-80 supplemented with 1%w/v Na CMC
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: dose suspension of MTHP in vehicle were preapred at 3, 12 and 50 mg/mL
- Amount of vehicle (if gavage): dose volume 10 mL/kg bg
- Lot/batch no.:
- Vehicle component 1: Carboxymethyl cellulose sodium (Na CMC); lot/batch no.: WKK2269
- Vehicle component 2: Water for injection; lot/batch no.: 9E70
- Vehicle component 3: Tween 80; lot/batch no.: ZBT2C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test doses were prepared weekly. Test article was weighed and suspended in 0.1% v/v Tween-80 supplemented with 1%w/v Na CMC. The sponsor confirmed that the test article was stable in the chosen vehicle at concentrations 0.1 and 100 mg/mL when stored at room temperature or refrigerated for 7 days.

The test dose suspensions perpared in weeks 1 and 4 of administration were analysed for concetration and homogeneity.

Duration of treatment / exposure:

28 days, with a further 6 animals/sex for the control and high dose group included for recovery purposes, with a 2 week withdrawal at the end of the administration period

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

0.1% v/v Tween-80 supplemented with 1%w/v Na CMC

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:

6/sex/gp

Control animals:

yes, concurrent vehicle

Details on study design:

After an acclimatisation period of ca. 8 days, rats were screened for ill health, weighed and allocated to treatment four groups of 6 animals/sex according to a randomised block design. A further 6 animals/sex for the control and high dose group were included for recovery purposes, with a 2 week withdrawal at the end of the administration period to examine any potential reversible effects. The test article was administered orally via gavage at concentrations of 0, 30, 120 or 500 mg/kg bw/day, employing a dose volume of 10 mL/kg bw. Six animals/sex/group were sacrificed at day 29.

Dose level selection was based on a range-finder experiment where 3 animals/sex were administered the test article orally via gavage at concentrations of 0, 15, 60, 250, 1000 mg/kg bw/d for 14 days. At doses of 1000 mg/kg bw/d decreased group-mean body weight and anesthetic effects in males and emaciation immediately before death and anesthetic effects in females were observed. Therefore, 500 mg/kg bw/d was deemed to be the maximum tolerated dose (MTD).

Positive control:

n/a

Observations and examinations performed and frequency:

Examined for clinical signs including external appearance, nutritional condition, posture, behaviour and excretment before dosing, immediately after and approximately 2 h post dosing. On the day of functional examinations (week 4 of dosing and once during the recovery period) observation frequency was reduced to twice daily (before and immediately post dosing). Recovery animals were examined once daily during the recovery period

Sacrifice and pathology:

- Gross pathology: All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ.

- Histopathology: the following tissues were examined from all control and high dose group animals, with the liver examined from all low and mid dose groups:
adrenals, brain (cerebrum, cerebellum), epididymides, eyeballs, femur (+ bone marrow), gastrointestinal tract (stomach, duodenum, jejunum, ileum (+Peyer's patch), caecum, colon, rectum), heart, kidneys, liver, lungs (+bronchus), lymph nodes (submandibular, mesenteric), mammary glands (inguinal region), ovaries, pituitary, prostate, sciatic nerve, seminal vesicles (+coagulating gland), skeletal muscle (femoral), skin (inguinal region), spinal cord (together with vertebra in the thoracic region), spleen, sternum (+bone marrow), testes, thymus, thyroid (+parathyroid), trachea, uterus (+cervix), urinary bladder, vagina

The following tissues were collected, preserved and not examined: aorta (thoracic), Harderian glands, oesphagus, optic nerves, pancreas, salivary glands (submandibular, sublingual)

Other examinations:

Refer to "Any other information on materials and methods incl. tables"

Statistics:

Bartlett's test (to determine homogeneity of variance between groups). If variances were homogenous, data were analysed by the Dunnett test, whereas heterogenous data were analysed by the Dunnett-type mean tank test between the control group and each dose level.
For the recovery groups, homogeneity of variance was tested for each group by the F-test. For homogenous data, the difference in the mean values between the control and treatment groups were analysed by the Student's t-test. Heterogenous data were analysed by the Aspin-Welch t-test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the high dose group (500 mg/kg bw/d), decreased motor activity was observed early on in males from day 1 (M: 2/12) ; females from on day 2 and 3 (F: 2/12). By day 2 and day 4 for males and females, respectively all animals showed no abnormalities.

Mortality:

no mortality observed

Description (incidence):

No test article related effects on mortality were observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test article related effect on body weight or body weight gain were observed over the entire treatment period including the recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test article related effects on food consumption were observed.

Food efficiency:

not examined

Description (incidence and severity):

n/a

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

effects observed, treatment-related

Description (incidence and severity):

High water intake in high dose females. Refer to "Urinalysis findings" for further details

Ophthalmological findings:

not examined

Description (incidence and severity):

Not conducted.

Haematological findings:

no effects observed

Description (incidence and severity):

No test article related effects on haematological parameters were observed.

In the recovery group, high dose group males showed a statistically significant lower value in mean haemoglobin concentration and females showed a statistically significant lower value in white blood cell count. High dose males showed a statistically significant increase in monocytes when expressed as a percentage; whilst females showed a statistically significant reduction in large unstained cells (expressed both as a percentage and actual number).

The haematological changes in the recovery group were considered to be incidental and not treatment related as the changes observed were minimal, were not consistent between both sexes and were not evident at the end of treatment (refer to "Any other information on results incl. tables" Table CA 7.5.1/01-1).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test article related effects on serum chemistry parameters were observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

A high water intake and low urine osmolality were observed in females in the 500 mg/kg bw/d gp in the wk 4 administration. No such effects were observed in the recovery group. It therefore can be concluded that reversibility of the changes after withdrawal of administration was observed (refer to "Any other information on results incl. tables" Table CA 7.5.1/01-2)

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In the detailed clinical observsations, functional examinations and grip strength there were no treatement related effects observed.

Immunological findings:

not examined

Description (incidence and severity):

n/a

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in relative liver and kidney weights were observed in high dose males only at the end of treatment. These changes were not observed after withdrawal of treatment. Histopathological changes were observed in the liver only.

High dose males in the recovery group exhibited statistically significant decreases in absolute brain and seminal vesicle weights. These were not reflected when organ weights were corrected for body weights. The marginal reduction in absolute weights of these organs were considered incidental and not treatment related as they were: i) minimal; ii) only evident in the recovery period; iii) not replicated in females (where relevant); iv) no associated histopathology (refer to "Any other information on results incl. tables" Table CA 7.5.1/01-3)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

End of administration period:
- Dark red foci in the lungs (2M control, 1M mid and 2M high dose gp)
- Dysplasia of the spleen (1M in the mid dose gp)
- Diverticulum in the ileum (1F control gp)

Recovery period:
- Dark red focus in the eyeballs (1F control gp)
- Small testes (1M high dose gp)

The gross pathological changes were sporadically, and deemed to not be treatment related based on incidence, lack of dose response and no associated histopathology

Neuropathological findings:

no effects observed

Description (incidence and severity):

No central or peripheral nervous system histopathological changes observed.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer to Table CA 5.7.1/01-4.
Hypertrophy of centrilobular hepatocytes (graded as minimal) was observed in 5/6 and 2/6 high dose males and females, respectively, with no effect observed in the respective concurrent control groups. Hypertrophy of centrilobular hepatocytes were not observed in the recovery group. Liver effects that were observed in treated animals in the recovery group (hepatocyte vacuolation in the periportal region) occurred with the same frequency in both concurrent control groups and test article treated animals, and therefore was not considered treatment related.

Tubular regeneration (graded minimal) was observed in high dose males (3/6), however this observation was also observed in 5/6 male control animals, and therefore was not considered treatment related. Tubular regeneration was not observed in either control or test article treated females. The kidney was not examined in recovery animals.

Other histopathological changes were considered not treatment related based on incidence and lack of dose response.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

n/a

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Motor activity:
Some inter-group variation was noted during the motor activity investigations (refer to Table CA 5.7.1/01-5). Main differences included:
- Low values were recorded at 40 – 50 and 50 – 60 minutes after the start of measurement and the total value from 0 – 60 minutes after the start of measurement in males in the high dose group in week 4 of administration. These low values were not replicated in the recovery group, and whilst there is evidence of treatment related effects, no associated peripheral or central nervous system histopathology was evident.
- High values in the measurement was recorded at 10 – 20 minutes after the start of measurement in males and a low value in the measurement recorded at 0 – 10 minutes after the start of measurement in females in the low dose group. These changes were judged to be incidental as they were minimal changes and there were no similar changes in the high dose group.
- High dose group females showed high values in the measurement values at 40 – 50 minutes and 50 – 60 minutes and in the total value from 0 – 60 minutes. These changes were however deemed to be incidental as they were slight and also observed in the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table CA 7.5.1/01-1:
Overview of 28-day repeat dose toxicity in rats treated orally (via gavage) with MTHP: selected serum haematology parameters (group mean values)

Parameters	Sample Time	/m (mg/kg bw/d)					F (mg/kg bw/d)
		0	30	120	500		0	30	120	500
Haematology
- MCHC (g/dL)	ET ER	34.8 33.3	34.8 -	34.8 -	34.8 32.9^*		36.0 33.9	35.7 -	35.4 -	35.9 34.2
- WBC (x10^2/uL)	ET ER	96.7 111.9	95.0 -	120.4 -	93.4 120.1		105.4 91.1	87.3 -	84.4 -	85.1 65.4^*
Selected differential leukocyte counts expressed as a %
Monocytes (%)	ET ER	1.8 1.5	1.8 -	2.0 -	1.7 2.0^*		1.5 1.7	1.2 -	1.5 -	1.4 2.1
LUC (%)	ET ER	1.5 0.5	1.0 -	1.0 -	1.0 0.4		1.2 0.5	0.9 -	0.9 -	1.0 0.3^*
Selected differential leukocyte counts expressed as a conc. (x10^2/uL)
Monocytes (x10^2/uL)	ET ER	1.8 1.7	1.7 -	2.5 -	1.6 2.4		1.7 1.4	1.0 -	1.3 -	1.2 1.4
LUC (x10^2/uL)	ET ER	1.5 0.6	0.9 -	1.2 -	1.0 0.5		1.3 0.5	0.8 -	0.8 -	0.8 0.2^*
*p=0.05 ET: end of treatment (wk 4) ER: end of recovery (wk 6, i.e. 2 wks post dosing)						MCHC: Mean cell haemoglobin concentration WBC: white blood cells LUC: large unstained cells

 

Table CA 7.5.1/01-2:
Overview of 28-day repeat dose toxicity in rats treated orally (via gavage) with MTHP: selected urinalysis parameters (group mean values)

Parameters	Sample Time	M (mg/kg bw/d)					F (mg/kg bw/d)
		0	30	120		500	0	30	120	500
Water intake	ET ER	34 31	35 -	36 -		39 44	28 25	28 -	29 -	34^* 26
Urine vol. (mL/24h)	ET ER	11.0 10.0	8.8 -	10.4 -		13.8 13.2	7.6 5.2	7.7 -	7.0 -	9.7 6.1
Osmolality (mOsm/kg)	ET ER	1983 2172	2150 -	2074 -		1697 1831	2191 2368	1975 -	2052 -	1727^* 2110

 Table CA 7.5.1/01-3:
Overview of 28-day repeat dose toxicity in rats treated orally (via gavage) with MTHP: selected organ weights (group mean values)

Parameters	M (mg/kg bw/d)					F (mg/kg bw/d)
	0	30	120	500		0	30	120	500
End of administration (wk 4)
Ter. body wt (g)	369	368	353	338		225	221	223	226
Organ weights (rel: g%; abs: g)
- Liver:  abs                rel % relvscon	10.93 2.96 -	11.20 3.05 103%	10.13 2.86 97%	11.34 3.36^** 114%		6.57 2.62 -	6.37 2.89 110%	6.42 2.87 110%	6.98 3.09 118%
- Kid:     abs                rel % relvscon	2.75 0.74 -	2.75 0.75 101%	2.70 0.77 104%	2.80 0.83^* 112%		1.76 0.78 -	1.66 0.75 96%	1.73 0.78 100%	1.84 0.81 104%
- Brain: abs                rel % relvscon	1.98 0.54 -	1.98 0.54 100%	1.96 0.56 104%	1.93 0.57 106%		1.86 0.83 -	1.84 0.83 100%	1.86 0.84 101%	1.80 0.80 96%
- S.V:     abs                rel % relvscon	2.08 0.56 -	2.21 0.60 107%	2.22 0.63 113%	1.97 0.58 104%		-	-	-	-
End of recovery (wk 6, i.e. 2 wks post dosing)
Ter. body wt (g)	438	-	-	421		246	-	-	238
- Liver:  abs                rel % relvscon	11.70 2.67 -	-	-	11.92 2.82 106%		6.61 2.69 -	-	-	6.57 2.76 103%
- Kid:     abs                rel % relvscon	2.95 0.68 -	-	-	2.91 0.69 101%		1.74 0.71 -	-	-	1.75 0.74 104%
- Brain: abs                rel % relvscon	2.11 0.49 -	-	-	2.02^** 0.48 98%		1.87 0.77 -	-	-	1.88 0.80 104%
- S.V:     abs                rel % relvscon	2.84 0.65 -	-	-	2.48^* 0.59 91%		-	-	-	-
*p=0.05; **p=0.01 Ter. body wt: Terminal body weight abs: absolute					rel: relative Kid: kidney S.V: seminal vesicles (including prostate and coagulating glands)

 

Table CA 7.5.1/01-4:
Overview of 28-day repeat dose toxicity in rats treated orally (via gavage) with MTHP: Non-neoplastic histopathological findings

Parameters	M (mg/kg bw/d)					F (mg/kg bw/d)
	0	30	120	500		0	30	120	500
Liver(incidence / total no. examined [minimal)
-Cen. hep. hyper.	0/6 [0]	0/6 [0]	0/6 [0]	5/6 [5]		0/6 [0]	0/6 [0]	0/6 [0]	2/6 [2]
Kidney(incidence / total no. examined [minimal)
-Reg, tubul.	5/6 [5]	0/6 [0]	0/6 [0]	3/6 [3]		0/6 [0]	0/6 [0]	0/6 [0]	0/6 [0]
Cen. hep. hyper.: Centrilobular hepatocyte hypertrophy					Reg. tubul.: regeneration, tubular

 

Table CA 5.7.1/02-5:
Overview of 28-day repeat dose toxicity in rats treated orally (via gavage) with MTHP: locomotor activity – mean beam break scores

Dose level (mg/kg bw/d)	Time (minutes)
	0-10	10-20	20-30		30-40	40-50	50-60	Total
Males: end of administration (wk 4)
0	426	357	349		330	330	318	2110
30	432	403^**	348		383	406	392	2364
120	432	386	347		353	328	301	2147
500	421	385	332		281	183^**	61^**	1864^**
Females: end of administration (wk 4)
0	418	364	270		277	211	222	1761
30	378^*	313	247		236	241	295	1709
120	396	342	324		302	312	298	1972
500	439	371	320		327	335^*	288^*	2080^*
Males: end of recovery (wk 6, i.e. 2 wks post dosing)
0	422	356	321		313	319	285	2016
500	410	365	326		340	305	277	2022
Females: end of recovery (wk 6, i.e. 2 wks post dosing)
0	385	307	234		276	272	190	1664
500	403	323	298		290	262	240	1815
*p=0.05; *p=0.01

 

Conclusions:

Under the conditions of this study, NOAEL for MTHP was 120 mg/kg bw/day following dosing for 28-daysviaoral gavage for males and females based on increased relative liver weights (exceeding 110% of control), with associated histopathology (minimal hypertrophy of centrilobular hepatocyte). High dose males also showed increased relative kidney weight, without any associated histopathology and reduced motor activity (without associated histopathology), with high dose group females showinga significant increase in water intake, with significantly reduced urine osmolality. These effects were all reversible following withdrawal of administration.

Executive summary:

In a 28-day sub-acute study, MTHP was administered orally via gavage to male and female rats at dose levels of 0, 30, 120 or 500 mg/kg bw/day, employing a dose volume of 10 mL/kg. Group sizes were 6 animals/sex, with a further 6 animals/sex/ group for control and high dose groups in order to assess recovery following withdrawal of treatment.

 

In addition to standard parameters measured (body weight, food consumption, blood haematology, clinical chemistry, urinalysis), a neurobehavioral test battery consisting of motor activity, functional observational battery (FOB) and detailed clinical observations were conducted on all rats onstudy week 4 of administration and in week 2 of recovery. At termination, all control and high dose animals were subjected to animals were subjected to histopathological examination including brain, spinal cord and sciatic nerve. The liver was examined from all low and mid dose groups.

 

There were no test article-related clinical findings noted at any exposure level.Overall (week 0-4) bodyweight gain was low vs.controls (11%) for high dose males. These differences from control were most apparent from ca. day 4 of treatment, but did not achieve statistical significance. These effects were not carried through to the recovery group.

 

No test article-related effects were noted on FOB parameters, including home cage, handling, open field, sensory, neuromuscular, and physiological observations, in the all test article treated dose groups during the study. Low motor activities were recorded for high dose males at 40-50 and 50-60 minutes after the start of measurement and the total value from 0-60 minutes after the start of measure in the week 4 administration. No associated histopathology was evident in the central or peripheral nervous system and there were no changes in week 2 of recovery.

 

A slight, but statistically significant increase in water intake, with significantly reduced urine osmolality was observed in high dose group females.

 

Hypertrophy of centrilobular hepatocytes (graded as minimal) was observed in 5/6 and 2/6 high dose males and females, respectively, with no effect observed in the respective concurrent control groups. Hypertrophy of centrilobular hepatocytes were not observed in the recovery group. Liver effects that were observed in treated animals in the recovery group (hepatocyte vacuolation in the periportal region) occurred with the same frequency in both concurrent control groups and test article treated animals, and therefore was not considered treatment related.

 

Tubular regeneration (graded minimal) was observed in high dose males (3/6), however this observation was also observed in 5/6 male control animals, and therefore was not considered treatment related. Tubular regeneration was not observed in either control or test article treated females. The kidney was not examined in recovery animals.

 

Under the conditions of this study, NOAEL for MTHP was 120 mg/kg bw/day following dosing for 28-days via oral gavage for males and females based on increased relative liver weights (exceeding 110% of control), with associated histopathology (minimal hypertrophy of centrilobular hepatocyte). High dose males also showed increased relative kidney weight, without any associated histopathology and reduced motor activity (without associated histopathology), with high dose group females showinga significant increase in water intake, with significantly reduced urine osmolality. These effects were all reversible following withdrawal of administration.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Comparison with the CLP criteria

The only significant toxic effects of relevance to humans were seen in the liver of rats; however, these occurred at dose levels well in excess of the specified guidance values for classification with STOT-RE Category 2 (oral =30 mg/kg bw/d, when converted from a 90 day study guidance value of =10 mg/kg bw/day).

 

On this basis, classification of MTHP with STOT-RE is not warranted