(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-09-23 to 2003-04-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline compliant GLP compliant

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

EC Number:

201-210-7

EC Name:

(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

Cas Number:

79-50-5

Molecular formula:

C6H10O3

IUPAC Name:

(±)-dihydro-3-hydroxy-4,4-dimethylfuran-2(3H)-one

Details on test material:

- Name of test material (as cited in study report): dl-Lactone
- Physical state: white crystalline mass
- Analytical purity: 99.8 %
- Purity test date: not reported
- Lot/batch No.: BX226
- Expiration date of the lot/batch: 31 May 2003
- Stability under test conditions: Stable under storage conditions
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar, Crl: (WI) BR (outbred, SPF-Quality).
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks
- Weight at study initiation: not reported
- Fasting period before study: no
- Housing: upon arrival 5 animals/sex/cage in suspended stainless steel cages, during the mating females and males caged together 1:1 in
suspended stainless steel cages with wire mesh floors; mated females and males individually housed in labelled polycarbonate cages containing sawdust (SAWI bedding, Jelu Werk, Rosenberg, Germany); from arrival until mating, males and females were housed in separate rooms; offspring kept with the dam until termination; during the final stage of the pregnancy period (from approximately day 16 of gestation onwards) and during lactation,
paper (Enviro-dri, BMI, Helmond, The Netherlands) provided as nesting material;
- Diet (e.g. ad libitum): ad libitum, standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.1-24.2
- Humidity (%): 33 -76%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Temporary deviations from the maximum level for relative humidity (with a maximum of 6%) and light/dark cycle (with a maximum of 1 hour) occurred due to cleaning procedures or performance of functional observations in the room. Based on laboratory historical data these deviations are considered not to affect the study integrity.

IN-LIFE DATES:
Acclimatisation: 18 September 2002
Start treatment: 23 September 2002
Start mating: 07 October 2002
Necropsy males: 21 October 2002
Necropsy females and pups: 21 October - 18 November 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Water (Milli-Q) of 37°C adjusted to pH 4. Acetic acid was added to milli-Q water to obtain pH 4.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
23 September -01 October 2002:
Sufficient ammounts of Milli-Q water of 37°C adjusted to pH 4 was added to the weighted test item. Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. The formulations were kept continuously at 37°C.
02 October -17 November 2002:
The test item was heated at 100°C, melted and divided into seven portions. For every week of study, milli-Q water at pH 4 and of 37°C was added to one of these portions. For every dose group, a stock-solution was prepared. The stock solutions and formulations were stirred and kept continuous at 37°C. From 08 October 2002 onwards, the stock-solution of group 1 was not heated, but was kept at room temperature due to practical reasons. Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to visually acceptable levels.
Storage conditions:
At 37°C, except for the stock-solution of group 1 which was kept at room temperature from 08 October 2002 onwards.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and on information provided by the sponsor. During NOTOX Project 257568 (Determination of the hydrolysis of dl-lactone as a function of pH) it was determined that dllactone was hydrolytically stable at pH 4 and 50°C.
- Concentration in vehicle: 0, 8, 40, 200 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg bw
- Stability: Formulations in Milli-Q water are stable for 4 hours at room temperature and formulations in Milli-Q water adjusted to pH 4 are stable for 4 hours at room temperature and for 8 days at 37°C (determined during this project).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of DL-Lactone was determined by High Performance Liquid Chromatography with Mass Spectrometry detection (LC-MS-MS):
Column: Lichrospher 100 RP-18, (250 * 4 (I.D.) mm; dp=5 pm (Merck, Darmstadt, Germany)
Mobile phase: 20/80/0.1 (v/v/v) Acetonitrile / Milli-Q water/ formic acid
Flow: 1 ml/min
Split: 1:1.5
Detection: SCIEX MSMS system API-300 mass spectrometer (Perkin Elmer, Biosystems, Foster City, Canada),
Interface: Turbo ionspray at 350°C; N2 flow rate of 6000 ml/min.; operated in positive ion mode
Monitored masses: MRM test substance m/z 131.1 --> 113.1, MRM internal standard m/z 127.1 --> 99.0
Injection volume: 100 µL
Column temperature: 20 °C
Autosampler temperature: 4 °C
- Detection limits (LOD, LOQ) (indicate method of determination/calculation):
linear relationship between response and concentration in the concentration range of 0.995 25. 0 mg/1 (r= 0.9989, n=6, weighted 1/concentration²)
LOD = 0.28 mg/L at an injection volume of 100 µl (i.e. 0.28 ng absolute)

Duration of treatment / exposure:

males:
2 weeks prior to mating, during mating, and upto termination (28 d for all males).
females:
2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation. The mean duration of treatment of females: 43 d, minimum: 28 d, maximum: 56 d

Frequency of treatment:

5 d/wk, single daily oral application via gavage

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 40, 200, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a dose range finding study
- Rationale for animal assignment: computer-generated random algorithm
- Section schedule rationale: not reported

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality - twice daily, clinical signs: once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, in addition once prior to start of treatment and once a week thereafter, this was also performed outside the home cage in a standard arena during the pre-mating period.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter; mated females on days 0, 7, 14 and 21 of gestation, and during lactation on days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly, for males and females; suspended during the mating; food consumption of mated females measured on gestation days 0, 7, 14 and 21, and during lactation on days 1 and 4.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination,
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, over night
- How many animals: 5 males and 5 females, randomly selected
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
assigned males were tested during week 4 of treatment
assigned females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
The following tests were performed in 5 males and 5 females, randomly selected from each group:
- hearing ability (score 0 = normal/present, 1 = abnormal/absent).
- pupillary reflex (score 0 = normal/present, 1 = abnormal/absent).
- static righting reflex (score 0 = normal/present, 1 = abnormal/absent).
- grip strength (score 0 = normal/present, 1 = abnormal/absent).
In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England). During the motor activity test, males were caged individually and females were caged with their offspring.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see table 2
HISTOPATHOLOGY: Yes, see table 2
- Of the selected 5 males/group of the control and high dose group: additional slides of the testes prepared to examine staging of spermatogenesis; testis processed, sectioned at 3-4 microns, and stained with PAS/hematoxylin.
- The following slides were examined by a pathologist:
The preserved organs and tissues of the selected animals of groups 1 and 4.
The additional slides of the testes of the selected 5 males/group of groups 1 and 4 to examine staging of spermatogenesis
The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis all gross lesions of all animals (all dose groups)
The preserved organs and tissues of all non-pregnant females and animals suspected of infertility

Other examinations:

Observations offspring:
- numbers of live and dead pups at the First Litter Check (= check at day 1 of lactation) and daily thereafter (if possible, defects or cause of death evaluated)
- individual weight of all live pups on days 1 and 4 of lactation
- sex of all pups (by assessment of the ano-genital distance)
- number of pups with physical or behavioural abnormalities daily

Macroscopic examination of offspring:
- all offspring sexed and externally examined if practically possible
- examination of stomach for the presence of milk
- descriptions of all macroscopic abnormalities recorded; defects or cause of death were evaluated, if possible
- no pups preserved

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data could not be assumed to follow a normal distribution.
The exact Fisher-test was applied for 2x2 tables if variables could be dichotomized without loss of information.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Aggressive and restless behaviour in females during days 5 to 15 of treatment;

Mortality:

mortality observed, treatment-related

Description (incidence):

Aggressive and restless behaviour in females during days 5 to 15 of treatment;

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

serum potassium level of males of the highest dose group statistical significant increased

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

, but no dose response, therefore regarded as not treatment related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Females of the highest dose group showed aggressive and restless behaviour during days 5 to 15 of treatment.

BODY WEIGHT AND WEIGHT GAIN
- body weights and body weight gain unaffected by treatment up to and including 1000 mg/kg body weight/day.
- males of the 200 mg/kg bw group:
statistical significant decreased body weights on day 1 of the mating period, but not toxicological relevant due to lack of dose response relationship
- males of the 1000 mg/kg bw group:
on day 8 of the pre-mating period, a very slight statistical significant increased body weight gain, incidental and not toxicological relevant

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
unaffected by treatment up to and including 1000 mg/kg bw/day.

HAEMATOLOGY
- not affected by treatment

CLINICAL CHEMISTRY
- males of the 1000 mg/kg bw group:
statistical significant increse of serum potassium levels, but only slight and therefore of no toxicological significance
effects on glucose and inorganic phosphate without dose-response and therefore of no toxicological significance

NEUROBEHAVIOUR
- no changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the treated animals
- variation in motor activity did not indicate a relation with treatment
- females of the highest dose group showed a decreased motor activity at the low sensors; but absence of similar changes of the high sensors; therefore considered to be of no toxicological relevance.

ORGAN WEIGHTS
- no treatment-related changes
- males of the 200 mg/kg bw group: statistically significantly decreased absolute and relative adrenals weight
- females of the 40 mg/kg bw group: statistically significantly increased relative adrenals weight
both considered to be not related to treatment due to lack of dose response

GROSS PATHOLOGY
- No treatment related alterations

HISTOPATHOLOGY: NON-NEOPLASTIC
- No treatment related alterations
- The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Effect levels

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of any adverse effect at this dose level

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

- Table 1: Animal body weight development

Males
PRE-MATING		GROUP 1 CONTROL	GROUP 2 40 MG/KG	GROUP 3 200 MG/KG	GROUP 4 1000 MG/KG
DAY 1	MEAN	332	333	---	326
WEEK 1	ST.DEV.	9.7	9.5	---	11.4
	N	10	10	0 x	10
DAY 8	MEAN	354	359	344	356
WEEK 2	ST.DEV.	13.0	11.0	17.5	15.6
	N	10	10	10	10
MATING
DAY 1	MEAN	380	383	361 *	381
	ST.DEV.	16.4	15.9	15.3	16.1
	N	10	10	10	10
DAY 8	MEAN	---	407	---	380
	ST.DEV.	---	16.0	---	---
	N	0	3	0	1
POST MATING
	MEAN	398	401	383	386
	ST.DEV.	16.5	12.2	18.9	27.3
	N	10	7	10	9
	MEAN	417	425	405	405
	ST.DEV.	19.3	9.4	22.4	25.1
	N	10	7	10	9
Females
PRE-MATING		GROUP 1 CONTROL	GROUP 2 40 MG/KG	GROUP 3 200 MG/KG	GROUP 4 1000 MG/KG
DAY 1	MEAN	235	231	237	235
WEEK 1	ST.DEV.	9.3	14.5	10.2	11.4
	N	10	10	10	10
DAY 8	MEAN	242	245	239	239
WEEK 2	ST.DEV.	9.0	10.5	9.8	11.4
	N	10	10	10	10
MATING
DAY 1	MEAN	253	255	252	254
	ST.DEV.	10.0	10.7	9.2	12.4
	N	10	10	10	10
DAY 8	MEAN	---	292	---	290
	ST.DEV.	---	10.1	---	---
	N	0	3	0	1
POST COITUM
DAY 0	MEAN	252	264	250	251
	ST.DEV.	15.0	24.4	5.5	9.0
	N	8	9	8	9
DAY 7	MEAN	284	295	280	284
	ST.DEV.	15.5	23.4	8.0	15.7
	N	8	9	8	9
DAY 14	MEAN	318	334	324	317
	ST.DEV.	15.3	26.5	10.3	21.6
	N	8	9	8	9
DAY 21	MEAN	409	440	429	404
	ST.DEV.	33.3	40.1	22.4	36.3
	N	8	9	8	9
LACTATION
DAY 1	MEAN	312	317	313	313
	ST.DEV.	22.4	29.0	11.6	23.0
	N	8	9	8	9
DAY 4	MEAN	312	325	319	318
	ST.DEV.	10.8	30.1	8.8	22.5
	N	7	9	8	8

x: The body weight determination on day 1 of the pre-mating period of the males of the 200 mg/kg dose group was performed incorrectly.

* / ** : Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Applicant's summary and conclusion

Conclusions:

RS-Pantolactone (named DL-LACTONE in the study report) was tested for it's repated dose toxicity in rats according to OECD TG 422.
In conclusion, gavage treatment of male and female Wistar rats with RS-Pantolactone at dose levels of 40, 200 or 1000 mg/kg body weight/day for at least 28 days (during premating ,mating, post-coitum, and lactation), revealed slight parental toxicity in animals receiving 1000 mg/kg b.w./day.

Executive summary:

RS-Pantolactone (named DL-LACTONE in the study report) was tested for it's repated dose toxicity in rats according to OECD TG 422. The test item was administered by daily oral gavage to male and female Wistar rats at dose levels of 40, 200 or 1000 mg/kg body weight/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (28 days for all males). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation. The mean duration of treatment of females was 43 days, with a minimum of 28 days and a maximum of 56 days.

Parental toxicity was assessed by observing mortality, clinical signs, body weights, food consumption, functional observations, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination.

At 40 mg/kg b.w./day, no parental toxicity was observed.

At 200 mg/kg b.w./day, no parental toxicity was observed.

At 1000 mg/kg b.w./day, parental toxicity consisted of clinical symptoms (aggressive and restless behaviour) in females during days 5 to 15 of treatment, and increased serum potassium level in males.

In conclusion, gavage treatment of male and female Wistar rats with RS-Pantolactone at dose levels of 40, 200 or 1000 mg/kg body weight/day for at least 28 days (during premating ,mating, post-coitum, and lactation), revealed slight parental toxicity in animals receiving 1000 mg/kg b.w./day.

Based on the results in this combined repeated dose toxicity study with reproduction/developmental screening test, the definitive parental No Observed Adverse Effect Level (NOAEL) was established as being 200 mg/kg body weight/day.