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

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

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)

Deviations:

yes

Remarks:

The number of implantation sites and corpora lutea was not recorded.

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 wks;
- 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 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).

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 1 - 2 wks (depending on mating success, females 53, 56, 57 (group 2), and 78 paired with proven males of the same treatment group after unsuccessfull mating during first week of cohabitation)
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility of the respective dose group.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually housed in labelled polycarbonate cages containing sawdust; from approximately day 16 of gestation onwards and during lactation, paper was supplied as nest material
- Any other deviations from standard protocol: no

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

Details on study schedule:

not applicable

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

Parental animals: Observations and examinations:

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.
- Cage debris of pregnant females was examined to detect abortion or premature birth. Signs of difficult or prolonged parturition were recorded.

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.

OTHER:
Other examinations are described under Beekhuijzen (2003) / Rep. No. 349469 / key (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) in chapter 7.5.1

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after the mating period, when the minimum total dosing period of 28 days had been completed.
- Maternal animals: All surviving animals, females with litter were at day 4 post partum or shortly thereafter, females without litter around the same time.

GROSS NECROPSY
- Gross necropsy consisted of cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs (see Table 1 for details).

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
- 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

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem macroscopic examination as follows:
sexed, externally examined, stomach analyzed for milk, macroscopic abnormalities were recorded, if possible, defects or cause of death evaluated.

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.

Reproductive indices:

Percentage mating = (Number of females mated / Number of females paired) x 100

Fertility index = (Number of pregnant females / Number of females paired) x 100

Conception rate = (Number of pregnant females / Number of females mated) x 100

Gestation index = (Number of females bearing live pups / Number of pregnant females) x 100

Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100

Percentage of postnatal loss days 0-4 post partum = (Number of dead pups on day 4 post partum / Number of live pups at First Litter Check) x 100

Viability index = (Number of live pups on day 4 post partum / Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No unscheduled deaths during the study period
- Agressive behaviour of females of the highest dose group during days 5 to 15 of treatment

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- 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 unaffected by treatment up to and including 1000 mg/kg bw/day.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
not applicable (gavage application)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- no adverse effects at any dose level (organ weights, gross necroscopy and histology)
- Staging of spermatogenesis:
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired
spermatogenesis.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No adverse effects at any dose level
- Reproduction parameters unaffected by treatment up to 1000 mg/kg body weight/day.
- control group: two females non-pregnant
40 mg/kg dose group: one female not mating, two females mated in the second mating period
200 mg/kg dose group: two females non-pregnant
1000 mg/kg dose group:, one female non-pregnant
- Mating performance, duration of gestation, fertility parameters, and number of pups at birth similar for the control and treated groups

ORGAN WEIGHTS (PARENTAL ANIMALS)
- 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 (PARENTAL ANIMALS)
- No treatment related alterations

HISTOPATHOLOGY (PARENTAL ANIMALS)
- No treatment related alterations

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of any adverse effect at this dose level

2

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Aggressive and restless behaviour in females during days 5 to 15 of treatment; Increased serum potassium level in males

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- Breeding parameters unaffected by treatment up to 1000 mg/kg body weight/day
- number of dead and living pups at first litter check, postnatal loss between days 0-4 post-partum, living pups at day 4 post-partum, and the viability index similar for control and treated groups.

CLINICAL SIGNS (OFFSPRING)
- No treatment related effects reported
- Incidental clinical symptoms: (very) small, little or no milk, and bruise on the head or snout

BODY WEIGHT (OFFSPRING)
- Mean body weights of pups similar for control and treated groups

OTHER FINDINGS (OFFSPRING)
Macroscopic examination:
- tip of tail missing, tip of tail discoloured dark red, autolyse, small appearance, and no milk; no relationship with treatment detectable for these observations or considered to be within the normal biological variation for rats of this age and strain

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: absence of any adverse effect at this dose level

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 2: Reproduction Processes F0-Generation - Post Coitum

Female Number	Male Number	Mating Date	Pregnant	Schedule	Delivery recorded	Necropsy
GROUP 1 (CONTROL)
41	1	08-OCT-02	Yes	Breeding	29-OCT-02	04-NOV-02
42 (A)	2	13-OCT-02	No	Breeding		04-NOV-02
43	3	11-OCT-02	Yes	Breeding	01-NOV-02	06-NOV-02
44	4	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
45	5	08-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
46	6	08-OCT-02	Yes	Breeding	29-OCT-02	04-NOV-02
47	7	10-OCT-02	Yes	Breeding	31-OCT-02	04-NOV-02
48	8	11-OCT-02	Yes	Breeding	02-NOV-02	07-NOV-02
49	9	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
50 (A)	10	08-OCT-02	No	Breeding		04-NOV-02
GROUP 2 (40 MG/KG)
51	11	08-OCT-02	Yes	Breeding	29-OCT-02	04-NOV-02
52	12	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
53	13	<**>
53	18	19-OCT-02	Yes	Breeding	09-NOV-02	14-NOV-02
54	14	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
55	15	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
56	16	<**>.
56	19	20-OCT-02	Yes	Breeding	11-NOV-02	18-NOV-02
57	17	<**>
57	20	<**>	-	---		21-OCT-02
58	18	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
59	19	11-OCT-02	Yes	Breeding	01-NOV-02	06-NOV-02
60	20	08-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
GROUP 3 (200 MG/KG)
61 (A)	21	08-OCT-02	No	Breeding		04-NOV-02
62	22	10-OCT-02	Yes	Breeding	01-NOV-02	06-NOV-02
63 (A)	23	08-OCT-02	No	Breeding		04-NOV-02
64	24	08-OCT-02	Yes	Breeding	29-OCT-02	04-NOV-02
65	25	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
66	26	11-OCT-02	Yes	Breeding	02-NOV-02	07-NOV-02
67	27	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
68	28	08-OCT-02	Yes	Breeding	29-OCT-02	04-NOV-02
69	29	08-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
70	30	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
GROUP 4 (1000 MG/KG)
71	31	08-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
72	32	09-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
73	33	11-OCT-02	Yes	Breeding	01-NOV-02	06-NOV-02
74	34	08-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
75	35	08-OCT-02	Yes	Breeding	29-OCT-02	04-NOV-02
76	36	10-OCT-02	Yes	Breeding	01-NOV-02	06-NOV-02
77	37	08-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02
78	38	<**>.
78 (A)	36	18-OCT-02	No	Breeding		04-NOV-02
79	39	09-OCT-02	Yes	Breeding	31-OCT-02	04-NOV-02
80	40	08-OCT-02	Yes	Breeding	30-OCT-02	04-NOV-02

(A) non-pregnant

<**> No mating

- Table 3: Number of Pups (Dead + Living) F0-Generation - Post Coitum, Females Scheduled for Breeding

GROUP 1		GROUP 2		GROUP 3		GROUP 4
CONTROL		40 MG/KG		200 MG/KG		1000 MG/KG
Female Number	Number of pups	Female Number	Number of pups	Female Number	Number of pups	Female Number	Number of pups
41	6	51	14	61 (A)	-	71	9
42 (A)	-	52	17	62	11	72	11
43	17	53	14	63 (A)	-	73	15
44	11	54	15	64	15	74	7
45	10	55	17	65	15	75	14
46	8	56	21	66	12	76	13
47	16	58	12	67	19	77	11
48	12	59	16	68	14	78 (A)	-
49	13	60	11	69	12	79	4
50 (A)				70	15	80	12
Mean	11.6		15.2		14.1		10.7
St.dev.	3.7		3.0		2.5		3.5
N	8		9		8		9

Non pregnant females were not used for calculation of Mean, St.dev. and N (A) non-pregnant

+/++   U-test significant at 5% (+) or 1% (++) level

- Table 4: Mating Performance F0-Generation - Post Coitum, Number Of Females Mated During The First Pairing Period

Day of the pairing period	GROUP 1 CONTROL	GROUP 2 40 MG/KG	GROUP 3 200 MG/KG	GROUP 4 1000 MG/KG
1	4	2	5	5
2	2	4	3	2
3	1	-	1	1
4	2	1	1	1
6	1	-	-	-
Median precoital time	2	2	2	1
Mean precoital time	2.5	2.0	1.8	1.8
N	10	7	10	9

- Table 5: Number of Females Mated during the Second Pairing Period

Day of the	GROUP 1	GROUP 2	GROUP 3	GROUP 4
pairing period	CONTROL	40 MG/KG	200 MG/KG	1000 MG/KG
4	-	-	-	1
5	-	1	-	-
6	-	1	-	-
Median precoital time		6		4
Mean precoital time		5.5		4.0
N	0	2	0	1

+/++ e U-test significant at 5% (+) or 1% (++) level

- Table 6: Fertility, F0-Generation - Post Coitum, Females Scheduled for Breeding

	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	40 MG/KG	200 MG/KG	1000 MG/KG
Percentage mating	100.0	90.0	100.0	100.0
Fertility index (%)	80.0	100.0	80.0	90.0
Conception rate(%)	80.0	100.0	80.0	90.0
Gestation index (%)(Breeding)	100.0	100.0	100.0	100.0

Percentage mating = (Females mated / Females paired) * 100

Fertility index = (Females achieving a pregnancy / Females paired) * 100

Conception rate = (Females achieving a pregnancy / Females mated) * 100

Gestation index = (Number of females with living pups / Number of females pregnant) * 100

# / ## : Fisher's Exact Test significant at 5% (#) or 1% (##) level

- Table 7: Breeding Data per Group F0-Generation - Lactation

	GROUP 1 CONTROL	GROUP 2 40 MG/KG	GROUP 3 200 MG/KG	GROUP 4 1000 MG/KG
LITTERS TOTAL	8	9	8	9
DURATION OF GESTATION MEAN (+)	21.3	21.2	21.4	21.7
ST.DEV	0.46	0.44	0.52	0.50
N	8	9	8	9
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (8)	1	1	0	1
TOTAL	2	1	0	1
MEAN  (+)	0.3	0.1	0.0	0.1
ST.DEV	0.71	0.33	0.00	0.33
N	8	9	8	9
LIVING PUPS AT FIRST LITTER CHECK % OF MALES / FEMALES (8)	59 / 41	50 / 50	48 / 52	57 / 43
TOTAL	91	136	113	95
MEAN  (+)	11.4	15.1	14.1	10.6
ST.DEV	3.46	2.76	2.53	3.57
N	8	9	8	9
POSTNATAL LOSS DAYS 0 - 4 P.P.
% OF LIVING PUPS	2.2	3.7	6.2	1.1
LITTERS AFFECTED (8)	2	3	2	1
TOTAL (8)	2	5	7	1
MEAN  (+)	0.3	0.6	0.9	0.1
ST.DEV	0.46	0.88	2.10	0.33
N	8	9	8	9
LIVING PUPS DAY 4 P.P.
TOTAL	89	131	106	94
MEAN (+)	11.1	14.6	13.3	10.4
ST.DEV	3.14	2.88	3.06	3.54
N	8	9	8	9
VIABILITY INDEX (8)	97.8	96.3	93.8	98.9

# / ## : Fisher's Exact Test significant at 5% (8) or 1% (#8) level

Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) * 100

- Table 8: Mean Body Weights of Pups Per Group (Gram) F0-Generation - Lactation (Lactation)

DAY	SEX		GROUP 1 CONTROL	GROUP 2 40 MG/KG	GROUP 3 200 MG/KG	GROUP 4 1000 MG/KG
1		MEAN	6.6	6.5	6.6	7.1
		ST.DEV.	0.58	0.32	0.79	0.78
		N	8	9	8	9
	F	MEAN	6.3	6.1	6.3	6.8
		ST.DEV.	0.47	0.36	0.75	0.70
		N	8	9	8	9
	M+F	MEAN	6.5	6.3	6.5	7.0
		ST.DEV.	0.47	0.36	0.76	0.74
		N	8	9	8	9
4	M	MEAN	9.6	9.3	9.1	10.6
		ST.DEV.	1.20	0.81	1.93	1.59
		N	8	9	8	9
	F	MEAN	9.3	8.5	8.6	9.9
		ST.DEV.	1.10	0.84	1.53	1.33
		N	8	9	8	9
	M+F	MEAN	9.4	8.9	8.8	10.3
		ST.DEV.	1.09	0.84	1.67	1.45
		N	8	9	8	9

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

Applicant's summary and conclusion

Conclusions:

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. The definitive reproductive, breeding and developmental NOAEL was established as being 1000 mg/kg body weight/day.

Executive summary:

RS-Pantolactone (named DL-LACTONE in the study report) was tested for its 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.

Reproductive toxicity was assessed by observing the mating performance, fertility indices, and number of pups at birth. No reproductive toxicity was observed up to 1000 mg/kg b.w./day. Breeding toxicity was assessed by observing the number of postnatal and breeding loss during lactation. No breeding toxicity was observed up to 1000 mg/kg b.w./day.

Developmental toxicity was assessed by observing clinical signs, body weights and macroscopic examination of the pups during their lactation period. No developmental toxicity was observed up to 1000 mg/kg b.w./day.

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. Reproductive, breeding, and developmental parameters were unaffected up to 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. The definitive reproductive, breeding and developmental NOAEL was established as being 1000 mg/kg body weight/day.