Tetrahydrofurfuryl alcohol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

not specified

GLP compliance:

yes

Remarks:

Certificate is not included in the study report (see also Overall Remarks)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydrofurfuryl alcohol

EC Number:

202-625-6

EC Name:

Tetrahydrofurfuryl alcohol

Cas Number:

97-99-4

Molecular formula:

C5H10O2

IUPAC Name:

tetrahydrofuran-2-ylmethanol

Details on test material:

Test substance name: Tetrahydrofurfuryl alcohol
Supplier: not reported
Lot. no.: not reported
Purity: 99.5%
Melting point: less than -80°C
Appearance and property: Colorless liquid and freely soluble in water and acetone

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMAL:
-Source: Charles River Laboratories Japan Inc. (795 Shimofurusawa, Atsugi-shi, Kanagawa)
-Age at purchase: 8 weeks old
-Age at study initiation: 10 weeks old
-Acclimation period: male/female; 13 days
-Mean body weight at study initiation: male; 393 g (364-431 g), female; 234 g (208-275 g)
-Diet: Solid feed, labo MR stock, Nosan Corporation, Lot. No.021072, 021255 ad libitum
-Water: Tap water sterilized by filtration with 1 µm diameter cartridge filter and UV radiation, ad libitum

ENVIRONMENTAL CONDITION:
-Housing: Test animals were bred in a stainless steel metal wire cage (260W x 380D x 180H mm) in animal room (No.2) with barrier system.
- Temperature: 22 ± 3°C
- Humidity: 55 ± 10%
- Air change: more than 10 times per hour
- Photoperiod: 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Remarks:

Japanese pharmacopoeia purified water (KYOEI Pharmaceutical Industries, Ltd. Lot. No.181376)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS;
The dosing formulation was prepared to be the aqueous test formulation of the pre-determined dose concentration using Japanese pharmacopoeia purified water (KYOEI Pharmaceutical Industries, Ltd., Lot No. 181376).
The stability test was performed for 20% and 0.2% test substance solution, as a results, the test substance solution was stable at least for 7 days under the cold and dark conditions (4°C). Therefore, the expiration period for use of the dosing formulation was set within 7 days after preparation of dosing formulation, and each prepared formulation was subdivided by daily consumption, sealed, shaded and stored in a cold place (4°C). Moreover, nominal concentration of the test substance in the dosing formulation was confirmed by analyses using first prepared dosing formulation.

VEHICLE;
The test substance is freely soluble in water. Therefore water is selected as vehicle.

TREATMENT METHODS;
For administration method, the dosing formulation of 5 mL/kg body weight was treated by oral gavage using a syringe with stomach tube made by Teflon. Control group was administered with Pharmacopoeia purified water which was used as the vehicle in the same manner. Dosages were calculated based on the most recent measured body weight individually.

Details on mating procedure:

After the completion of 2-week administration before mating (in the afternoon of day 15 of administration), treated animals were paired on a 1 male: 1 female basis in each dose group and housed in the same cage up to 2 weeks until copulation was noted. During the mating period, copulation check was performed approximately on the constant time every morning (about 9:30). The copulation was confirmed by presence of a vaginal plug or presence of sperm in the vaginal smear and the day when copulation was confirmed was designated as day 0 of gestation.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Treatment period;
Both sexes;
2 weeks of before mating period
Male;
47 days including 2 weeks before mating period
Female;
Shortest 42 days – longest 52 days including 2 weeks before mating period, a cohabitation period, a gestation period and a lactation day 4 after delivery

Frequency of treatment:

Frequency of treatment;
Treatment was performed once daily approximately in the morning (9:00 – 12:00)

Details on study schedule:

After the completion of 2-week administration before mating (in the afternoon of day 15 of administration), treated animals were paired on a 1 male: 1 female basis in each dose group and housed in the same cage up to 2 weeks until copulation was noted.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale;
In the single oral dose toxicity study, lethal dose level of tetrahydrofurfuryl alcohol was more than 2000 mg/kg. In the 14-day dose range-finding study in 5 weeks old rats at oral dose levels of 50, 100, 200, 500 and 1000 mg/kg/day, there were no treatment-related changes in the 50 mg/kg group. In the 100mg/kg group, an increase in motor activity was noted in only 1 female. In the 200 mg/kg group, an increase in motor activity in female and the lower value in absolute and relative weight of thymus and pituitary in female were noted. In the 500 mg/kg group, the increase and decrease in motor activity, the lower value in food consumption and enlargement of the cecum in both sexes, and piloerection in some males, a tendency to suppressed body weight gain in male, and the lower value in absolute and relative weight of thymus and pituitary in female were noted. In the 1000 mg/kg group, piloerection and the lower value in absolute and relative thymus weights in female were noted in addition to the changes noted in 500 mg/kg group.
According to the above results, the maximum dose level in the present study was set at 500 mg/kg/day which is supposed to cause the toxic effect and the minimum dose level was set at 15 mg/kg/day which is supposed to cause the no toxic effect. Between these 2 doses, dose levels of 50 and 150 mg/kg/day were set (4 doses in total).
Five test groups were used in the study with the composition as follows; (1) vehicle administration group (control group hereinafter), (2) 15 mg/kg/day of the test substance administration group (15 mg/kg group), (3) 50 mg/kg/day of the test substance administration group (50 mg/kg group), (4) 150 mg/kg/day of the test substance administration group (150 mg/kg group) and (5) 500 mg/kg/day of the test substance administration group (500 mg/kg group).

Positive control:

not used

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS:
- All animals were observed at least twice a day, i.e., in the morning and evening, for the general appearance, behavior, morbidity and mortality. Especially, state of gestation, delivery and lactation of rats were closely observed.

BODY WEIGHT:
- Males were weighed on treatment day 1 (the initiation day of the treatment) , 8, 15, 22, 29, 36, 43 and 47. Females were weighed on treatment day 1, 8 and 15, gestation day 0, 7, 14 and 20, lactation day 0 and 4.

FOOD CONSUMPTION:
- For the food consumption, 24 hours intake per cage until the next day was measured on the same day of the body weight measurement. The final measurement of the food consumption was performed on the treatment day 46 for male and on the lactation day 3 for female. The measurement of the food consumption were not performed on treatment day 15 during the cohabitation period for all animals in both sexes, on treatment day 22 for males and females who’s copulation was not confirmed.

WATER CONSUMPTION: No data

Oestrous cyclicity (parental animals):

For all the females, vaginal smears specimens stained with Giemsa were prepared, and determined into each stage of estrus cycle by microscopy during acclimatization and quarantine period and until confirmation of copulation.

Sperm parameters (parental animals):

no data

Litter observations:

(1) Number of newborns, sex ratio and external observation;
After confirmation of delivery completion, each litter size (total of live and dead pups) were counted and delivery index (%) [(total number of pups delivered / number of implantation sites) x 100] was determined. The individually sex was determined by anogenital distance and the sex ratio was calculated ever group. Any external abnormalities including the oral cavity in pups were observed.
(2) General condition;
General condition, morbidity and mortality were examined daily and live birth index (%) [(number of live birth / total number of newborns) x 100] and newborn viability index (%) [(live pups on lactation day 4 / number of live birth) x 100] were determined.
(3) Body weight measurement;
Each litter was weighed per sex on lactation day 0 and 4 and mean value per 1 animal was calculated.
(4) Gross examination;
Rats were euthanized by diethyl ether anesthesia and exsanguination. The main organs in the thoracic and abdominal cavity of gross examinations were performed within the day of being found dead for dead animals and on lactation day 4 for living pups.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals:
On the next day of the treatment 47 for all surviving male animals.
- Female animals:
On the lactation day 5 for females which showed normal delivery and lactation.
On the 24 days after the end of the mating period (the next day of the treatment day 52) for female which was not confirmed its copulation in the 15 mg/kg group.
Within the 4 days that all litter loss was confirmed for all litter loss 8/12 females in the 150 mg/kg group.
On the next day, following the continuous observation was carried out until the 4 day after the expected day of delivery for 2 females in the 150 mg/kg group and all females in the 500 mg/kg group those delivery were not confirmed after the expected day of delivery.
GROSS NECROPSY
Each rats were euthanized by diethyl ether anesthesia and exsanguination. The body surface, mucous membrane of orifices and internal organs of gross examinations were performed.
HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weight;
The pituitary, thymus, kidney in both sexes, testis and epididymis in male were weighed (absolute weight), and the ratio to the body weight (relative weight) was calculated based on the body weight measured on the day of necropsy. For females, number of corpora lutea and number of implantation sites were counted, and implantation index (%) [number of implantation sites / number of corpora lutea x 100] was determined.The pituitary, thymus, kidney in both sexes, testis and epididymis in male were weighed (absolute weight), and the ratio to the body weight (relative weight) was calculated based on the body weight measured on the day of necropsy. For females, number of corpora lutea and number of implantation sites were counted, and implantation index (%) [number of implantation sites / number of corpora lutea x 100] was determined.
Histopathology;
The following were collected from the animals, fixed by immersion in a 10% neutral phosphate-buffered formalin solution (the testes and epididymis were pre-fixed in Bouin’s solution) and were stored in a 10% neutral phosphate-buffered formalin solution.
brain, pituitary, thyroid (include the parathyroid), thymus, trachea, lung, stomach, intestine, heart, liver, spleen, kidney, adrenal gland, bladder, testis, epididymis, prostate gland, seminal vesicle, ovary, uterus, spinal cord (cervix, thorax, lumbar), sciatic nerve, bone marrow (femur), lymph node (neck and mesentery), mammary gland and gross regions.
For the testis, epididymis, ovary, pituitary, thymus and spleen with gross region which was considered to be the test substance treatment change of animals from vehicle control group and 500 mg/kg, the histopathological examination was performed. For the spleen, selected 5 animals which were allocated by stratified random sampling method at the initiation of treatment and 12 animals with gross region which was considered to be the test substance treatment related change were examined. As the results, effect of the test substance treatment was noted in testis, epididymis, thymus and spleen, therefore, these organs were received a histopathological examination on the animals in the 15, 50 and 150 mg/kg group which were allocated by stratified random sampling method at the initiation of treatment. Moreover, in the 15 mg/kg group, for a male of a pair which copulation was not confirmed and died after the mating period, (animal No. 022), testis, epididymis, pituitary, thymus, brain, spinal cord, stomach, intestine (include the Peyer's patch), adrenal gland, spleen, heart, liver, kidney, thyroid (include parathyroid), trachea, lung, bladder, sciatic nerve, bone marrow, lymph node (neck and mesentery) and for a female of pair mentioned above, ovary and uterus were histopathologically examined. In the 15 and 150 mg/kg group, for the male paired with the non-pregnant female, testis, epididymis, prostate gland and seminal vesicle and for the non-pregnant female, ovary, uterus and pituitary were histopathologically examined. For the female which became pregnant but no delivery was observed, ovary, uterus and pituitary were examined, and moreover for the female which were delivered but all litter was loss during lactation period, mammary gland was also examined histopathologically. According to a conventional method, organs were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and examined microscopically.

Postmortem examinations (offspring):

SACRIFICE:
- For living pups;
On lactation day 4
Rats were euthanized by diethyl ether anesthesia and exsanguination.
- For dead pups;
Within the day of being found

HISTOPATHOLOGY / ORGAN WEIGTHS:
Rats were euthanized by diethyl ether anesthesia and exsanguination. The gross examinations were performed for the main organs in the thoracic and abdominal cavity of the dead animals on the day when the animal was found dead and of live pups on day 4 of lactation.
Organ weight: no data

Statistics:

The obtained mean value and incidence was statistically analyzed significant difference (significance level: < 5%) between the vehicle control group and each test substance treatment group using following methods.
The Bartlett’s test was performed on the parametric data (body weight, food consumption, organ weight, number of corpora lutea and implantation sites, gestation length and litter size). When homogeneous variances were found, a one-way analysis was performed, then, when significant differences were found, the comparative test between the vehicle control group and each test substance treatment group was performed using the the Scheffe’s test. When heterogeneous variances were detected, and for the nonparametric data (implantation index, birth rate, delivery index, viability index of newborn, incidence of external and visceral findings of pups), Kruskal-Wallis test was performed. As a results, when significant differences were found, the Scheffe’s type was performed. For the categorical data examination), Fisher’s exact probability test(clinical signs, incidence of the abnormal findings in necropsy and histopathological or Chi-square test (copulation index, fertility index, gestation index and sex ratio) was performed. For litter parameters, the litter mean was used as the experimental unit.

Reproductive indices:

no description

Offspring viability indices:

no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 150 mg/kg/day and more, the increase of motor activity was noted in the both sexes.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day and more, the body weight gain was significant decrease was noted in the both sexes. [SIAR, 2005: reduced body weight gain in males at 500 and females at >=150 mg/kg bw/day. Reduced food consumption, both sexes at >= 150 mg/kg bw/day.]

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day and more, the body weight gain was significant decrease was noted in the both sexes. [SIAR, 2005: reduced body weight gain in males at 500 and females at >=150 mg/kg bw/day. Reduced food consumption, both sexes at >= 150 mg/kg bw/day.]

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The effect related to the test substance treatment was noted in testis, epididymis, thymus and spleen. {SIAR, 2005: these effects at 150 mg/kg bw/day and above.]

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

In the 150 mg/kg/day, the tendency of prolonged estrus cycle was noted. [SIAR, 2005: significant increase in oestrus cycle at 500 mg/kg bw/day.]

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day and more, prolonged gestation period. 150 mg/kg group, parturition was noted in 9 out of 11 females, but the Parturition of 5/9 dams were not observed. [SIAR, 2005: no live births at 500 and reduced live births at 150 mg/kg bw/day.]

Details on results (P0)

1. Repeated dose toxicity;
(1) General condition and mortality:
No abnormalities were noted in the general condition of the vehicle control group, 15 and 50 mg/kg groups.
In the 150 mg/kg group, slight increase in motor activity was noted in both sexes. Particularly in males, slight decrease in motor activity was sporadically noted following the increase in motor activity during the first half of the treatment period.
In the 500 mg/kg group, slight increase in motor activity and accompanied decrease in motor activity was noted in many animals through the treatment period. Males showed tendency to have high incidence of decrease in motor activity compared to females. In 2 females of the 500 mg/kg group, slight vaginal hemorrhage was noted in the latter gestation period. In the 15 mg/kg group, one male (animal number 022) which did not achieve copulation showed moderate decrease in motor activity from treatment day 20 and died on treatment day 22.
(2) Body weight:
In 15 and 50 mg/kg group of both sexes, the body weight and body weight gain showed no significant differences compared to vehicle control group. In the male of the 150 mg/kg group, although there were no significant differences, body weight gain showed the tendency of suppression after treatment day 36 and body weight gain during treatment period significantly decreased. For females, the body weight on gestation day 20 showed significant lower value compared to the control group and body weight gain during the gestation period significantly decreased.
In the 500 mg/kg group, the significant suppression of body weight gain and significant decrease of body weight gain during treatment period were noted in male from treatment day 8. For female, significant suppression of the body weight gain and significant decrease of the body weight gain during the gestation period were noted from gestation day 14.
(3) Food consumption:
In the 15 mg/kg group of both sexes, no significant change was noted in food consumption during the study period. In the male of the 50 mg/kg group, the significant decrease was noted in food consumption only on treatment day 22 compared to the control group. In the 150 mg/kg group, significant decrease of food consumption was noted on treatment day 8 in male and on gestation day 14 and 20 in female. In the 500 mg/kg group, significant decrease of food consumption was noted on treatment day 1, 8 and 22 in male and on treatment day 1, gestation day 0, 14 and 20 in female.
(4) Necropsy:
On terminal killed males, small-sized testis and epididymis were noted in one animal of the control group, 15 and 150 mg/kg group, respectively. For the 500 mg/kg group, these findings were noted in 10 animals. The males which showed small-sized testis and epididymis in the 15 and 150 mg/kg group did not achieve pregnancy in females. The rough surface and white spot (s) / area of spleen were noted, and the incidence of rough surface was noted in 6 animals in the 500 mg/kg group and white spot (s) / area was noted in one and 4 animals in the 150 and 500 mg/kg groups, respectively. Dark redness in lung, grayish white spots in heart and small-sized spleen and thymus were noted in one male which did not achieve copulation and died in the 15 mg/kg group.
On terminal killed females, small-sized thymus was noted in one animal in the control group and 50 mg/kg group, respectively, and red spots of thymus was noted in one female in the 15 mg/kg group. No other changes were noted. No change was noted in one animal which did not achieve copulation in the 15 mg/kg group, one animal which did not achieve pregnancy in the 15 and 150 mg/kg groups, respectively. In 8 females which lost all litter after delivery in the 150 mg/kg group, atrophy of thymus was noted in one animal and rough surface of spleen was noted in 2 dams. In 2 dams in the 150 mg/kg group and 12 dams in the 500 mg/kg group which lost all litter, early resorption of rice grain or smaller size and mummified fetus in uterus were noted in one female of the 150 mg/kg group, and early resorption was noted in uterus of all female including the 500 mg/kg group. Moreover, in the 500 mg/kg group, small-sized thymus was noted in 5 females. Additionally, rough surface of spleen in 11 females and white spot (s) / area in 5 females were noted, and on a part of spleen, adhesion with neighboring organ / tissue was accompanied in 2 females.
(5) Organ weight:
No significant change was noted in the final body weight and measured organ weight in the 15 and 50 mg/kg group. In the 150 mg/kg group, tendency to decrease of the final body weight in male, significant decrease in female were noted. The absolute weight of pituitary showed significant decrease in both sexes, but significant change was not noted in relative weight. For the kidney weight of female, absolute weight showed tendency to slight decrease and relative weight showed significant increase. In male of the 500 mg/kg group, significant decrease of the final body weight was noted, and significant decrease was noted in the relative and absolute weight of thymus, testis and epididymis. A significant decrease was also noted in the absolute weight of kidney and pituitary but no significant change was noted in the relative weight.
(6) Histopathological examination:
(a) The organs excluding the genital system;
Atrophy of thymus was noted also in one female of the vehicle control group, but the incidence of atrophy of thymus showed tendency to increase in female of the 150 mg/kg group and in both sexes of the 500 mg/kg group, and the significant difference was noted in the incidence of this finding in males of the 500 mg/kg group. In spleen, the grade of the extramedullary hematopoiesis of splenic red pulp tended to decrease in both sexes of the 150 and 500 mg/kg group, significant difference was noted in female of the 150 and 500 mg/kg group, and atrophy of splenic red pulp was indicated. In spleen, inflammation such as cellular infiltration and fibrosis of capsule was noted in 3 / 5 males and in 2 / 5 females in the 150 mg/kg group, and in 11 / 12 males and 12 / 12 females in the 500 mg/kg group. Dead animal in the 15 mg/kg group (animal number 022) showed the necrosis/mineralization of the myocardial, congestive edema of lung, atrophy of thymus and spleen and basophilic tubules of kidney. It was estimated that this death was mainly due to the necrosis/mineralization of the myocardial, congestive edema of lung. No changes were noted in mammary gland of the females which lost all litter after delivery.
(b) Genital systems;
In the males which successfully induced pregnancy in females, atrophy of seminiferous tubule was noted in all 12 males of the 500 mg/kg group and 10 of them also showed hyperplasia of the interstitial cell. These animals showed decrease of sperm in lumen of the seminiferous tubules and germ cell was disappeared excluding Sertoli cell in the severely affected regions. Atrophy of seminiferous tubule was also noted in 3 males of the vehicle control group (including 2 males with focal change) and hyperplasia of the interstitial cell was also noted in one of them. Decrease of sperm in lumen of epididymis and cell debris was noted in epididymis of the animals which showed atrophy of seminiferous tubule, including the animals of the vehicle control group. Atrophy of seminiferous tubule in testis, hyperplasia of the interstitial cell and decrease of sperm in lumen of epididymis and cell debris were noted also in one animal in the 15 mg/kg group and one animal in the 150 mg/kg group which failed inducing pregnancy in females, and infiltration of interstitial cell was additionally noted in prostate gland in the animal of the 150 mg/kg group.
Placental remnant in uterus was noted in 2 females of the 150 mg/kg group and all 12 females in the 500 mg/kg group with total fetal death. No change was noted in ovary.
2. Reproductive and developmental toxicity:
(1) Estrus cycle;
From the time of grouping to before mating, the estrus cycle of females in the vehicle control group was approximately 4 days. However, one female (animal number 501) showed 6 days and one female (animal number 512) showed 5 days, and the mean length of the estrus cycle was 4.3 days. On the other hand, in the 15 and 50 mg/kg groups, the estrus of estrus were approximately 4 days and mean length of the estrus cycle were 4.0 and 4.1 days, respectively. In the groups of 150 mg/kg/day and above, the estrus cycle of animals tended to prolong compared to the vehicle control group. The estrus cycle of 4 females in the 150 mg/kg group and 7 females in the 500 mg/kg group was more than 5 days. In the 150 mg/kg group, mean length of the estrus cycle was 4.5 days. In the 500 mg/kg group, mean length of the estrus cycle was 4.8 days and significantly longer compared to the vehicle control group. Moreover, sporadically some individual showed metestrus stage I (III) with consecutive 2 or 3 days.
(2) Copulation index and fertility index;
Copulation was successful in all pairs except for a pair in the 15 mg/kg group, and no significant difference was noted in the period to achieve copulation between each test substance treatment group and the vehicle control group. The male (animal number 022) of a pair which did not achieve copulation died on treatment day 22 and the female of this pair was continuously mated with another male (animal number 023) which achieved copulation with another female, but copulation was not confirmed. Infertility was noted in one animal each in the 15 and 150 mg/kg groups but no significant difference was noted in the fertility index between the vehicle control group and the test substance treatment group.
(3) Number of corpora lutea and implantation sites and implantation index;
There was no significant difference in number of corpora lutea, implantation sites and implantation index between each test substance treatment group and the vehicle control group.
(4) Delivery index and gestation length;
Delivery index was 100% in the vehicle control group, 15 and 50 mg/kg group. In the 150 mg/kg group, delivery index was 36.4% and significantly decreased. In the 500 mg/kg group, no females delivered. There was no significant difference in the gestation length between each group of 15 and 50 mg/kg groups and the vehicle control group. The significant prolongation in gestation length was noted in the 150 mg/kg group.
(5) Parturition and lactational condition;
No abnormality was noted in parturition and lactational condition in the vehicle control group, 15 and 50 mg/kg groups. In the 150 mg/kg group, parturition was noted in 9 / 11 females. The initiation of parturition was confirmed in 5 females of them in the evening but lactation was not observed and all litters were dead or cannibalized in the next morning. Lactation was noted in other 4 dams but the amount of milk in the stomach which was transparent over the abdominal wall of pups was small.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: repeat dose toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

3

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In the 150 mg/kg group, 4 dams had live pups on checking of delivery, and the number of total newborn per litter, delivery index, litter size and birth rate significantly decreased.

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The systemic edema was noted in one pup out of 28 newborns from 4 litters in 150 mg/kg group but no external and visceral abnormalities were noted in pups of any other groups. [SIAR, 2005: no significant increase in morphological abnormalities.]

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Thymic remnant in neck was noted in each group as the visceral variations. Persistent left umbilical artery was noted in the control group (1.2 %) and in the 50 mg/kg group (2.2 %) but no significant difference was noted between the groups.

Details on results (F1)

1. Viability and body weight:
There was no significant difference in number of total newborn per litter, delivery index, litter size, birth rate, sex ratio, body weight on day 0 of lactation and viability index and body weight on day 4 of lactation of the 15 and 50 mg/kg group compared to the vehicle control group. No abnormality was noted in general condition of the newborns. In the 150 mg/kg group, 4 dams had live pups on checking of delivery, and the parameter of number of total newborn per litter, delivery index, litter size and birth rate was significantly decreased. More than half of newborns died on day 0 of lactation. Pups survived up to day 4 of lactation were only 4 from a litter and all of them showed the lowered body weight. In the 500 mg/kg group, no female delivered and no newborn was obtained.
2. Morphology:
The systemic edema was noted in one pup out of 28 newborns from 4 litters in 150 mg/kg group but no external and visceral abnormalities were noted in pups of any other groups. Thymic remnant in neck was noted in each group as the visceral variations. In the 50 mg/kg group, this incidence was slightly high (8.4 %) but no significant difference was noted compared to the control group (3.2 %). Additionally, persistent left umbilical artery was noted in the control group (1.2 %) and in the 50 mg/kg group (2.2 %) but no significant difference was noted between the groups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

viability

mortality

other: development

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Tetrahydrofurfuryl alcohol showed the toxicity mainly to testis, epididymis, spleen and thymus in the repeated treatment in rats.

It was judged that the no observed adverse effect level in repeated administration in the parental animal was 50 mg/kg bw/day in both males and females. For reproduction of male parent animal the NOAEL was 150 mg/kg bw/day and for reproduction of female parent animal and development of the pups 50 mg/kg bw/day.

Table 1 - Reproductive Results

Dose (mg/kg/day)	0	15	50	150	500
Estrous cycle (mean days)	4.3	4.0	4.1	4.5	4.8*
No. pairs mated	12	12	12	12	12
No. pairs with successful copulation	12	11	12	12	12
Copulation index (%)	100	91.7	100	100	100
Mean days until copulation	2.7	2.5	2.9	2.3	3.7
No. of pregnant females	12	10	12	11	12
Fertility index (%)	100	90.9	100	91.7	100
Mean No. of corpora lutea	17.7	16.5	17.8	16.4	17.0
Mean No. of implantation sites	15.6	15.3	16.1	13.7	14.5
Mean Implantation index (%)	88.8	93.5	90.7	84.5	87.9
No. of pregnant females with parturition	12	10	12	9	0
Gestation length (mean days)	22.6	22.7	22.9	24.0**	-
No. of pregnant females with live pups	12	10	12	4	-
Gestation index (%)	100	100	100	36.4**	-
No. of pregnant females with live pups on Day 4	12	10	12	1	-

* p<0.05

** p<0.01

Table 2 - Litter Results

Dose (mg/kg/day)	0	15	50	150	500
Mean No. of pups born	14.8	14.5	14.8	7.0**	-
Delivery index	95.3	94.7	91.9	46.4*	-
Mean No. pups alive on Day 0            Total            Male            Female	14.8 7.2 7.7	14.5 7.2 7.3	14.6 6.8 7.8	3.0** 1.5** 1.5**	- - -
Live birth index (%)	100	100	98.8	43.1*	-
Sex ratio (male/female)	0.93	0.99	0.90	1.15	-
Mean No. pups alive on Day 4            Total            Male            Female	14.7 7.0 7.7	14.4 7.2 7.2	14.3 6.7 7.6	1.3** 0.7** 0.7**	- - -
Viability index (%)	98.9	99.3	97.7	26.7	-
Mean body weight live pups Day 0            Male            Female	7.3 7.0	7.4 7.0	7.1 6.9	5.9 6.3	- -
Mean body weight live pups Day 4            Male            Female	11.8 11.2	11.5 10.9	11.0 10.7	9.1 8.4	- -

 * p<0.05

** p<0.01

Applicant's summary and conclusion

Conclusions:

An oral combined repeated dose/reproductive and developmental screening test in the rat, conducted ina manner similar to the current guideline and in accordance with GLP, identified an NOAEL of 150 mg/kg bw/day for the parental generation males, and of 50 mg/kg bw/day for dams and pups.