Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Three good quality oral studies are available; one a 28 -day reproductive/developmental screening test, and two repeated dose oral studies (13-week dietary and 28-day gavage). Good quality 13-week repeated dose inhalation and dermal studies are also available. Of these studies the oral reproduction/developmental screening test was chosen as the key study.

Link to relevant study records

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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
Reason / purpose for cross-reference:
reference to same study
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
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
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.
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
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
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
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: 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.]
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.


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
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)
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)
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.
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.
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
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

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

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.
Endpoint:
fertility, other
Remarks:
effects on reproductive organs in 90-day repeated inhalation toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994-10-03 TO 1995-01-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Guideline:
other: OECD 413
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
clean air
Details on mating procedure:
no mating
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6hr/day, 5 days/week, at least 65 exposures (equivalent to 13 weeks)
Frequency of treatment:
daily on weekdays
Details on study schedule:
exposure of both sexes, no mating
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
150 ppm (nominal)
Dose / conc.:
500 ppm (nominal)
No. of animals per sex per dose:
10, with a further 4 males per concentration to examine spermatogenic effects.
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEC
Effect level:
50 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: reproductive effects
Dose descriptor:
NOAEC
Effect level:
< 50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general systemic effects
Reproductive effects observed:
not specified

PARENTAL ANIMALS

CLINICAL SIGNS AND MORTALITY

1/10 females died in at 50 ppm during week 0, the cause of death was not found to be treatment-related. The predominant clinical finding was intermittent whole-body spasms in all treated groups that were frequent and exposure-related. Hypoactivity and excessive grooming were occasionally observed in males and females at 500 ppm. One hour after exposure, concentration-related hyperactivity was reported in all treated groups, and yellow urogenital matting and salivation at 500 ppm.

 

BODY WEIGHT AND WEIGHT GAIN

Mean body weight gains were lower than controls in males at 150 and 500 ppm (9.2% and 13.3%, respectively).

 

FOOD CONSUMPTION

Mean food consumption was generally lower in mid and high exposure males throughout the study

 

OPHTHALMOSCOPIC EXAMINATION

No treatment-related observations.

 

HAEMATOLOGY

Several statistically significant treatment-related changes were reported after exposure to 500 ppm. Platelet and haemoglobin means were significantly reduced in both sexes (study weeks 3 and/or 13). MCH (mean cell haemoglobin) was reduced in males (weeks 3 and 13) and females (week 13); MCHC (mean corpuscular haemoglobin concentration) was reduced in males (week 13) and females (week 3).

 

CLINICAL CHEMISTRY

No treatment-related effect.

 

ORGAN WEIGHTS

Prostate weight (absolute and relative) were lower than controls in 150 and 500 ppm groups. The weights of seminal vesicles (absolute) and epididymides (absolute and relative) were lower at 500 ppm.

 

GROSS PATHOLOGY

No macroscopic lesions in any exposure group.

 

HISTOPATHOLOGY: NON-NEOPLASTIC

Spermatogenic findings: reduced epididymal sperm numbers and motility, and a higher incidence of morphological abnormalities were seen in 500 ppm males at weeks 6 and 13. Multifocal atrophy of the testes was reported in a single male at 500 ppm.

Conclusions:
A 13-week rat inhalation study, conducted according to the current guideline (OECD 413) and in accordance with GLP, reported overt toxicity at the lowest tested nominal concentration of 50 ppm (around 0.2 mg/l) and male reproductive effects at 150 ppm (around 0.6 mg/l), and above.
Endpoint:
fertility, other
Remarks:
effects on reproductive organs in 90-day repeated dermal toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994-09-08 to 1994-12-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD 411
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
not mated
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6h/day, 5 days/week, 13 weeks
Frequency of treatment:
daily (weekdays)
Details on study schedule:
application to both sexes, no mating
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
17 males, 12 females (5 males at each dose terminated at 7 weeks for examination of spermatogenic endpoints)
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: spermatogenic effects at 300 mg/kg bw/day
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Reproductive effects observed:
not specified

PARENTAL ANIMALS

SPERMATOGENIC ENDPOINTS:

Administration of up to 1000 mg THFA/kg bw/day for 7 weeks (interim necropsy) had no effect on spermatogenic endpoints (testicular epididymal sperm numbers, sperm production rate, sperm motility, sperm morphology).

 

Mean numbers of sperm present in testicular homogenates and mean sperm production rate were lower than controls in 300 and 1000 mg/kg bw/day males after 13 weeks treatment (p<0.01). One male in the 1000 mg/kg bw/day group had markedly lower values for these parameters but the the group mean was still significantly reduced (p<0.01) when the values for this male were omitted. Sperm motility was reduced at 1000 mg/kg bw/day (p<0.05).

 

BODY WEIGHT AND WEIGHT GAIN

8-10% lower weight gain in 1000 mg/kg bw/day males comapred to controls (p<0.05 or 0.1; weeks 9-13). 7% lower weight gain in 1000 mg/kg bw/day females compared to controls (p<0.05; weeks 12, 13).

 

ORGAN WEIGHTS

No treatment-related effect on organs including: epididymis, testis, female mammary gland, ovary.

 

GROSS PATHOLOGY

No treatment-related observations.

 

HISTOPATHOLOGY: NON-NEOPLASTIC

No treatment-related effects at any dose on tissues including: epididymis, female mammary gland, ovary, prostate, seminal vesicles, testis, vas deferens.

Conclusions:
A 13 week dermal study, conducted according to the current guideline (OECD 411) and GLP, identified spermatogenic effects at 300 mg/kg bw/day but not at 100 mg/kg bw/day. These effects were not detected after 7 weeks treatment.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two further good quality repeated dose studies recorded reproductive effects.

In a 13-week dietary study (WIL, 1993) there were clear effects on the male reproductive system (e.g. reduced testis and epididymis weight, soft testes, changes to germinal epithelium of the seminiferous tubules) at 5000 ppm in the diet (mean measured dose 339 mg/kg bw/day). The NOAEL for clear reproductive effects was 1000 ppm (69 mg/kg bw/day). This study was well conducted and reported, and complied with GLP but not meeting the requirements of a standard reproduction study it was judged to be reliability 2 (reliable with restrictions).

A 28-day oral study (MHLW, 2004a) also identified effects on the male reproductive system (e.g. necrosis of seminiferous epithelium, reduced ratio of spermatid:sertoli cells) at 150 mg/kg bw/day, or higher. The necrotic effect tended to worsten during the 14-day untreated recovery period. Again this study was well conducted and reported, with GLP, but was not a standard reproduction study and as such was judged to be reliability 2 (reliable with restrictions).
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
200 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
No further inhalation studies were identified.
Effect on fertility: via dermal route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
No further dermal studies were identified.
Additional information

In a 28 -day reproductive/developmental toxicity screening test, THFA was administered by oral gavage to male and female rats at doses of 0, 15, 50, 150 or 500 mg/kg/day for around 50 days. In males, effects on the testes, epididymis and seminiferous tubules (including reduced weights of testes and epididymides, atrophy of seminiferous tubules, reduced intraluminal sperm) at 500 mg/kg/day were noted. In females effects including prolonged gestation length and oestrus cycle at 150 and 500 mg/kg/day, were noted. Based on these results the No-Observed-Adverse-Effect-Level for reproductive effects in the parental generation was 150 mg/kg/day for males, and 50 mg/kg/day for females. The NOAEL for maternal toxicity was also 50 mg/kg/day based on decreased body weight gain, haematological and endocrine effects.

Further information on reproductive effects of THFA comes from repeat dose toxicity studies in which effects on the male reproductive organs were noted (see Section 5.6.3 for full details).

Two further repeat dose oral studies, one a 90-day dietary study, the other a 28-day oral gavage study, resulted in similar effects which included lower testes and epididimal, prostate and seminal vesicle weights, degeneration or necrosis of germinal epithelium of seminiferous tubules and interstitial (peritubular) oedema in the testes and empty or fluid filled tubules with accumulation of cellular debris in the epididymides.

A 90-day inhalation study was conducted in which THFA was administered to rats at 0, 50, 150 or 500 ppm. Prostate, seminal vesicle and epididymides weight were lower at 150 and/or 500 ppm. Microscopically, reduced epididymal sperm numbers and motility and multifocal atrophy of the testes were recorded at 500 ppm. Based on the effects on the male reproductive organs No-Observed-Adverse-Effect-Level for male reproductive effects was considered to be 50 ppm.

A 90-day dermal study was conducted in which THFA was administered to rats at 0, 100, 300 or 1000 mg/kg/day. Mean numbers of sperm present in testicular homogenates and mean sperm production rate were lower than controls in 300 and 1000 mg/kg/day males and sperm motility was reduced at 1000 mg/kg/day. Based on the spermatogenic effects the No-Observed-Adverse-Effect-Level for male reproductive effects was considered to be 100 mg/kg/day.

No data are available from an extended one-generation study with THFA.

Further testing for reproductive toxicity is not required if a substance already meets the criteria for classification as toxic for reproduction category 1A or 1B. Fertility and development must be considered separately. No further testing of THFA is required for developmental toxicity, as THFA is classified category 1B for developmental toxicity. Therefore, the main aim of an EOGRTS would be to consider whether a more severe classification for fertility is appropriate.

Animal testing should be avoided if possible; the information derived from any animal study should justify the sacrifice of animals. An EOGRTS requires at least ~1400 animals; the extension to the second generation and inclusion of Cohorts 2A, 2B and 3 could increase this number to as many as 2600 animals. It is questionable whether the use of these experimental animals would provide a higher degree of protection for human health. DNELs and risk characterisation are already based on repeated dose systemic effects which occur at the same doses as or lower doses than reproductive or developmental effects.

Information has been considered for structural analogues, 2-methoxyethanol (CAS 109-86-4) and 2‑ethoxyethanol (CAS 110-80-5), both of which are classified for toxicity to reproduction, category 1b, with the hazard phrases ‘May damage the unborn child’ and ‘May damage fertility (H360FD)’, according to Regulation (EC) 1272/2008.

The systemic toxicity and reproductive effects seen with THFA are very similar to those reported for the structural analogues 2-methoxyethanol and 2-ethoxyethanol, and occur at similar doses. In view of the consistency of effects and the structural similarities it is considered appropriate to conclude that THFA has a similar mechanism of toxicity to the structural analogues 2-methoxyethanol and 2-ethoxyethanol and therefore should be classified consistently with 2-methoxyethanol and 2-ethoxyethanol. Therefore, the requirement for extended one-generation testing is waived. The similarities between THFA and the analogues 2-methoxyethanol and 2-ethoxyethanol are discussed in detail in the document attached to this endpoint summary.





Effects on developmental toxicity

Description of key information

A 28 -day reproductive and developmental toxicity screening study conducted by the oral route is selected as the key study. An oral range finding developmental toxicity study is also available.

Link to relevant study records
Reference
Endpoint:
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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 421 (Repeated Dose Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
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
Route of administration:
oral: gavage
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.
Analytical verification of doses or concentrations:
not specified
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.
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:
Once daily in the morning
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
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).
Maternal 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
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
After the completion of delivery was confirmed, each litter size (total of live and dead pups) were counted and parturition index (%) [(number of total newborn / number of implantation sites) x 100] was determined. The sex was determined individually by ano-genital distance and the sex ratio was calculated for each group. Any external abnormalities including the oral cavity in pups were observed.

General condition and mortality were examined daily and live birth index (%) [(number of live birth on checking of delivery / total number of newborns) x 100] and newborn viability index (%) [(live pups on lactation day 4 / number of live birth on checking of delivery) x 100] were determined.

Each litter was weighed per sex on days 0 and 4 of lactation and mean value was calculated per animal.

Pups 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.
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.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
General condition and mortality
No abnormalities were noted in the general condition of the control group, 15, and 50 mg/kg groups. In the 150 mg/kg group, slight increase in motor activity was noted after 5 - 15 minutes of administration. In the 500 mg/kg group, slight increase in motor activity and accompanied decrease in motor activity was noted in many animals through the administration period. In 2 females of the 500 mg/kg group, slight vaginal hemorrhage was noted in the latter gestation period.

Body weight: In 15 and 50 mg/kg group the body weight and body weight gain showed no significant differences compared to vehicle control group. For females in the 150 mg/kg group, 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, for females, 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.

Food consumption: In the 15 mg/kg group, no significant change was noted in food consumption during the study period. In the 150 mg/kg group, significant decrease of food consumption was noted 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, gestation day 0, 14 and 20 in female



Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
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 control group. No abnormality was noted in general condition of the newborn. 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. 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.

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.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Sex:
male/female
Basis for effect level:
changes in postnatal survival
Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
An oral combined repeated dose/reproductive and developmental screening test in the rat, conducted in a manner similar to the current OECD 422 guideline and in accordance with GLP, identified a maternal and developmental NOAEL of 50 mg/kg bw/day based on adverse effects on delivery indices, litter sizes, pup survival to Day 4 and other indices at 150 mg/kg/day and above.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The database for developmental toxicity is somewhat limited. Only a combined repeated-dose and reproductive/developmental toxicity screening test and a range-finding developmental study were identified.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the 28 -day reproductive/developmental toxicity screening test conducted by the oral route, the NOAEL for developmental effects was 50 mg/kg/day, with adverse effects on delivery indices, litter sizes, survival to Day 4 and other indices at 150 mg/kg/day and above.

In a further oral range-finding study, general maternal toxicity (including reduced weight gain) was reported at 500 mg/kg/day, and developmental toxicity (reduced mean foetal weight and increased malformations) was identified at 100 mg/kg/day. Based on these effects the maternal NOAEL was considered to be 100 mg/kg/day and the developmental NOAEL was considered to be 50 mg/kg/day.


Justification for classification or non-classification

A 28 -day reproductive/developmental toxicity study, together with four repeated dose studies give clear evidence that the male reproductive system is the main target organ in rats exposed to THFA via the oral, inhalation or dermal routes. The combined study supported by a limited (range-finding) developmental study also provides evidence of developmental but not clear teratogenic effects.

THFA is classified for toxicity to reproduction, category 2 for fertility and category 1b for development according to the sixth adaptation to technical progress to CLP 2008 with the hazard phrases 'May damage the unborn child' and 'Suspected of damaging fertility' (H360Df) according to Annex VI of Regulation (EC) 1272/2008.

THFA is structurally similar to the substances 2-methoxyethanol (CAS 109-86-4) and 2-ethoxyethanol (CAS 110-80-5), both of which are classified as toxic to reproduction, category 1b for fertility and category 1b for development with the hazard phrases ‘May damage the unborn child’ and ‘May damage fertility (H360FD) according to Regulation (EC) No 1272/2008. Although these substances do not include a ring structure, both are low molecular weight glycol ethers containing the -C-O-C-C-OH group which is also present THFA. It is therefore considered that THFA would be subject to similar metabolic pathways and produce similar effects to these substances. This is reflected in the nature of the effects reported in the disseminated dossier for 2-methoxyethanol and the RAC Opinion on 2-ethoxyethanol, which are similar to those observed in the studies with THFA. 

Substance

Tetrahydrofurfuryl alcohol

2-methoxyethanol

2-ethoxyethanol

CAS No.

97-99-4

109-86-4

110-80-5

EC No.

202-625-6

203-713-7

203-804-1

Structure

Structures are shown in the attached document

Structures are shown in the attached document

Structures are shown in the attached document

Molecular weight

102.13 g/mol

76.10 g/mol

90.12 g/mol

Canonical SMILES

OCC1CCCO1

COCCO

CCOCCO

Based on findings in males, including lower prostate, epididymal and testes weights, necrosis of the seminiferous tubular epithelium and lower sperm production and findings in females including prolonged oestrus cycle and gestation length together with developmental findings of increased incidence of foetal resorption or mummification and dead pups on PND 1, it is considered that the substance should be classified for toxicity to reproduction, category 1b, with the hazard phrases ‘May damage the unborn child’ and ‘May damage fertility (H360FD)’, according to Regulation (EC) 1272/2008.

Additional information