Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Aug 2005 - 9 Nov 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted Jan 2001
Deviations:
yes
Remarks:
result tables are missing in the study report
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
1998
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (MAFF), 12 NohSan No. 8147, 2-1-17
Version / remarks:
2000
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: BrlHan:WIST@Jcl[GALAS]
Details on species / strain selection:
The Wistar Hannover GALAS rat was selected because the rat is a commonly used mammalian species for reproduction toxicity studies of agricultural chemicals and because this strain of rats yields stable reproduction results.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Fuji Breeding Center, CLEA Japan, Inc., Shizuoka, Japan
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 116 - 133 g (males), 89 - 103 g (females)
- Housing: Pre-mating: groups of 3 animals per sex per cage in wire-mesh stainless steel cages, Mating: 1:1 in an aluminum cage with wire-mesh floor and front and a subfloor tray (W260 x D400 X H240 mm), Gestation and lactation: individually in aluminum breeding boxes (W260 x D400 x H200 mm) with nesting materials (Sunflake: Oriental Yeast Co., Ltd., Tokyo, Japan), after weaning, offspring were housed by litter in the breeding boxes; males that accomplished mating and females that accomplished lactation were housed until necropsy in wire-mesh stainless steel cages
- Diet: certified pulverized feed (MF Mash, Oriental Yeast Co., Ltd., Tokyo, Japan), ad libitum
- Water: well water purified with a rapid filtration-activated carbon adsorption system and sterilized with sodium hypochlorite, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: 10 times during the study, once in one to 6 weeks
- Storage temperature of food: The prepared test diets were sealed in plastic bags and stored in a dark and cold room (1 - 10 °C)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: presence of vaginal plug / sperm in vaginal smears referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually in aluminum breeding boxes (W260 x D400 x H200 mm) with nesting materials
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In the dose range-finding study previously performed, it was confirmed that the test substance was stable in the basal feed at concentrations of 2 and 2000 ppm for 56 days after preparation (5 weeks in a cold and dark room with a tightly-packed condition, and 21 days in an animal room).
Chemical analyses for homogeneity and concentration of the test substance in the basal feed were performed at the first and 6th preparation samples taken from the top, middle, and bottom portions of the mixer for each dose level. Storage conditions of test diets were monitored at the 3rd and 8th preparations. Samples taken from the middle portion of the mixer immediately after preparation and from the last batch of the preparations after use were analyzed for the concentration of test substance in the basal feed at each dose level. Control diets were also analyzed in the same manner to ensure that the basal feed was not contaminated by the test substance. All scheduled analyses at the diet preparation were performed before the first use of the prepared diets except for monitoring storage condition. In the homogeneity study, the coefficient of variation of mean test substance concentration in the diet for each dose level ranged 3.0 - 7.9%, being confirmed a homogeneous distribution of the test substance in basal feed. In the concentration analyses of test diets in each dose group, the concentration of test substance in the diet at dose level of 2 ppm prepared at the 8th preparation were slightly low, 80% to the target concentration. However, in the analyses of the final batch in the same lot, an analytical value of the sample at dose level of 2 ppm was 95% to the target concentration, being concluded no problem in the test diet and storage condition of that lot. In other concentration analyses just after the preparation, the concentration of test diets in each dose group ranged 95 - 107% to the target concentrations, indicating the good condition of preparation. Furthermore, in the analyses of the final batches divided in each dose group, all analytical values ranged 85 - 104% to the target concentrations, being considered no problem in the storage condition of test diets prepared.
Duration of treatment / exposure:
approximately 18 weeks (P and F1 generations)
Frequency of treatment:
continuously (via diet)
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 - 25 days of age
Doses / concentrationsopen allclose all
Dose / conc.:
2 ppm
Remarks:
equivalent to 0.126, 0.202, 0.142 and 0.204 mg/kg bw/day in (P) males and females and (F1) males and females, respectively
Dose / conc.:
20 ppm
Remarks:
equivalent to 1.25, 2.03, 1.40 and 2.03 mg/kg bw/day in (P) males and females and (F1) males and females, respectively
Dose / conc.:
200 ppm
Remarks:
equivalent to 13.1, 20.4, 14.6 and 20.9 mg/kg bw/day in (P) males and females and (F1) males and females, respectively
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a reproduction toxicity dose range-finding study in rats (report no. IET 04-0031). In the study, the test substance was administered to 8 SPF Wistar Hannover (BrlHan:WIST@Jcl[GALAS]) rats per sex per group at dietary concentrations of 2, 20, 200, and 2000 ppm for a period of 3 weeks prior to mating and until weaning of their F1 offspring. In the results, the body weights of male and female parental animals at 2000 ppm decreased statistically significantly and the body weight of male parental animals at 200 ppm also decreased slightly with no statistical significance. In the offspring, the body weights of males at 200 ppm and higher dose groups and females at 2000 ppm decreased statistically significantly. In addition, both parental animals and offspring showed opacity of the eyeball, and the finding which was the most sensitive in female parental animals were noted in 2, 6, and 6 animals at 20, 200, and 2000 ppm, respectively. On the other hand, increases in the liver weights and other findings were noted in both the parental animals and their offspring, whereas no remarkable toxicological effects in any items suspected in relation with the treatment of test substance were noted in the parental animals and their offspring at 2 ppm. Based on the results, a dose level of 2 ppm was selected as the low-dose level, and dose levels of 20 ppm and 200 ppm were selected as the middle-dose and high-dose levels, respectively.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once on holidays)
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Parental animals of both sexes were observed from cage-side twice daily (once on holidays) for clinical signs. Each animal was also examined in detail for the presence of abnormalities such as excitement, convulsion, sedation, and abnormal gait when it was weighed. During breeding, females were examined for the status of pregnancy and parturition. Any abnormalities were recorded along with the date of onset, nature, degree, and duration.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on the day of initiation of dosing, weekly during the pre-mating growth and breeding periods and on the day of necropsy.
Females were weighed on the day of initiation of dosing, weekly during the pre-mating growth period, on Days 0, 7, 14, and 20 of gestation and on Days 0, 7, 14, and 21 of lactation during the breeding period and on the day of necropsy. These measurements were made on females that lost all pups during the lactation period. Females not producing offspring were weighed weekly on Day 25 and thereafter of post coitus and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption of lactating females was expressed as the total amounts of food consumed by maternal animals and their offspring. During the mating period, food consumption was not determined for either sex.
- Compound intake calculated as time-weighted averages from the consumption and body weight data: Yes

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Vaginal smears were taken from females in each group, stained with Giemsa solution, and examined microscopically. Regularity in microscopic changes of vaginal smears associated with various stages of the estrous cycle in females was checked for more than 2 weeks. Females periodically showing estrous vaginal smears were considered to be normal and the percentage of normal females was calculated for each group. Estrous cycle length of each female was expressed as mean number of days from the day of estrous to the day before next estrus. Mean estrous cycle length was calculated for each group. When estrous continued 2 days, calculation of estrous cycle length was based on the first day of estrous.
Sperm parameters (parental animals):
Parameters examined in all P and F1 male parental generations:
At necropsy, sperm heads and sperm were collected from the testis and cauda epididymis (right side in principle), respectively, of all (P) and (F1) parental males. The number of testicular sperm heads was enumerated by using a hemacytometer. The number and motility of cauda epididymal sperm were examined by a computer-assisted sperm analysis system. Morphology of cauda epididymal sperm was performed microscopically after fixation in 10% neutral-buffered formalin.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible)

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: still births, live births, postnatal mortality, clinical signs, complete external examinations, body weight, number and sex of pups. The number of pups surviving in each litter was counted on Days 0, 4, 7, 14, and 21 of lactation. In each litter, pups were weighed on Days 0, 4, 7, 14, and 21 of lactation. Body weight measurement was performed by sex on lactation day 0 and individually on lactation day 4 and thereafter. Then mean body weights were calculated for each sex for each group.

GROSS EXAMINATION OF DEAD PUPS:
yes, for gross findings
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals after weaning of F1 pups
- Maternal animals: all surviving animals after weaning of F1 pups

GROSS NECROPSY
- Gross necropsy consisted of external examinations. Parental females were examined through vaginal smears for estrous cycle stage before necropsy and the animals showing metestrus or diestrus were necropsied in order because the effects of estrous cycle stage on the changes of uterus weight were reduced as small as possible.

HISTOPATHOLOGY / ORGAN WEIGHTS
After necropsy, the following organs from all P and F1 parental animals were weighed: brain, thyroid, pituitary, liver, kidneys, adrenals, spleen, ovaries, uterus (with cervix and oviducts), testes, epididymides, seminal vesicles with fluids (including coagulating glands), and prostate (ventral lobe).

The eyeballs (both sides), vagina and organs with gross abnormalities as well as the organs weighed were fixed and preserved in 10% neutral-buffered formalin with the exception of the testes which were fixed in a FSA (formalin-sucrose-acetic acid) solution.

Histopathology of the following organs was performed for 10 pairs of P and F1 parental animals selected randomly from each in the control and high dose groups, that accomplished the rearing of offspring until weaning: reproductive organs (ovaries, oviducts, uterus (horns and cervix), vagina, testis (left side), epididymis (left side), seminal vesicles, coagulating glands, and prostate), pituitary, and adrenals. The testis of all P males was examined histopathologically because the relative weights of the testes in all treated groups increased significantly in P generation. In addition, the epididymis of remaining P males in the control and high-dose groups was examined histopathologically because the relative weight of the epididymides in each treated group increased. In these histopathological examinations, abnormalities of spermatogenesis in the testis were examined in detail. The examination of the epididymis was performed on its head, body, and cauda. The ovaries of F1 females were examined for the number of primordial follicles.
Histopathology of the reproductive organs, pituitary and adrenal was carried out in pairs of animals that failed to mate or pregnancy and in maternal females that showed abnormal parturition or whole litter losses. In addition, the eyeballs (both sides) and liver in all P and F1 parental animals were examined histopathologically because these organs were considered to be the target organs.
On the other hand, the kidneys and thyroids in male parental animals were examined histopathologically because weights of these organs increased in relation with the treatment of test substance. The kidneys in the control and high-dose groups were examined in 24 animals of each group in P generation and 10 animals of each group in F1 generation. The thyroids were examined histopathologically in all P and F1 parental male animals. Furthermore, histopathology of the organs showing gross abnormalities at necropsy was performed when the study director deemed it necessary.
Postmortem examinations (offspring):
SACRIFICE
- The F1 and F2 offspring not selected on Day 4 of lactation were sacrificed and subjected to necropsy.
- The F1 offspring not selected as parental animals and all F2 weanlings were sacrificed at 26 days of age and gross findings were recorded.

GROSS NECROPSY
Of F1 and F2 weanlings in each litter in each group, one male and one female that were ranked first by sex were subjected to body weight and organ weight measurements on the day of necropsy. Organ weight measurement was performed on the brain, spleen, thymus, liver, and uterus.
The measured organs were fixed and preserved in 10% neutral-buffered formalin along with the reproductive organs, eyeballs (both sides), and organs showing gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
The eyeballs (both sides) and liver of all F1 and F2 weanlings whose organs were weighed were subjected to histopathology.
Statistics:
Please see entry under "Any other information on materials and methods incl. tables".
Reproductive indices:
Male mating index (%) = (number of copulations / number of males used for mating) x 100
Female mating index (%) = (number of copulations / number of females used for mating) x 100
Fertility index (%) = (number of pregnancies / number of females copulated) x 100
Gestation index (%) = (number of normal parturitions / number of pregnancies) x 100
Duration of gestation = days from detection of copulation to completion of parturition
Number of implantation sites = number of implantation sites in the uterus
Offspring viability indices:
Viability index on lactation day 0 (%) = (number of pups alive on lactation day 0 / number of pups delivered) x 100
Viability index on lactation day 4 (%) = (number of pups alive on lactation day 4 / number of pups alive on lactation day 0) x 100
Viability index on lactation day 7 (%) = (number of pups alive on lactation day 7 / number of pups selected on lactation day 4) x 100
Viability index on lactation day 14 (%) = (number of pups alive on lactation day 14 / number of pups selected on lactation day 4) x 100
Viability index on lactation day 21 (weaning index, %) = (number of pups alive on lactation day 21 / number of pups selected on lactation day 4) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In clinical observation, opacity of the eyeballs and coarsened corneal surface were noted in males and females at 20 and 200 ppm. In addition, swelling of the mammary gland was noted in one to 3 females at 20 and 200 ppm just after weaning. On the other hand, no findings were noted in the eyeballs and mammary gland at 2 ppm.
At 200 ppm, the incidence of loss of fur in females was statistically significantly higher than that in the control group. However, the incidences of loss of fur in F1 females in each treated group were not significantly different from those in the control group, concluded that the significant increase of this incidence would be unrelated to the treatment of test substance.
Furthermore, eye discharge, supernumerary incisor and wound were noted in one to 2 males and females in each group. However, there were no statistical significant differences of those incidences between the treatment groups and the control group, being not suspected in relation with the treatment of test substance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights and body weight gain of males and females at 2 and 20 ppm group were similar to those in the control group.
At 200 ppm, the body weights were reduced in males and females reaching statistically significance in males at Week 17 of treatment and in females at Week 8 of treatment, on GD 14 and on Day 7 and 14 of lactation. The body weight gains were slightly reduced in males from Week 0 until 17 of treatment and significantly in females at Week 3 and thereafter in the growth period and on Days 0-7 and 0-14 of lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumptions of males in each treated group were similar to those in the control group throughout the treatment period except for accidental significant high values at 2 or 200 ppm from Week 1 to 2 of treatment. Significant reduced food consumption was observed in females at 20 and 200 ppm on Days 0-7 of lactation and during the lactation period, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
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 liver and eyeballs in all animals were examined histopathologically because those organs were considered to be a common target of the test substance in both sexes. In the liver, centrilobular hypertrophy of the hepatocytes was observed in males at 20 and 200 ppm and the incidences were statistically significantly higher than those in the control group. However, this finding was not observed in males at 2 ppm and females in all treated groups. In addition, single cell necrosis or focal necrosis of the hepatocytes, microgranuloma and hepatodiaphragmatic nodule were observed in males and females in each treated group including the control group and those findings occurred singly in each treated group. In the eyeballs, opacity and coarsened corneal surface noted grossly were diagnosed histopathologically as keratitis. In addition, the incidences of the findings in males and females at 20 and 200 ppm groups were statistically significantly higher than those in the control group. On the other hand, no keratitis was observed in any males and females at 2 ppm. In the eyeballs, furthermore, calcification in the cornea was observed in each treated group including the control group, whereas the incidence in each group was one or 2 animals per group. Additionally, the thyroids, kidneys, testes, epididymides and seminal vesicle with coagulating gland with suspected relations with the treatment of test substance and weight gain were examined histopathologically. In the thyroid, colloidal alteration, hypertrophy of the follicular cell and increased large-sized follicles were observed in each treated group including the control group and the statistically significant differences in these incidences were occasionally noted at 20 and 200 ppm. Hydropic degeneration of the follicular cell, which has been known as one of spontaneously occurring lesions in this strain of rats, was also observed in one to 3 animals in each group, whereas there were no significant differences of the incidences between each treated group and the control group. In the kidneys, there were no findings suspected the relation with the treatment of test substance in all animals at 200 ppm. The testes, epididymides and seminal vesicle with coagulating gland were examined in all animals selected. In results, atrophy of the seminiferous tubule was observed in one to 3 animals in each group including the control group, whereas there were no abnormal findings in relation with the cause of increased organ weight.
In addition to the organs described above, the reproductive organs, pituitary and adrenals were histopathologically examined in each 10 males and females selected from the control and high-dose groups. However, there were no findings suspected in relation with the treatment of test substance in these organs. On the other hand, other gross lesions were examined histopathologically as necessary, whereas there were no abnormal findings in relation with the treatment of test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In sperm examination, sperm head count in the testis, sperm counts in the cauda epididymis and sperm motility in each group were not statistically significantly different from those in the control group. Statistically significant low incidences of sperm with normal morphology were noted at 20 and 200 ppm. However, these values were 97.9 and 98.1%, respectively, and ranged within the background data (97.4-99.2%), being considered to be of no toxicological significance.
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant differences of mating index between each treated group and the control group, and mating was noted in all pairs. There were also no statistically significant differences of the number of days until mating and mating was completed in almost all animals for 1 day or 2 days after pairing. On the other hand, 6 pairs were completed to mate for 3 and more days, whereas those pairs belonged to each treated group including the control group at a low incidence such as one or less in each group.
Fertility indices of females in each treated group including the control group were favorable ranging from 87.5 to 100.0% and there were no statistically significant differences between each treated group and the control group. The numbers of females without gestation were one female at 20 and 200 ppm, respectively.
Gestation indices in each treated group including the control group were 100%.
Durations of gestation in females in each treated group were similar to those in the control group and almost all females having live pups delivered at Day 22 of gestation. Other remaining females delivered at Day 21 or 23 of gestation.
Numbers of implantation sites in females in each treated group were similar to those in the control group.
The mean numbers of pups delivered in each treated group were similar to those in the control group.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
2 ppm
Based on:
test mat.
Remarks:
equivalent to 0.126 and 0.202 mg/kg bw/day in males and females, respectively
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 200 ppm
Based on:
test mat.
Remarks:
equivalent to 13.1 and 20.4 mg/kg bw/day in males and females, respectively
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
20 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
20 ppm
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Almost all animals at 200 ppm showed either opacity of the eyeballs or coarsened corneal surface.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of parental males and females at 2 ppm were similar to those in the control group.
At 20 ppm, the body weight and body weight gain of F1 parental males was slightly lower without statistical significance.
At 200 ppm, the body weights were reduced in males and females reaching statistically significance in males throughout the treatment period and in females from the initiation of treatment to Week 4 of treatment. The body weight gains were significantly reduced in males during the treatment period and in females on Days 0-7 and 0-14 of lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was significantly reduced in males at 20 ppm in Week 14 and thereafter of treatment and at 200 ppm in Week 1, Week 4-7 and Week 13 and thereafter of treatment, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males in each treated group, statistically significant differences of the absolute and/or relative weights of the organs except for the prostate were noted occasionally between each treated groups and the control group. In these changes, the relative weights of the liver, kidneys and adrenals increased significantly at 20 and 200 ppm. Those changes were related to the dose levels, being considered to be related to the treatment of test substance. There were no statistically significant differences of these organ weights observed at 2 ppm. Otherwise, the relative weights of the testes at 20 and 200 ppm increased significantly, being suspected to be related to the treatment of test substance. In addition, the absolute weights of the epididymides and seminal vesicles at 20 and 200 ppm and were slightly higher than those in the control group. The relative weights of the seminal vesicles at 200 ppm reached statistically significance. Changes of the organ weights in females were similar to those in males. The absolute weights of the liver at 2 and 20 ppm and the relative weights at 20 and 200 ppm were statistically significantly different from those in the control group. In addition, the adrenal weights at 20 and 200 ppm were increased slightly. The absolute weights at 20 ppm and the relative weights at 20 and 200 ppm were statistically significantly different from those in the control group, respectively. Otherwise, the absolute weights of the brain at 20 and 200 ppm were significantly decreased and the relative weights at 200 ppm.
No statistically significant differences of other organ weights were observed between each treated group and the control group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 20 and 200 ppm, opacity and coarsened corneal surfaces of the eyeballs were frequently noted in males and females, either or both incidences of those findings were statistically significantly higher than those in the control group. On the other hand, enlargement of the thyroid, hepatodiaphragmatic nodule, coarse surface and pelvic dilatation of the kidneys, luminal dilatation of the common bile duct, and others were noted in each group including the control group, whereas there were no statistically significant differences of the incidences of these findings between each treated and control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The liver and eyeballs in all animals were examined histopathologically because those organs were considered to be a common target of the test substance in both sexes. In the liver, centrilobular hypertrophy of the hepatocytes was observed in males at 20 and 200 ppm and the incidences were statistically significantly higher than those in the control group. However, this finding was not observed in males at 2 ppm and females in all treated groups. In addition, single cell necrosis or focal necrosis of the hepatocytes, microgranuloma and hepatodiaphragmatic nodule were observed in males and females in each treated group including the control group and those findings occurred singly in each treated group. In the eyeballs, opacity and coarsened corneal surface noted grossly were diagnosed histopathologically as keratitis. In addition, the incidences of the findings in males and females at 20 and 200 ppm groups were statistically significantly higher than those in the control group. On the other hand, no keratitis was observed in any males and females at 2 ppm. In the eyeballs, furthermore, calcification in the cornea was observed in each treated group including the control group, whereas the incidence in each group was one or 2 animals per group. Additionally, the thyroids, kidneys, testes, epididymides and seminal vesicle with coagulating gland with suspected relations with the treatment of test substance and weight gain were examined histopathologically. In the thyroid, colloidal alteration, hypertrophy of the follicular cell and increased large-sized follicles were observed in each treated group including the control group and the statistically significant differences in these incidences were occasionally noted at 20 and 200 ppm. Hydropic degeneration of the follicular cell, which has been known as one of spontaneously occurring lesions in this strain of rats, was also observed in one to 3 animals in each group, whereas there were no significant differences of the incidences between each treated group and the control group. Chronic nephropathy was observed in two (one of them showing coarse surface) of 12 animals (10 selected animals and two animals with gross findings) at 200 ppm. The testes, epididymides and seminal vesicle with coagulating gland were examined in all animals selected. In results, atrophy of the seminiferous tubule was observed in one to 3 animals in each group including the control group, whereas there were no abnormal findings in relation with the cause of increased organ weight.
In females at 200 ppm, the number of primordial follicles was similar to that in the control group. The mammary gland which was grossly hypertrophic was confirmed histopathologically as incomplete recovery from lactation phase, whereas there were no findings indicating toxicological effects.
In addition to the organs described above, the reproductive organs, pituitary and adrenals were histopathologically examined in each 10 males and females selected from the control and high-dose groups. However, there were no findings suspected in relation with the treatment of test substance in these organs. On the other hand, other gross lesions were examined histopathologically as necessary, whereas there were no abnormal findings in relation with the treatment of test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In sperm examination, sperm head count in the testis, sperm counts in the cauda epididymis and sperm motility in each group were not statistically significantly different from those in the control group.
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant differences of mating index between each treated group and the control group, and mating was noted in all pairs except for one pair at 200 ppm. Mating of this pair was not completed until 2 weeks after pairing, whereas mating was completed by exchanging the male by another which was already successful in mating. There were also no statistically significant differences of the number of days until mating and mating was completed in almost all animals for 1 day or 2 days after pairing. On the other hand, 6 pairs were completed to mate for 3 and more days, whereas those pairs belonged to each treated group including the control group at a low incidence such as one or less in each group.
Fertility indices of females in each treated group including the control group were favorable ranging from 87.5 to 100.0% and there were no statistically significant differences between each treated group and the control group. The numbers of females without gestation were 2 females at 20 ppm and 3 females at 200 ppm.
Gestation indices in each treated group including the control group were 100% except for 95.2% at 200 ppm. In one female which parturition was not confirmed, implantation site was noted in the uterine horns.
Durations of gestation in females in each treated group were similar to those in the control group and almost all females having live pups delivered at Day 22 of gestation. Other remaining females delivered at Day 21 or 23 of gestation.
Numbers of implantation sites in females in each treated group were similar to those in the control group.
The mean numbers of pups delivered in each treated group were similar to those in the control group.

Effect levels (P1)

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Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
2 ppm (nominal)
Based on:
test mat.
Remarks:
equivalent to 0.126 and 0.202 mg/kg bw/day in males and females, respectively
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 200 ppm (nominal)
Based on:
test mat.
Remarks:
equivalent to 13.1 and 20.4 mg/kg bw/day in males and females, respectively
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

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Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
20 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
20 ppm
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At observation on Days 15-21 of lactation, opacity of the eyeballs was noted in 2 animals at 200 ppm.
In the treated group, dark-coated skin, concavity of the head, wound of the ear, bending of the tail and loss of fur in the back were noted in one or 2 pups during the lactation period; this was not considered to be treatment-related.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
At observation on Day 0 of lactation, there were a few pups found dead in each treated group, whereas there were no statistically significant differences of the incidences between each treated group and the control group. At observation on Days 1-4 of lactation, one pup died at 200 ppm. In addition, there were one to 4 animals with lost of pups in each group including the control group. However, there were no statistically significant differences of the incidences between each treated group and the control group. At observation on Days 5-7 and Days 8-14 of lactation, lost of pups was noted at 2 ppm.
At 20 ppm, one animal with malocclusion and eye discharge died after weaning. At necropsy of the animal, these findings were concluded to be caused by fracture of the nasal bone.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the pups at 2 ppm were similar to those in the control group during the lactation period. At 20 ppm, the body weights of male and female pups were significantly lower than those in the control group in the later half of lactation period. At 200 ppm, the body weights were lower than those in the control group during the lactation period and there were statistically significant differences of the values on Day 7 and thereafter of lactation.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
At observation of sexual development, the age at completion of preputial separation in males was delayed at 20 and 200 ppm in relation with the dose levels of test substance and there were statistically significant differences of these values between those treatment groups and the control group. However, the body weights at completion of preputial separation were similar among each group and the control group. On the other hand, the age at completion of preputial separation in males at 2 ppm was similar to that in the control group. In sexual development in females, there were no statistically significant differences of the age and body weights at completion of vaginal opening between each treated group and the control group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute weights of the liver increased in male and female weanlings in each treated group, similar to their parental animals. The relative weights of these changes were closely related to the dose levels and there were statistically significant differences of the changes between male and female weanlings at 20 and 200 ppm. In addition, the absolute weights of the brain, spleen and thymus of both sexes often showed significant low values at 20 and 200 ppm. However, the relative weights of these organs were similar to or significantly higher than those in the control group, being considered to be the changes caused by low body weights.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At necropsy of pups found dead on Day 0 of lactation, there were no findings except for left umbilical artery in one pup at 2 ppm. At necropsy of pups excluded on Day 4 of lactation, left umbilical artery was noted in 5 to 12 animals in each group including the control group and testicular hemorrhage, hepatodiaphragmatic nodule, white spots on the liver, and thymic remnant in the neck were noted in one to 3 animals in each group including the control group. However, there were no statistically significant differences of the incidences of these findings between each treated group and the control group. At necropsy of weanlings, opacity of the eyeballs and coarsened corneal surface were noted in 22 and 7 pups, respectively, at 200 ppm. However, none of these findings were noted at 2 and 20 ppm. Dilatation of renal pelvis was noted in weanlings in each group including the control group. There was no tendency of increasing incidence of this finding at 2 and 20 ppm. On the other hand, cyst of the ovary, hepatodiaphragmatic nodule, and red sebum were noted occasionally without statistical significance.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
The eyeballs and liver being weighed were examined histopathologically in all weanlings, which revealed keratitis in some weanlings in the groups treated at 20 ppm and above. The incidences of this finding in male weanlings at 20 and in male and female weanlings at 200 ppm were significantly higher than those in the control group. On the other hand, there were no abnormal findings in the eyeballs of any weanlings at 2 ppm. In the liver, focal necrosis of the hepatocyte was observed in one or 2 males and female in each treated group, whereas there were no statistically significant differences of the incidence between each treated group and the control group.
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Sex ratio and the mean viability indices of pups on any lactation days were similar in each treated group to that in the control group.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
2 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
sexual maturation
body weight and weight gain
gross pathology
Remarks on result:
other: the delayed preputial separation in male was observed in offspring of females with a reduced body weight gain during lactation day 0-7

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 200 ppm, intraocular opacity was noted in 2 animals in the same litter, whereas no gross abnormalities were noted in the cornea, considered to be unrelated to the treatment of test substance.
In the treated group, dark-coated skin, concavity of the head, wound of the ear, bending of the tail and loss of fur in the back were noted in one or 2 pups during the lactation period, being not suspected the relation with the treatment of test substance.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
At observation on Day 0 of lactation, there were a few pups found dead in each treated group, whereas there were no statistically significant differences of the incidences between each treated group and the control group. At observation on Days 1-4 of lactation, one pup died in the control group. In addition, there were one to 4 animals with lost of pups in each group including the control group and exceptionally all pups were lost in a litter at 2 ppm. However, there were no statistically significant differences of the incidences between each treated group and the control group. At observation on Days 5-7 and Days 8-14 of lactation, lost of pups was noted at 20 ppm.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the pups at 2 ppm were similar to those in the control group during the lactation period. The body weights at 20 ppm were not significantly different from those in the control group. At 200 ppm, the body weights were lower than those in the control group during the lactation period and there were statistically significant differences of the values on Day 7 and thereafter of lactation.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
The absolute values and relative body weight ratio of anogenital distance in male and female pups in each treated group were similar to those in the control group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute weights of the liver increased in male and female weanlings in each treated group, similar to their parental animals. The relative weights of these changes were closely related to the dose levels and there were statistically significant differences of the changes between male and female weanlings in all treated groups and those in the control group. In addition, the absolute weights of the brain, spleen and thymus of both sexes often showed significant low values at 20 and 200 ppm. However, the relative weights of these organs were similar to or significantly higher than those in the control group, being considered to be the changes caused by low body weights. Furthermore, the relative weight of the brain in male weanlings at 2 ppm was significantly low, whereas there were no significant differences at 20 and 200 ppm, being considered to be an accidental change.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No abnormalities of pups found dead on Day 0 of lactation were observed at necropsy. At necropsy of weanlings, opacity of the eyeballs and coarsened corneal surface were noted each in 36 pups at 200 ppm. However, none of these findings were noted at 2 and 20 ppm. Dilatation of renal pelvis was noted in weanlings in each group including the control group reaching statistically significance at 200 ppm. There was no tendency of increasing incidence of this finding at 2 and 20 ppm. On the other hand, cyst of the ovary, hepatodiaphragmatic nodule, and red sebum were noted occasionally without statistical significance.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
The eyeballs and liver being weighed were examined histopathologically in all weanlings, which revealed keratitis in some weanlings in the groups treated at 20 ppm and above. The incidences of this finding in male and female weanlings at 200 ppm were significantly higher than those in the control group. On the other hand, there were no abnormal findings in the eyeballs of any weanlings at 2 ppm. In the liver, focal necrosis of the hepatocyte was observed in one or 2 males and female in each treated group, whereas there were no statistically significant differences of the incidence between each treated group and the control group.
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

Sex ratio and the mean viability indices of pups on any lactation days were similar in each treated group to that in the control group.

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F2
Effect level:
2 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
20 ppm (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion