4,4'-methylenedi-2,6-xylenol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Weight of evidence information based on information of various test chemical.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Approximately 68 days

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Data is from a J-check report.

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The above experiment was performed in order to assess and eveluate the effects of the test chemical on the reproductive parameters of the male and female test animals.

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No Data Available

Specific details on test material used for the study:

- Molecular weight (if other than submission substance):340.51 g/mole
- Substance type:organic
- Physical state:solid
- Impurities (identity and concentrations):1.8%(identity and concentration not given)

Species:

rat

Strain:

other: Crj:CD (SD) IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 10 week old
- Weight at study initiation: 332-383 g for male, 206-238 g for female.
- Fasting period before study: Not available
- Housing: Stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.
- Diet (e.g. ad libitum): The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.)
- Water (e.g. ad libitum): The drinking water was freely taken in all of the tap water
- Acclimation period: Not available

ENVIRONMENTAL CONDITIONS .
- Temperature (°C): 20 to 26 ° C.
- Humidity (%): 40 to 70%
- Air changes (per hr): 12 times / hour.
- Photoperiod (hrs dark / hrs light): Light and dark each for 12 hours (lighting: 6 am to 6 pm)

Route of administration:

oral: gavage

Vehicle:

other: Five percent gum Arabic

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in 5% aqueous gum arabic solution and it was stored in refrigerator under light-protected conditions for max. 7 days.The purity and the stability of the test chemical was confirmed previously.The concentartion of the test chemical in the administration solution during the administration period was checked and confirmed. Doses given to animals were based on the initial body weights.

DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A

VEHICLE
- Justification for use and choice of vehicle (if other than water): 5% gum arabic
- Concentration in vehicle: 12.5, 50, 200 or 800 mg/kg/day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy vaginal plug
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility. Not available
- Further matings after two unsuccessful attempts: [no / yes (explain)] Not available
- After successful mating each pregnant female was caged (how):Not available
- Any other deviations from standard protocol:Not available

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No Data Available

Duration of treatment / exposure:

Male: 50-52 days
Female: 40 to 48 days.

Frequency of treatment:

Daily

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters.: Not available
- Selection of parents from F1 generation when pups were [...] days of age.: Not available
- Age at mating of the mated animals in the study: [...] weeks: Not available
-No other details given.

Remarks:

Doses / Concentrations:
12.5, 50, 200, 800 mg/kg/day (in 5% gum arabic)
Basis:
no data

No. of animals per sex per dose:

Control-12 female and 12 male
12.5 mg/kg/day- 12 female and 12 male
50 mg/kg/day-12 female and 12 male
200 mg/kg/day-12 female and 12 male
800 mg/kg/day-12 female and 12 male

Control animals:

yes, concurrent vehicle

Details on study design:

No data Available

Positive control:

No Data Available

Parental animals: Observations and examinations:

Males:
Clinical Signs: The general condition and the presence or absence of death were observed twice a day before and after administration.

Body weights: Body weight was measured twice a week.

Food Consumption: Food consumption was measured twice a week from 14 days before the start of the mating and after the end of the mating cycle.

Necropsy and Histopathology: On the day after the final administration, the animals were sacrificed by abdominal aorta under ether anesthesia, necropsied, and the tail weight of testis, epididymis and epididymis was measured. The relative weight was also calculated by dividing each organ weight by the final body weight. Testis and epididymis heads were fixed in Bouin's solution. Prostate and seminal vesicles were fixed in 20% neutral buffered formalin. Testis and epididymis were paraffin-embedded specimens prepared according to a conventional method. HE stained tissue specimens were prepared for the control group and the testis and epididymis heads in the 800 mg / kg group, and seminal vesicles with abnormality at necropsy (6 subjects in the 800 mg / kg group), and histopathology An inspection was carried out. For testes and epididymis heads considered to be different in number of animals showing abnormalities compared with the control group in the 800 mg / kg group examination, the 12.5, 50 and 200 mg / kg group was also examined did.

Sperm paramters: The tail of the right epididymis was divided in sperm culture (0.5% bovine serum albumin plus Medium 199) warmed to 37 ° C. and used as a sperm stock solution. The sperm concentrate was used to examine sperm activity, sperm viability and sperm morphology.Sperm activity was determined by diluting the sperm diluted solution with sperm culture solution in sperm culture solution and incubating the sperm diluted solution with HTM-IVOS (Hamilton Thorne Research) after incubating for about 30 minutes (culture conditions: 37 ° C., 5% carbon dioxide, 95% The active spermatozoa ratio was obtained and the reference point moving speed, the shortest moving speed, the total moving speed and the number of crossings of the head were calculated for the active sperm.
As the viability of sperm, according to the method 2) of Kato et al., The sperm stock solution was diluted about 3 times with the sperm culture solution in the microwell plate, then 16 μM calcein acetoxy methyl ester and 8 μM ethidium homodimer-1 After incubation and staining for about 2 hours (culture conditions: 37 ° C, 5% carbon dioxide, 95% air), spermatozoa were classified as living sperm, mid-dead sperm and killed sperm under a fluorescence microscope and surviving sperm ratio and survival The sperm ratio was determined. For survival, mid-death, and death sperm determination, green fluorescent color development is observed in the head to tail area, living spermatozoa, red fluorescent coloring in the head and green fluorescent coloring in the tail are observed in the head Somewhat dead sperm, sperm on the head, red fluorescent coloring was observed in the head, but those that did not show fluorescent coloring in the tail were regarded as dead sperm.
The morphology of the sperm was prepared by mixing a sperm stock solution and a 10% eosin stain solution, and smears were prepared and observed under a microscope.
Sperm counts were calculated using HTM-IVOS after homogenizing the left epididymis tail. The number of spermatozoa per 1 g of the tail of the left epididymis was also calculated.

Females:
Clinical Signs: The general condition and the presence or absence of death were observed twice a day before and after administration.

Sex cycle: The sex cycle was observed once a day from the administration start date to the mating confirmation date. In addition, when , the estrus period was observed over 2 consecutive days it was counted as 1 time.

Body Weights: Body weights were measured twice a week during the 14 days before the mating and during the mating period, at 0, 7, 14 and 21 gestation during gestation, on 0 and 4 days of feeding during the feeding period, respectively .

Food Intake: Food consumption was measured twice a week until 14 days before the start of the mating. Also, during pregnancy, gestation was measured on 2, 9, 16 and 21 gestation, and 4 days after nursing during gestation period.

Delivery Status: Mating females were allowed to spontaneously deliver, and the presence or absence of abnormality in labor condition and confirmation of end of delivery were confirmed once a day from day 21 of pregnancy until 25th day of pregnancy. If delivery was completed at 10:00 am, that day was taken as nursing day 0. Females who did not deliver until 25th day of pregnancy were necropsied after lethality from the abdominal aorta under ether anesthesia.

Necropsy and Histopathology: Maternal animals were observed for nursing condition once a day until 4th day of nursing and autopsied after death from the abdominal aorta under ether anesthesia on the day when all newborn cases died or on nursing 4th day, necropsied, and the number of implantation traces and I counted the corpus luteum. The ovaries were weighed and fixed in 20% neutral buffered formalin. The relative weight was also calculated by dividing the ovarian weight by the final body weight. For the ovary, a paraffin-embedded specimen was prepared according to an ordinary method. HE stained tissue specimens were prepared for the control group and the ovaries of the 800 mg / kg group and histopathological examination was performed.

Oestrous cyclicity (parental animals):

the estrus period was observed over 2 consecutive days

Sperm parameters (parental animals):

testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, viability, sperm morphology

Litter observations:

Number of pups, live birth index, body weights of both sexes, number of stillbirths.

Postmortem examinations (parental animals):

Yes. Male animals: Yes. Male animals: Animals after the last administration day were sacrificed and necropsied. The weight of testis and epididymis head as well as the epididymis tail were measured, and the relative weight was also calculated by dividing each organ weight by the final body weight. Testis and epididymis heads were fixed in Bouin's solution. Prostate and seminal vesicles were fixed in 20 % of buffered formalin. From the testis and epididymis paraffin-embedded specimens were prepared according to a conventional method. Hematoxylin-eosin staining of samples of testis and epididymis heads was performed from the control group and the 800 mg/kg group, and detailed histopathological examination of the seminal vesicles was carried out (6 subjects in the 800 mg / kg group). Since several animals from the high-dose group showed abnormalities in testes and epididymis heads compared with the control group, further histopathological examinations were performed in 12.5, 50 and 200 mg / kg groups. Females animals: Maternal animals were observed for nursing conditions once a day until 4th day of nursing when all animals together with newborns were sacrificed and necropsied. An in uterine examination was performed and the number of implantations and number of corpus luteum were calculated. The ovaries were weighed and fixed in 20% of buffered formalin. The relative ovary weight was calculated by dividing the ovarian weight by the final body weight. For the ovary, a paraffin-embedded specimens were prepared according to conventional ordinary method. Hematoxylin-eosin staining of ovary specimens from control and 800 mg / kg group was carried out and histopathological examination was performed.

Postmortem examinations (offspring):

The surviving pups was sacrificed by abdominal aorta from the abdominal aorta under necropsy under ether anesthesia on 4th day of nursing and then necropsied.

Statistics:

For statistical analysis, homodispersity was tested by the Bartlett method. In the case of equi-variance, variance analysis was performed by one-way method, and if significant, it was performed by the Dunnett method. On the other hand, when it was not recognized as equal variance, we performed analysis by the one-way method using rank order (Kruskal-Wallis test), and if significant, we used Dunnett type test method using ranking.

Mating rate, conception rate and birth rate were determined by χ ^ 2 test.

In histopathological examination, the toxicological effects were suggested in the 800 mg / kg group, and findings of organs and tissues examined for other groups were ranked using comparison between groups with the control group Dunnett type test method was used. Therefore, when a significant difference was observed with the control group, the dose reactivity test was conducted using Cochran · Armitage trend test.

Reproductive indices:

Implantation Index, Resorption Index, Mating Index, Gestational Index

Offspring viability indices:

Pups viability index, Litter Index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In males, transient salivation was observed in the 200 and 800 mg / kg group in general condition observation. In addition, loose stools were observed in each group including the control group, but no relation with the dose was observed.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No Mortality was observed in either of males and female animals of treated groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, there was no significant difference in body weight at any measurement day in each administration group compared with the control group. In females, Before the mating, there was no significant difference in body weight at any measurement day in each administration group compared with the control group.
During the gestation period, there was no significant difference in body weight at any measurement day in the 12.5, 50 and 200 mg / kg group compared with the control group. In the 800 mg / kg group, there was a significant lower value of body weight on 0 to 21 days of pregnancy than in the control group.
During the nursing period, there was no significant difference in body weight at any measurement day in the 12.5 and 50 mg / kg group compared to the control group. In the 200 and 800 mg / kg group, a significant lower value of body weight was seen on 4th day of nursing compared with the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, in the 12.5, 50 and 200 mg / kg groups, there was no significant difference in food intake on any measurement day compared to the control group. In the 800 mg / kg group, a significant low value of food intake was seen on the 3rd day of administration compared with the control group. In females, Before the mating, there was no significant difference in food consumption on any measurement day in the 12.5 and 50 mg / kg group compared with the control group. In the 200 mg / kg group, a significant lower value of food intake was observed on days 3 and 6 compared to the control group. In the 800 mg / kg group, a significant low value of food intake was seen on the 3rd day of administration compared with the control group. During the gestation period, there was no significant difference in food intake on any measurement day in the 12.5 and 50 mg / kg group compared with the control group. In the 200 and 800 mg / kg group, a significant lower value of food intake was seen on the 2nd day of pregnancy than in the control group. There was no significant difference in food intake in the 12.5 and 50 mg / kg group during the nursing period compared with the control group. In the 200 and 800 mg / kg group, a significant low value of food intake was seen on the 4th day of nursing compared with the control group.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males, In the testes, the atrophy of seminiferous tubules was observed in 6 patients in the 200 mg / kg group and in 12 cases in the 800 mg / kg group. One case of degeneration of seminiferous tubules was observed in the 200 mg / kg group. A reduction in sperm was observed in the 200 mg / kg group in one case. Two cases of giant cells appeared in 50 mg / kg group and two cases in 200 mg / kg group. In the epididymis, nine cases of sperm decrease in 200 mg / kg group and 12 cases in 800 mg / kg group were observed. In the testes, atrophy of seminiferous tubules and spermatozoa in epididymis was significantly different from the control group in the 200 and 800 mg / kg group, and the dose correlation was also confirmed. No abnormalities were found in the seminal vesicle (at 6 months in the 800 mg / kg group) who showed atrophy at necropsy. In females, in the ovary, no abnormalities were observed in the control group and the 800 mg / kg group.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no significant difference in the number of estrus counts during the pre-mating period (14 days) compared with the control group in each administration group.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

In the 12.5 mg / kg group, compared with the control group, there were no significant differences in activity sperm ratio, reference point moving speed, shortest distance traveling speed, crossing number of head crossings, malformed sperm rate, survival sperm rate, surviving sperm rate, sperm count, epididymis There was no significant difference in sperm count per 1 g of tail. In the 12.5 mg / kg group, a significant increase in the total migration rate was observed compared to the control group, but it was not a change related to the dose, the other items related to sperm activity (activity sperm ratio, The reference point moving speed, the shortest moving speed, and the number of crossings of the head), it was judged that it was not due to administration. In the 50 mg / kg group, there was a significant lower value of sperm count per g of active sperm ratio, survival sperm ratio, survival sperm ratio, sperm count and epididymal tail compared with control group, significant high value of malformed sperm ratio Was observed. In the 50 mg / kg group, a significant high value of the crossing number of the head was seen compared to the control group, but because it was not a change related to the dose, it was judged that it was not due to administration. In the 200 mg / kg group, there was a significant lower value of active sperm ratio, survival sperm ratio, survival sperm ratio, sperm count and sperm count per gram of epididymal tail compared to control group. In the 200 mg / kg group, the active spermatozoa rate in 6 cases was 0%, but no significant difference was found in the calculated animal's reference point moving speed, shortest distance moving speed and total moving speed. In the 200 mg / kg group, there was a significant lower value of the crossing number of the head compared to the control group, but it was very slight difference. In the same group, seven cases where sperm morphology observation was possible showed a significant high value of malformed sperm ratio as compared with the control group. Significant lower values ​​of sperm count and sperm count per gram of epididymal body tail were observed in the 800 mg / kg group compared to the control group. In the 800 mg / kg group, the active spermatozoic rate was 0% in all animals, a significant difference was recognized as compared with the control group, and the reference point moving speed, the shortest moving speed, the total moving speed and the crossing number of the head Can not be measured. In the 800 mg / kg group, only one animal was able to observe morphology of spermatozoa, but most of it was malformation sperm. In the same group, observable spermatozoa could not be obtained, so it was not possible to calculate surviving sperm ratio and survival sperm ratio.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was no significant difference in the number of estrus counts during the pre-mating period (14 days) compared with the control group in each administration group. Since mating was confirmed in all cases used for mating, the mating rates of each group were all 100%. There was no significant difference in the number of days required for copulation between the control group and each administration group. One unfertilized female was found in the 200 mg / kg group, but no significant difference was observed in the conception rate between each administration group and the control group.

Male
At 50 mg/kg/day:
Giant cell formation in the testis. Decrease in sperm motility ratio and number of sperms in the cauda epididymis. Increase in abnormal sperm ratio.
At 200 mg/kg/day:
Atrophy of the testis and the epididymis. Decrease in the absolute and relative testis and epididymis weights. Atrophy of seminiferous tubules. Degeneration of seminiferous tubules. Decrease in number of sperm in the cauda epididymis. Giant cell formation.
At 800 mg/kg/day:
Transient decrease in food consumption. Atrophy of the testis, the epididymides and the seminal vesicle. Decrease in the absolute and relative testis and epididymis weights. Atrophy of seminiferous tubules in the testis. Observation of no motile sperm. Increase tendency in number of abnormal sperm. Decrease in number of sperm in the cauda epididymis.
Female
At 200 mg/kg/day:
Suppression of body weight gain during the lactation period. Lower food consumption during pre-mating, pregnancy and lactation periods.
At 800 mg/kg/day:
Suppression of body weight gain during the pregnancy and the lactation periods. Lower food consumption during pre-mating, pregnancy and lactation periods..

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

12.5 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: neoplastic

reproductive function (sperm measures)

reproductive performance

other: Testicular atrophy and impaired spermatogenesis were observed at 50, 200 and 800 mg/kg/day.

Dose descriptor:

LOAEL

Remarks:

Reproductive

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Increase of percentage of sperms with abnormal morphology, atrophy of seminal vesicles of testis and epididymis, giant cell formation in the testis were observed at 50 mg/kg and above.

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect was seen on female gonad function, fertility, mating behaviour and conception. The fertility and copulation indexes were unchanged and no histopathological lesion of gonads were seen.

Dose descriptor:

NOAEL

Remarks:

Maternal developmental

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The number of pups born, number of live pups born and the number of live pups on lactation day 4, as well as the delivery index were significantly decreased at 200, 800 mg/kg.

Dose descriptor:

NOAEL

Remarks:

Systemic maternal

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Critical effects observed:

not specified

System:

other: Not Specified

Clinical signs:

no effects observed

Description (incidence and severity):

There was no abnormality in each group in the observation of the external table of the newborn. Also, there was no abnormality in each group in the general condition of the newborn.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

In the 12.5 and 50 mg / kg groups, there was no significant difference in the number of surviving children and survival rate on 4th day of nursing compared to the control group. In the 200 mg / kg group, there was a significant low value of the number of surviving children on 4th day of nursing compared to the control group. In the 800 mg / kg group, although there was no significant difference compared with the control group, a low value trend of the number of surviving children on nursing 4th day was observed.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the 12.5 and 50 mg / kg group, there was no significant difference in body weights of sexes on 0 and 4 days of nursing compared with the control group. In the 200 mg / kg group, there was a significant high value of sex and body weight on 0th day of nursing compared to the control group, but because there was no significant difference in the 800 mg / kg group, it was not due to administration It is judged. In the 800 mg / kg group, there was no significant difference in male body weight on 4th day of nursing compared to the control group, but there was no significant difference in female body weight on 4th day of nursing though there was no significant difference.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormality was found in any of the groups.

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

There was no abnormality in each group in the observation of the external table of the newborn. Also, there was no abnormality in each group in the general condition of the newborn. In the 12.5 and 50 mg / kg groups, there was no significant difference in the number of surviving children and survival rate on 4th day of nursing compared to the control group. In the 200 mg / kg group, there was a significant low value of the number of surviving children on 4th day of nursing compared to the control group. In the 800 mg / kg group, although there was no significant difference compared with the control group, a low value trend of the number of surviving children on nursing 4th day was observed. In the 12.5 and 50 mg / kg group, there was no significant difference in body weights of sexes on 0 and 4 days of nursing compared with the control group. In the 200 mg / kg group, there was a significant high value of sex and body weight on 0th day of nursing compared to the control group, but because there was no significant difference in the 800 mg / kg group, it was not due to administration It is judged. In the 800 mg / kg group, there was no significant difference in male body weight on 4th day of nursing compared to the control group, but there was no significant difference in female body weight on 4th day of nursing though there was no significant difference. No abnormality was found in any of the groups.

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

other: The number of pups born, number of live pups born and the number of live pups on lactation day 4, and pup body weight were significantly decreased at 200 and 800 mg/kg.

Remarks on result:

other: no adverse effect level is estimared

Critical effects observed:

not specified

System:

other: Not Specified

Reproductive effects observed:

not specified

Conclusions:

Based on the findings of necropsy, organ weight measurements, sperm test findings and histopathology of male sexual organs, the reproductive NOAEL (No Observed Adverse Effect Level) for male SD rats was 12,5 mg/kg/day. The test chemical had no effect on female gonad function, fertility, mating behaviour and conception as it was evidenced by the unchanged fertility and copulation index and by the lack of histopathological findings. The reproductive NOAEL for female rats was 800 mg/kg/day. The developmental NOAEL was estimated to be 50 mg/kg/day for both maternal and F1 generations based on the reduced delivery index and viability of the pups.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was carried out to evaluate the toxic potential ofthe test chemicalon reproductive performance and the development of offspring in male and female Sprague-Dawley rats. The test chemical was administered by gavage daily at the concentrations of 12.5, 50, 200 and 800 mg/kg/ bw/day. Males were dosed for the total of 50-52 days (14 days before mating and 36 to 38 days after mating), while for females, the total administration period was 40 to 48 days (14 days before mating, 14 days of mating, 21 days pregnancy and 3 days of lactation). The test chemical was solved in 5% aqueous gum Arabic solution and employed as a vehicle in controls. The purity and the stability of the test chemical were confirmed, and it was stored in refrigerator under light-proof conditions.During the administration period, ratswere observed for general condition, body weight alteration and food consumption. After the last administration day, a complete necropsy of reproductive organs was carried out. The sperm parameters including sperm motion, morphology, viability and survivability were recorded in males. Detailed histopathological examination of testis and epididymis were performed, as well. In females the weight of ovaries, and uterus parameters including number of corpora lutea, implantation, gestation length and number of live pups born were recorded. Newborn pups were observed for general conditions, neonatal survival and body weights. In male rats, no difference in body weights and food intake values were observed during the experiment. However, in the 200 and 800 mg/kg groups the relative and absolute weights of testis and epididymis were significantly decreased.The results of sperm analysesshowedsignificantlyreduced ofsperm motility, viability, survivability and the number of sperms in the left epididymis in the group treated with 50 mg/kg or more of the test chemical compared with controls. The percentage of sperms with abnormal morphology significantly increased in 50, 200 and 800 mg/kg groups in comparison to control. The histopathological examination of the gonads revealed atrophy of seminiferous tubule of testis and the epididymis in the two highest dose groups (200 and 800 mg/kg). Giant cell formation in the testis, which is usually accompanies germinal celldegeneration/atrophy was observed at the dose of 50 mg/kg and above. No death and alterations in general conditions were observed among the female rats during the test chemical administration. However, the body weight gain was significantly decreased in the high-dose group between 0-21 day of pregnancy and during the lactation period in 200 and 800 mg/kg treated group compared to controls. Similarly, the food consumption in female rats is dropped significantly on day 3-6 of pre-mating period, on day 2 of pregnancy and day 4 of lactation in the two highest dose groups. No abnormalities were seen by necropsy and the weights of ovaries showed no significant alterations between any treated and control groups. The histopathological analyses showed no alterations of uterus parameters. Similarly, no changes in fertility and mating performance between any of the treated and control groups were observed. However, the number of pups born showed a significant and dose-dependent decrease in the 200 and 800 mg/kg group compared with controls. The number of live pups born and the number of live pups on lactation day 4 significantly dropped in the two high-dose groups. The necropsy of newborns revealed no abnormalities in any of the treated-group, although the body weight of pups dropped significantly in the two highest-dose group. In summary, the present study provides clear evidence that the repeated oral exposure ofthe test chemical exertedstrong adverse effect of testicular spermatogenesis and promotes the atrophy of male gonads in male Sprague-Dawley rats. The reproductive NOAEL (No Observed Adverse Effect Level) for male SD rats was 12,5 mg/kg/day based on findings of necropsy, organ weight measurement,sperm testfindingsand histopathology. The repeated exposure of the test chemical during pre-mating and mating period had no effect on female gonad function, fertility, mating behaviour and conception as it was evidenced by the unchanged fertility and copulation index and by the lack of histopathological findings. The reproductive NOAEL for maternal generation was 800 mg/kg/day. The number of pups born, number of live pups born and the number of live pups on lactation day 4, as well as the delivery index were significantly decreased at 200 mg/kg and remained reduced, but not significantly at 800 mg/kg, which was still considered important from toxicological point of view, when female SD rats were administeredthe test chemicalduring the period of pre-mating mating, pregnancy and early lactation. Hence, the developmental NOAEL was estimated to be 50 mg/kg/day for both maternal and F1 generations. The NOAEL for systemic maternal toxicity was considered as 50 mg/kg/day based on reduced body weight and feed consumption at the two highest dose. The NOAEL for general toxicity for males was 800 mg/kg/day as no alteration of bodyweight and food consumption was seen.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: evaluation of the testicular toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

no guideline available

Principles of method if other than guideline:

A reproductive toxicity study was performed for assessing the testicular toxic effect of the test chemicals.

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No Data Available

Specific details on test material used for the study:

- Molecular weight (if other than submission substance):340.504 g/mol
- Substance type: organic
- Physical state: solid
- Impurities (identity and concentrations): 5%(identity not known)

Species:

rat

Strain:

Fischer 344/DuCrj

Details on species / strain selection:

No Data Available

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 4-week-old
- Weight at study initiation: 57.7-70.7 g
- Fasting period before study: No data available
- Housing: Animals were housed individually in chip-bedded plastic and stainless steel suspended cages in a controlled environment.
- Diet (e.g. ad libitum): The standard chow diet CE-2
- Water (e.g. ad libitum): No data available
- Acclimatization period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 11-hrs light/13-hrs dark

Route of administration:

oral: feed

Vehicle:

other: CE-2 diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The experimental pellet diets were made from powdered CE-2 and MM using a mixer, a pellet maker and a dryer. The diets were kept in a refrigerator at 10°C before use. The basal CE-2 diet (crude protein: about 25%) was free of contamination by pesticides.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Basal diet CE-2
- Storage temperature of food: At 10°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet CE-2
- Concentration in vehicle: 0 or 0.06% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): E2070-UZ; E2063-T8; E2034-YA and E2104-OX.
- Purity: No data available

Details on mating procedure:

Mating was not performed

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No Data Available

Duration of treatment / exposure:

2 months

Frequency of treatment:

Daily

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters: No data available
- Selection of parents from F1 generation when pups were [...] days of age.: No data available
- Age at mating of the mated animals in the study: [...] weeks: No data available
-No other details given.

Remarks:

Doses / Concentrations:
0.06% per day (ca. 38.6-58.0 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

Control: 8 males
0.06% per day: 8 males

Control animals:

yes, concurrent vehicle

Details on study design:

No Data Available

Positive control:

No Data Available

Parental animals: Observations and examinations:

Clinical signs of toxicity were recorded daily. Body weight and food consumption were occasionally measured.

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

Testicular sperm content and daily sperm production were determined.

Litter observations:

No data available

Postmortem examinations (parental animals):

Hematological and serum biochemical examinations were conducted after 16 hrs starvation. All animals were studied for histological changes.

Postmortem examinations (offspring):

No data available

Statistics:

All quantitative data except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheffe's multiple comparison procedures. Histopathological data were statistically analyzed with the cumulative Chi-squares test which is known to be useful for analysis of categorical data.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in body weights among F344 rats administered with the test chemical at a level of 0.06% per day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences in body weights among F344 rats administered with the test chemical at a level of 0.06% per day.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Vacuolation of Sertoli cells, exfoliation, retention of elongated spermatids in the basilar region of a stage X, degeneration of spermatids, disappearance of basement membrane, and broken tails of elongated spermatids were, however, observed in the test chemical treated group.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Daily sperm production (DSP) and DSP/g testis were significantly decreased in the test chemical-treated rats.

Reproductive performance:

not examined

No significants differences in body weight gain compared to control, no effects on the absolute organ weights, decrease in relative testicular and epididymal weights, no weights of other sex accessory organs were reduced, DSP/g testis were significant decreased, no significant change in serum testosterone level.

Histopathological findings:
Vacuolation of Sertoli cells ,exfoliation ,retention of elongated spermatids in the basilar region of stage X ,degeneration of spermatids, giant cells ,multinucleated giant cells , disappearance of basement membrane , broken tails of elongated spermatids , necrotic spermatocytes and/or spermatogonia,disappearance of germ cells , disappearance of round spermatids were observed.
ERalpha competitive binding of the test chemical:
ER alpha competitive binding of the test chemical showed that the test substance did not inhibit E2-ER alpha binding (up to 10-3 M).Serum testosterone levels were not significantly changed after treatment with the test chemical.

Dose descriptor:

LOAEL

Effect level:

ca. 40 - ca. 60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathological changes in testis and reduces sperm production.

Remarks on result:

other: Not Specified

Critical effects observed:

not specified

System:

other: Not Specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

No data available

Remarks on result:

not measured/tested

Critical effects observed:

not specified

System:

other: Not Specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Conclusions:

Based on all the results and observations, it was concluded that, the reproductive LOAEL was considered to be 0.06 % (40-60 mg/kg/day) in male F344/DuCrj (Fischer) rats when exposed to the test chemical.

Executive summary:

When the reproductive toxicity of the test chemical and its estrogenic effect were estimated in male F344/Fischer rats, the animals were fed with basal diet (control) and basal diet containing the test chemical at levels of 0.06 % (40-60 mg/kg/day) for two months. During the drug administration period, the body weight gain and food intake values were measured occasionally. Clinical signs of toxicity were recorded daily. At the termination of the study, rats were sacrificed, and the blood samples were collectedto determine the testosterone level in the serum.Preputial glands, testis, epididymis, prostate glands, seminal vesicles with coagulation glands, kidneys, and liver were dissected out and weighed.Testis was fixed and stained routinely with hematoxylin-eosin solution. Detailed histopathological observations including features and appearance of seminiferous tubules such as disruption (exfoliation), sloughing, appearances of giant cell, granuloma and vacuolated/multinucleated spermatocyte, lumen dilatation, and Leidig cell vacuolization and proliferation were recorded. Testicular sperm content and daily sperm production (DSP) were also determined.The results showed no significant differences in body weight gain among the rats administered the test chemical at any level compared with control. No alteration in mean food intake and absolute organ weights were seen at the dose of 0.06 %. However, the relative weight of testis and epididymis significantly decreased after 0.06 % of exposure. No weights of other sex accessory organs were reduced. The DSP and DSP/g testis were significantly decreased in rats treated with 0.06 % of MM, while the serum testosterone levels were not significantly altered after the treatment. Histopathological observation of testes did not indicate severe injuries, although mildtissue degenerations including vacuolated Sertoli cells, exfoliation, retention of elongated spermatids, degenerated spermatids, disappearance of basement membrane, and broken tails of elongated spermatids were observedat the dose level 0,06 %. No estrogenic activity of the test substance was noted in an in vitro ER-binding assay. In summary,the test chemicalwas found to be moderately toxic to male reproductive organ and adversely affect sperm production when F344/Fischer rats were fed at the dose of 0.06 % for two months. Hence, the LOAEL (Lowest Observed Adverse Effect Level) is 0.06 % (40-60 mg/kg/day).

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: evaluation of the testicular toxicity and estrogenic activity

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from a publication.

Qualifier:

no guideline available

Principles of method if other than guideline:

A reproductive toxicity study was performed for assessing the testicular toxic effect of the test chemicals.

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No Data Available

Specific details on test material used for the study:

- Molecular weight (if other than submission substance): 340.504 g/mol
- Substance type:organic
- Physical state:White powder
- Impurities (identity and concentrations): 5%(identity not known)

Species:

mouse

Strain:

other: Crj:CD-1 (ICR)

Details on species / strain selection:

No Data Available

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 4-week-old
- Weight at study initiation: 19.3-22.8 g
- Fasting period before study: No data available
- Housing: Animals were housed individually in chip-bedded plastic and stainless steel suspended cages in a controlled environment.
- Diet (e.g. ad libitum): The standard chow diet CE-2
- Water (e.g. ad libitum): No data available
- Acclimatization period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 11-hrs light/13-hrs dark

Route of administration:

oral: feed

Vehicle:

other: CE-2 diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The experimental pellet diets were made from powdered CE-2 and MM using a mixer, a pellet maker and a dryer. The diets were kept in a refrigerator at 10°C before use. The basal CE-2 diet (crude protein: about 25%) was free of contamination by pesticides.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Basal diet CE-2
- Storage temperature of food: At 10°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet CE-2
- Concentration in vehicle: 0 or 0.25% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): E2070-UZ; E2063-T8; E2034-YA and E2104-OX.
- Purity: No data available

Details on mating procedure:

Mating was not performed

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No Data Available

Duration of treatment / exposure:

2 months

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters: No data available
- Selection of parents from F1 generation when pups were [...] days of age.: No data available
- Age at mating of the mated animals in the study: [...] weeks: No data available
-No other details given.

Remarks:

Doses / Concentrations:
0 or 0.25% per day (ca. 160-230 mg/kg bw/day)
Basis:
nominal in diet

No. of animals per sex per dose:

Control-8 male mice
0.25%-8 male mice

Control animals:

yes, concurrent vehicle

Details on study design:

Feeding Experiment:Male mice were fed 0.25% of the test substance in diet for 2 months,followed by various examinations.

Positive control:

No Data Available

Parental animals: Observations and examinations:

Clinical signs of toxicity were recorded daily. Body weight and food consumption were occasionally measured.

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

Testicular sperm content and daily sperm production were determined.

Litter observations:

No data available

Postmortem examinations (parental animals):

At termination of administration, mice were killed and blood was collected. Preputial glands, testes, epididymides, prostate glands, seminal vesicles with coagulation glands, kidneys and liver were dissected out and weighed. Testis was fixed with formalin, sectioned, stained routinely with hematoxylin and eosin and observed microscopically.

Postmortem examinations (offspring):

No data available

Statistics:

Variations of body and organ weights are routinely expressedas per standard deviation. Bartlett’s test, analysis of variance (ANOVA), Kruskal–Wallis test, and Dunnett’s parametric or non-parametric test were used as tests for significance of differences. Fisher’s exact test was used for histopathologic data. The limit for significance was set at P<0.05.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain was not significantly depressed in male mice fed with the test chemical.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Body weight gain was not significantly depressed in male mice fed with the test chemical..

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Sloughing of seminiferous tubules was found in 50% mice. Giant cells were abundantly observed in 75% mice. In addition, dilatated lumen and Leidig cells vacuolization was also observed.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not specified

No absolute sex accessory organ weights and liver and kidney weights were changed in mice fed the test substance.Sloughing of seminiferous tubules,Giant cells,Dilatated lumen,Leidig cells vacuolization.
ERalpha competitive binding of the test chemical in vitro:
the test substance did not inhibit E2 -ERalpha binding (up to 10-3M)
Serum testosterone levels were not significant changed after treatment

Dose descriptor:

LOAEL

Remarks:

Reproductive

Effect level:

ca. 160 - ca. 230 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathological lesions were observed in testis.

Critical effects observed:

not specified

System:

other: Not Specified

Not examined/No data

Remarks on result:

not measured/tested

Critical effects observed:

not specified

System:

other: Not Specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Conclusions:

Based on all the observations and results, the reproductive LOAEL was considered to be 0.25% (160-230 mg/kg/day) in male CRrj:CD-1 (ICR) mice when exposed to the test chemical.

Executive summary:

The reproductive toxicity of the test chemical and its estrogenic effect have been evaluated in CRj: CD-1 (ICR) mice. The animals were fed either with the basal diet (control) or the basal diet containing the test chemical at levels of 0.25% (160-230 mg/kg/day) for 2 months. During the experimental period, the body weight and food intake were measured occasionally. Clinical signs of toxicity were recorded daily. At the termination of the administration period, mice were sacrifice and blood was collected to determine the blood testosterone concentration. The male sex accessory organs (preputial glands, testis, epididymis, prostate glands, seminal vesicles with coagulation glands) and the kidneys and liver were dissected out and weighed. Testis was fixed and stained then detailed histopathological analysis were carried out on it. As seen by the results, the body weight gain and the food intake were not significantly depressed in male mice fed with the test chemical. Similarly, the testicular weight was also not affected by the drug administration. No absolute sex accessory organ weights, liver and kidney weights were changed. However, testicular histological examinations provided clear evidences for an increased appearance of giant cells and the sloughing of seminiferous tubules, which could be the indicators of testicular atrophy. No estrogenic activity of the test substance was noted in an in vitro ERα-binding assay. These observations indicated that the test chemical was weakly-moderately toxic to male reproductive organs. Hence, the LOAEL was considered to be 0.25% (160-230 mg/kg/day per day) when male CRrj: CD-1 (ICR) mice were fed with 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol for 2 months.

Cross-reference 4

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from a publication.

Qualifier:

no guideline available

Principles of method if other than guideline:

In a chronic feeding study, the toxic effect of the test chemical was assessed to evaluate the effects on the reproductive parameters in rats.

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No Data Available

Specific details on test material used for the study:

- Molecular weight (if other than submission substance):340.55 g/mole
- Substance type:organic
- Physical state:solid
- Impurities (identity and concentrations):Not available

Species:

rat

Strain:

Wistar

Details on species / strain selection:

No Data Available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SLC Co. (Hamamatsu, Japan)
- Age at study initiation: 4 weeks old
- Weight at study initiation: No data avilable
- Fasting period before study: No data available
- Housing: Animals were housed individually in aluminum hanging cages.
- Diet (e.g. ad libitum): Basal diet powder F-2 (Funabashi Farm, Japan)
- Water (e.g. ad libitum): No data available
- Acclimatization period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-hrs light/12-hrs dark

Route of administration:

oral: feed

Vehicle:

other: F-2 basal diet powder

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test chemical was mixed into the basal diet powder F-2 prior to pelleting at the desired concentrations.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): F-2 basal diet powder.
- Storage temperature of food: At 4°C until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): F-2 basal diet powder
- Concentration in vehicle: 0, 0.01, 0.03 or 0.1% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

Mating was not performed.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No Data Available

Duration of treatment / exposure:

18 months

Frequency of treatment:

Daily

Details on study schedule:

- Dose selection rationale: Because of the severe histopathological changes in the testis of the lowest-dose group (0.12% per day in the diet) males in the 12-week study, doses selected for the 18-month study were 0, 0.01, 0.03 and 0.1% per day in the diet.
Male and female Wistar rats were fed 0, 100, 300 and 1000 ppm of the test substance in diet for 18 months .Various examinations were done during and after the exposure period.

Remarks:

Doses / Concentrations:
0, 0.01, 0.03 or 0.1% per day
Basis:
nominal in diet

No. of animals per sex per dose:

Control: 30 males, 30 females
0.01% per day: 30 males, 30 females
0.03% per day: 30 males, 30 females
0.1% per day: 30 males, 30 females

Control animals:

yes, concurrent vehicle

Details on study design:

No Data Available

Parental animals: Observations and examinations:

Clinical signs were recorded daily. Body weight and food consumption were monthly.

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

No data available

Litter observations:

No data available

Postmortem examinations (parental animals):

Hematological and serum biochemical examinations were conducted after 16 hrs starvation at month 6, 12 and 18. Hematological parameters examined were Red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT) and white blood cells (WBC). Serum biochemical examinations included total protein (T-PRO), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), uric acid (UA), glucose (GLU), non-esterified fatty acids (NEFA), phospholipids (PL), triglycerides (T-GLY), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 7-glutamyl transpeptidase ( 7 -GTP) , 2-hydroxybutyrate dehydrogenase (HBDH), leucine aminopeptidase (LAP) and lactate dehydrogenase (LDH) and calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), chloride (CI) and magnesium (Mg).

At autopsy, the weights of the brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary and thyroid glands were measured. The above-mentioned organs and the salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiatic nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle and bone marrow (femur and sternum) were fixed in 10% buffered formalin solution for routine histological processing and examionation.

Postmortem examinations (offspring):

No data available

Statistics:

All quantitative data except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheffe's multiple comparison procedures. Histopathological data were statistically analyzed with the cumulative Chi-squares test which is known to be useful for analysis of categorical data.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs and mortality: No remarkalbe changes in general appearance were observed in any rat. survival rates in all treated animals were comparable to those of control (see Table below).
Body weight: Significant suppression of body weight gain was only observed in 0.1% per day males from month 6 and 0.1% per day females from month 1.
Mean food intakes per rat or per kg BW in all treated groups were comparable to those of controls.Mean efficiency of feed utilization was dose-dependently decreased in both sexes.

Organ weights (testis): Significant increases or tendencies for increase in relative liver weights were observed in the 0.1% per day animals of both sexes.
Significant decreases in absolute and relative testis weights were observed in the 0.1% group throughout the study. In contrast, no change in ovary weights was observed in any of the treated females. No changes in other organs were observed in any treated groups for either sex.

Histopathology (incidence and severity):
Histopathological lesions were only observed in the testis and epididymis of males.Atrophy of testicular tubules and spermatogenic arrest and epididymis hypospermia were observed limited to the 0.1% group, throughout the study. However, no interstitial cell tumors were apparent.
In the other organs of males, no changes induced by MBMBP were observed and no changes in any organs were apparent in females.
No neoplastic lesions which could be attributed to MBMBP were observed in any organs of either sex.
Male: Atrophy of testicular tubules, spermatogenic arrest and epididymis hypospermia were observed in the 1000 ppm group.
Female: No significant effect was observed.
Haematology:In the hematological and serum biochemical analyses, several parameters demonstrated significant alteration. However, none appeared to be of biological significance, since they did not show the same tendency throughout the experimental period and/or the degrees of change were very small.
Gross pathology incidence and severity: Organ weight changes:
Male: Increase in liver weight at 300 ppm (absolute (p<0.05) and relative (p<0.01)); decrease in testis weight at 300 ppm (absolute and relative) (p<0.01).
Female: Increase in liver weight at 1000 ppm (relative) (p<0.01). no changes in ovary weights were observed in any of the treated females. no changes in other organs were observed in any treated groups for males and females.

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

12.7 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

other: Significant decrease in absolute and relative testis weight was seen at 12.7 mg/kg/day. The atrophy of testicular tubules and spermatogenic arrest and epididymis hypospermia were evident at the highest dose group throughout the study.

Dose descriptor:

LOAEL

Remarks:

Reproductive

Effect level:

42.3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathological lesion of testis and decrease in relative and absolute testis weight.

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

54.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

other: No adverse effect on female reproductive organ was seen.

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Table: survival rate, body weight and food consumption: effects seen after 18 months

 

	Males				Females
Diet level (ppm)	0	100	300	1000	0	100	300	1000
Final mean body weight (g)	545	528	520	498*	375	368	353	278*
Mean food intake (g/rat/day)	16.6	16.7	16.6	16.2	12.2	12.7	12.2	11.5
Survival rate (%)	95	95	91	95	90	100	95	95

(* significant: not specified the degree)

Testis weights: 18 month

diet level (ppm)	0	100	300	1000
Testis (g)	3.28 ± 0.48	3.40 ± 1.10	3.98 ± 0.54	0.82 ± 0.24**
Testis (g%)	0.63 ± 0.10	0.68 ± 0.22	0.82 ± 0.11	0.17 ± 0.05**

significantly different from control, *P<0.05,**P<0.01

Table: Histopathological effects seen after 18 months

Diet level (ppm)	degree*	0	100	300	1000
No. of animals		19	19	18	19
Testis, tubules Atrophy	±	0	1	0	0
	+	0	0	0	0
	++	2	0	0	0
	+++	0	3	1	19
Spermatogenic arrest	++	2	0	0	0
	+++	0	1	1	19
Epididymis Hypospermia	++	2	0	0	0
	+++	0	1	1	19

Conclusions:

Based on the organ weight measurements and histopathological findings, the reproductive NOAEL was estimated as 0.03 % (12.7 mg/kg/body weight/day) for males and 0.1 % (54.2 mg/kg/body weight/day) for females when Wistar rats were exposed to the test chemical for 18 months. LOAEL was estimated as 0.1 % (42.3 mg/kg/body weight/day) for male Wistar rats.

Executive summary:

In a chronic feeding study, Wistar male and female rats were given pellets containing either 0 (control) or 0.01, 0.03 and 0.1%of test chemicalfor 18 months. General conditions were daily monitored, and body weights and food consumption values were recorded monthly,throughout the experiment. Groups of 5 animals at month 6, 12 and 18 underwent haematological and serum biochemical examinations. All animals,5 animals/group at month 6 and 12,and all surviving animals at month 18, as well as those dying during the experiment were studied for histological changes. Survival rates were assessed using the life table technique (Rose, 1959). No remarkable changes in general appearance were seen in any rats. Survival rates of all treated animals were comparable to those of controls. Mean food intakes per rat in all treated group was comparable to those of controls. Significant suppression of body weight gain wasobserved in 0.1 % males from 6 months and 0.1 % females from month 1. In thehaematological and serum biochemical analyses, several parameters demonstrated significant alterations, but none appeared to be biological significance and toxicologically relevant. Significant decrease in absolute and relative testis weights were observedin the 0.1 % group throughout the study. In contrast,no change in ovary weight was observed in any of the treated females.Significant increases or tendencies in relative liver weights were noted in the 0.1 %groupin both sexes. In males, at 18 month the relative liver weight increased significantly at middle and high doses (0.03 and 0.1%). The blood test results did not indicate serious liverlesionat any dose group. No changes in other organs were observed in any treated groups for either sex.Histopathological lesions were only observed in the testis and epididymis in males. Atrophy of testicular tubules and spermatogenic arrest and epididymis hypospermia were evident in the 0.1 % group throughout the study. However, no interstitial cell tumour was apparent. No changes in the other male organs were seen and no changes induced by the test chemical were apparent in females. In summary, chronic administration ofthe test chemicalfor 18 month induced suppression of body weight gain in both sexes at the 0.1%. Testicular toxicity was observed at the 0. 1%, but not the 0.03 % level. Nolesion of sexual organswasapparentin females. Based on the tissue integrity of the male and female sexual organs,the reproductive NOAEL for male animals wereestimated as 0.03 % (12.7 mg/kg/day) and 0.1% (54.2 mg/kg/day) for female rats whenthe test chemicalwas chronically administered to Wistar rats for 18 months.

Cross-reference 5

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: subchronic oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from a publication.

Qualifier:

no guideline available

Principles of method if other than guideline:

The above study was performed to assess the effect of the test chemical on the reproductive parameters of the rats.

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No Data Available

Specific details on test material used for the study:

- Molecular formula (if other than submission substance):C23H32O2
- Molecular weight (if other than submission substance):340.55 g/mol
- Substance type:organic
- Physical state:solid
- Impurities (identity and concentrations):Not available

Species:

rat

Strain:

Wistar

Details on species / strain selection:

No data Available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SLC Co. (Hamamatsu, Japan)
- Age at study initiation: 4 weeks old
- Weight at study initiation: No data avilable
- Fasting period before study: No data available
- Housing: Animals were housed individually in aluminum hanging cages.
- Diet (e.g. ad libitum): Basal diet powder F-2 (Funabashi Farm, Japan)
- Water (e.g. ad libitum): No data available
- Acclimatization period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-hrs light/12-hrs dark

Route of administration:

oral: feed

Vehicle:

other: F-2 basal diet powder

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test chemical was mixed into the basal diet powder F-2 prior to pelleting at the desired concentrations.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): F-2 basal diet powder.
- Storage temperature of food: At 4°C until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): F-2 basal diet powder
- Concentration in vehicle: 0, 0.12, 0.6 or 3.0% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

Mating was not performed

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No Data Available

Duration of treatment / exposure:

12 weeks

Frequency of treatment:

Daily

Details on study schedule:

Male and female Wistar rats were fed 0,0.12, 0.6 or 3.0% per day of the test substance in diet for 12 weeks .Various examinations were done during and after the exposure period.

Remarks:

Doses / Concentrations:
0,1200,6000,30000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

Control-10 male and 10 female
1200 ppm-10 male and 10 female
6000 ppm-10 male and 10 female
30000 ppm-10 male and 10 female

Control animals:

yes, plain diet

Details on study design:

Male and female Wistar rats were fed 0(control), 0.12, 0.6 or 3.0% of the test substance in diet for 12 weeks .Various examinations were done during and after the exposure period.

Positive control:

No Data Available

Parental animals: Observations and examinations:

Clinical signs were recorded daily. Body weight and food consumption were weekly.

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

No data available

Litter observations:

No data available

Postmortem examinations (parental animals):

Hematological and serum biochemical examinations were conducted after 16 hrs starvation at week 4 and 12. Hematological parameters examined were Red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT) and white blood cells (WBC). Serum biochemical examinations included total protein (T-PRO), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), uric acid (UA), glucose (GLU), non-esterified fatty acids (NEFA), phospholipids (PL), triglycerides (T-GLY), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 7-glutamyl transpeptidase ( 7 -GTP) , 2-hydroxybutyrate dehydrogenase (HBDH), leucine aminopeptidase (LAP) and lactate dehydrogenase (LDH) and calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), chloride (CI) and magnesium (Mg).

Urinalysis was also conducted in fresh urine for pH, protein, glucose, ketone bodies, occult blood, bilirubin and urobilin in the morning at weeks 4 and 12.

At autopsy, the weights of the brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary and thyroid glands were measured. The above-mentioned organs and the salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiatic nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle and bone marrow (femur and sternum) were fixed in 10% buffered formalin solution for routine histological processing and examionation.

Postmortem examinations (offspring):

No data available

Statistics:

All quantitative data except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheffe's multiple comparison procedures. Histopathological data were statistically analyzed with the cumulative Chi-squares test which is known to be useful for analysis of categorical data.

Reproductive indices:

No data available

Offspring viability indices:

Survival indices at wk 12

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Haemorrahage from the nasal cavity was observed from all the dead animals. Diarrhea was observed in two of the four of 3.0% of the males and one in four females from 3.0% dose group of dead animals.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality was observed in males of 0.6% and 3.0% dosed group and females of 3.0% dose group from weeks 3 to 4.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In both the sexes, severe suppression was observed in 0.6 and 3.0 % dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Decreased mean food intake was observed in males of 0.6 and 3.0% dose group and females of 3.0% dose group.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant decrease in the RBC was observed in 0.6% males at week 12, but not in treated females. Significant decrease in HGB at 3.0% dose group males in week 4, and 0.6% dose group males and all treated females at week 12. Although, not significant, a decrease in PLT in both the sexes of 0.3% were observed in week 12.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant decrease in GLU was observed in 3.0% dose groups males and 0.6 and 3.0% dose group females at week 12. PL was significantly increased in 0.6 and 3.0% males and 0.6% females at week 4 and 0.6% and 3.0% dose group females at week 12. T-CHO and F-CHO were significantly elevated elevated in both the sexes at 0.6 and 3.0% dose groups in week 4 and thereafer. CHE activity was significantly decreased in both males and females receiving 0.6 or 3.0% at week 4 and in 0.6% and 3.0% females at week 12. Gamma-GTP was significantly increased in 0.6 and 3.0% females at week 4 and 0.6% males and 0.6 and 3.0% females at week 12.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no changes observed in biochemical urine parameters from the test chemical treated rats.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In male rats, testicular atrophy and appearance of the giant cells were observed in the 0.6 and 3.0% dose groups in week 4. At week 12, testicular atrophy was observed and pronounced at all the dose groups and all the male treated animals. Decreased amount of spermatogenesis was observed in all treated males in week 4 and thereafter. Interstitial edema was also observed in testes of the male rats at all dose groups at week 12. At week 4 and 12, epididymis atrophy and hypospermia at 3.0% males and atrophy of the seminal vesicles and prostate in the 0.6% and 3.0% groups were observed. In females, atrophy of the ovaries and uterus were apparent in the 0.6% and 3.0% groups. In both the sexes, thymus atrophy and bone marrow hypoplasia were noted in the 0.6 and 3.0% groups. No histopathological changes were observeds in any other organs.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Decreased amount of spermatogenesis was observed in all treated males in week 4 and thereafter. Interstitial edema was also observed in testes of the male rats at all dose groups at week 12. At week 4 and 12, epididymis atrophy and hypospermia at 3.0% males and atrophy of the seminal vesicles and prostate in the 0.6% and 3.0% groups were observed.

Reproductive performance:

not specified

Clinical signs :Deaths were observed in the 0.6 and 3.0% per day in males and in 3.0% females from weeks 3 to 4. All mortalities was accompanied by hemorrhage from the nasal cavity. Diarrhea was observed in two of the four of the 3.0% per day males and in one of the four 3.0% per day females which died.
Body weight and food consumption:In both sexes, severe suppression of body weight gain was observed when treated with 0.6 and 3.0% per day. Decreases of mean food intake per rat were observed for the 0.6 and 3.0% per day males and for the 3.0% per day females.
Organ weights:Relative liver weights were dose-dependently increased in males and relative and absolute liver weights were dose-dependently increased in females. Absolute and relative testis weights were time- and dose-dependently decreased in all treated males. In females, absolute and relative ovary weights were significantly decreased at 3.0% per day at weeks 4 and 12.
Histopathology:In male rats, testicular atrophy and the appearance of giant cells were observed at 0.6 and 3.0% per day at week 4. At week 12, testicular atrophy was pronounced and observed in all-treated rats. Decrease of spermatogenesis was evident in all treated males at week 4 and thereafter. Interstitial edema in the testis was also apparent in all treated rats at week 12. At weeks 4 and 12, epididymis atrophy and hypospermia in the 3.0% per day males and atrophy of the seminal vesicles and prostate at 0.6% and 3.0% per day were observed.
In females, atrophy of the ovaries and uterus were apparent in the 0.6 and 3.0% per day groups. In both sexes, thymus atrophy and bone marrow hypoplasia were noted in the 0.6 and 3.0% per day groups.
No histopathological changes were observed in any other organs.
Hematology
Significant decrease in the RBC was observed in 0.6% per day males at week 12, but not in treated females. Significant decrease of HGB was observed in 3.0% per day males at week 4, and 0.6% per day males and all treated females at week 12. Although not significant, a decrease of PLT in both sexes of the 0.3% per day group noted at week 12.
Clinical chemistry
Significant decrease of GLU was observed in 3.0% per day males and 0.6 and 3.0% per day females at week 4 and 3.0% per day females at week 12. PL was significantly increased in 0.6 and 3.0% per day males and 0.6% per day females at week 4 and 0.6 and 3.0% per day females at week 12. T-CHO and F-'CHO were significantly elevated in both sexes of the 0.6 and 3.0% per day groups at week 4 and thereafter.
CHE activity was significantly decreased in both males and females receiving 0.6 or 3.0% per day at week 4 and in 0.6 and 3.0% per day females at week 12. 7-GTP was significantly increased in the 0.6 and 3.0% per day females at week 4 and the 0.6% per day males and 0.6 and 3.0% per day females at week 12.
There were no changes in the biochemical parameters for urine from MBMBP treated rats.

Dose descriptor:

LOAEL

Remarks:

Reproductive

Effect level:

88 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

other: Atrophy of testicular tubules, decrease of spermatogenesis and the significant reduction of testis weights were observed at all dose levels.

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

104 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Significant decrease of absolute and relative ovaries weights and the atrophy of ovaries and uterus were seen at middle and high doses.

Dose descriptor:

NOAEL

Remarks:

General

Effect level:

ca. 88 - ca. 104 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Critical effects observed:

not specified

System:

other: Not Specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Remarks on result:

not measured/tested

Critical effects observed:

no

System:

other: Not Specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Table: survival rate and food intake and mean chemical intake  Test substance in the diet (%)  0  0.12  0.6  3.0  Males          Survival rate at wk 12 (%)  100  100  45  20  Mean food intake (g/rat/day)  19.2  18.7  15.5  12.1  Mean test substance intake (mg/kg bw/day)  0  88  564  3120  Females          Survival rate at wk 12 (%) 100   100  100  36  Mean food intake (g/rat/day) 13.3   14.3  13.0  9.1  Mean test substance intake (mg/kg bw/day) 0   104  618  2610

Conclusions:

Based on all the results and observations, it was concluded that:
1. The reproductive NOAEL for female rats was considered as 0.12 % (104 mg/kg body weight/day).
2. No reproductive NOAEL for male rats was possible to determine as testicular atrophy and reduced spermatogenesis were observed at the lowest applied dose level. Hence, the reproductive LOAEL for male rats was considered as 0.12 % (88 mg/kg body weight/day).
3. The NOAEL for systemic toxicity was 0,12 % for both males and females, based on mortality, clinical signs of toxicity, decreased body weight, increased liver weight, thymus hypertrophy and bone marrow hyperplasia, when male and female Wistar rats were fed the test chemical for 12 weeks.

Executive summary:

In a subchronic study, Wistar rats were fed with diet the test chemical at the concentrations of 0, 0.12, 0.6 or 3.0 % for 12 weeks. The body weight and food consumption values were recorded weekly. During the entire course of the study, rats were daily observed. Haematological and serum biochemical examinations were conducted. All animals were studied for histopathological changes. Severe suppression of body weight gain was observed in both sexes of 0.6 and 3.0 % groups. Death accompanied by haemorrhage from nasal cavity was observed in 0.6 and 3.0 % males and 3.0 % females. Significant and dose-dependent decrease in the absolute and relative liver weight was noted in both sexes of the 0.6 and 3.0 % groups. The blood chemical test showed elevated parameters relevant for liver disfunction as well. Significant and dose-dependent decrease in absolute and relative testis weights were observed in 0.12, 0.6 and 3.0 % males. Similarly, the uterus weight dropped significantly and dose-dependently in the 0.6 and 3.0 % groups. Histopathological examination revealed testicular atrophy, the appearance of giant cells and decrease of spermatogenesis in a dose-and time-dependent manner in all treated males. Interstitial edema in the testis was also apparent in all treated rats at week 12. At weeks 4 and 12, epididymis atrophy and hypospermia in the 3 % males and the atrophy of the seminal vesicles and prostate in the 0.6 % and 3.0 % group were noted. In females, atrophy of the ovaries and uterus were apparent in the 0.6 and 3.0 % groups. In both sexes, thymus atrophy and bone marrow hyperplasia were noted in the 0.6 % and 3.0 % groups. No histopathological changes were observed in any other organs. In conclusion, oral administration ofthe test chemicalexerted toxicologically serious adverse effectson male and female reproductive organs. In males, the remarkable effectswerethe atrophy of testicular tubules, decrease of spermatogenesis and the significant reduction of testis weight at all doses applied. In females, significant decrease of absolute and relative ovaries weightwas observed, which was accompanied by the atrophy of ovaries and uterus at 0.6 and 3 %. Hence, the reproductive NOAEL for female rats was considered as 0.12 % (104 mg/kg body weight/day). No reproductive NOAEL for male rats was possible to determine as testicular atrophy and reduced spermatogenesis were observed at the lowest applied doseslevel. Hence, the reproductive LOAEL for male rats was considered as 0.12 % (88 mg/kg body weight/day). The NOAEL for systemic toxicity was 0,12 % for both males and females, based on mortality, clinical signs of toxicity, decreased body weight, increased liver weight, thymus hypertrophy and bone marrow hyperplasia, when male and female Wistar rats were fed the test chemicalfor 12 weeks.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1999

Reference 2

Reference Type:

publication

Title:

Male reproductive toxicity of four bisphenol antioxidants in mice and rats and their estrogenic effect.

Author:

Osamu Takahashi & Shinshi Oishi

Year:

2006

Bibliographic source:

Bibliographic source- Arch. Toxicol., 80, 225-241

Reference 3

Reference Type:

publication

Title:

Male reproductive toxicity of four bisphenol antioxidants in mice and rats and their estrogenic effect q

Author:

Osamu Takahashi & Shinshi Oishi

Year:

2006

Bibliographic source:

Bibliographic source Arch. Toxicol., 80, 225-241,

Reference 4

Reference Type:

publication

Title:

ACUTE, SUBCHRONIC AND CHRONIC TOXICITY STUDIES OF A SYNTHETIC ANTIOXIDANT, 2,2'-METHYLENEBIS(4-METHYL-6-TERTBUTYLPHENOL) IN RATS.

Author:

Atsuya TAKAGI, Koichi TAKADA, Kimie SAL Toshiaki OCHIAI, Kiyoshi MATSUMOTO, Kiyoshi SEKITA, Junko MOMMA, Yoshitaka AIDA, Minoru SAITOH, Katsushi NAITOH, Tsuyoshi FURUYA, Ryuichi HASEGAWA and Yuji KUROKAWA

Year:

1994

Bibliographic source:

The Journal of Toxicological Sciences, vol. 19, 77-89

Materials and methods

Principles of method if other than guideline:

The above experiments were performed to assess the effects of the test chemical on the reproduction parameters of the test animals.

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No Data Available

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenedi-2,6-xylenol

EC Number:

226-378-9

EC Name:

4,4'-methylenedi-2,6-xylenol

Cas Number:

5384-21-4

Molecular formula:

C17H20O2

IUPAC Name:

4-[(4-hydroxy-3,5-dimethylphenyl)methyl]-2,6-dimethylphenol

Details on test material:

Details on test material
- Name of test material (as cited in study report): 4,4'-Methylenedi-2,6-xylenol
- Molecular formula (if other than submission substance): C17-H20-O2
- Molecular weight (if other than submission substance): 256.343 g/mol
- Substance type: Organic
- Physical state: White Powder
- Impurities (identity and concentrations): No Data Available

Specific details on test material used for the study:

- Molecular weight (if other than submission substance): 256.343 g/mol
- Substance type: Organic
- Physical state: White Powder
- Impurities (identity and concentrations): No Data Available

Test animals

Species:

other: Study 1: no data; Study 2 and 3: rat; Study 4: mouse; Study 5 and 6: rat

Strain:

other: Study 1: no data; Study 2: Crj:CD (SD) IGS; Study 3: Fischer 344/DuCrj; Study 4: Crj:CD-1 (ICR); Study 5 and 6: Wistar

Details on test animals or test system and environmental conditions:

Study 2: TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 10 week old
- Weight at study initiation: 332-383 g for male, 206-238 g for female.
- Fasting period before study: Not available
- Housing: Stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.
- Diet (e.g. ad libitum): The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.)
- Water (e.g. ad libitum): The drinking water was freely taken in all of the tap water
- Acclimation period: Not available
ENVIRONMENTAL CONDITIONS .
- Temperature (°C): 20 to 26 ° C.
- Humidity (%): 40 to 70%
- Air changes (per hr): 12 times / hour.
- Photoperiod (hrs dark / hrs light): Light and dark each for 12 hours (lighting: 6 am to 6 pm)

Study 3: TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 4-week-old
- Weight at study initiation: 57.7-70.7 g
- Fasting period before study: No data available
- Housing: Animals were housed individually in chip-bedded plastic and stainless steel suspended cages in a controlled environment.
- Diet (e.g. ad libitum): The standard chow diet CE-2
- Water (e.g. ad libitum): No data available
- Acclimatization period: No data available
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 11-hrs light/13-hrs dark

Study 4: TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 4-week-old
- Weight at study initiation: 19.3-22.8 g
- Fasting period before study: No data available
- Housing: Animals were housed individually in chip-bedded plastic and stainless steel suspended cages in a controlled environment.
- Diet (e.g. ad libitum): The standard chow diet CE-2
- Water (e.g. ad libitum): No data available
- Acclimatization period: No data available
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 11-hrs light/13-hrs dark

Study 5: TEST ANIMALS
- Source: SLC Co. (Hamamatsu, Japan)
- Age at study initiation: 4 weeks old
- Weight at study initiation: No data avilable
- Fasting period before study: No data available
- Housing: Animals were housed individually in aluminum hanging cages.
- Diet (e.g. ad libitum): Basal diet powder F-2 (Funabashi Farm, Japan)
- Water (e.g. ad libitum): No data available
- Acclimatization period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-hrs light/12-hrs dark

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

Study 2: PREPARATION OF DOSING SOLUTIONS: The test chemical was prepared by suspending it in a 5% gum arabic solution. The test chemical was converted to purity, and the dose was expressed in terms of the original weight. Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation. Also, as a result of confirming the concentration of the test chemical in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test chemical.
DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A
VEHICLE
- Justification for use and choice of vehicle (if other than water): 5% gum arabic
- Concentration in vehicle: 12.5, 50, 200 or 800 mg/kg/day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Study 3: PREPARATION OF DOSING SOLUTIONS:
The experimental pellet diets were made from powdered CE-2 and MM using a mixer, a pellet maker and a dryer. The diets were kept in a refrigerator at 10°C before use. The basal CE-2 diet (crude protein: about 25%) was free of contamination by pesticides.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Basal diet CE-2
- Storage temperature of food: At 10°C
VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet CE-2
- Concentration in vehicle: 0 or 0.06% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): E2070-UZ; E2063-T8; E2034-YA and E2104-OX.
- Purity: No data available

Study 4: PREPARATION OF DOSING SOLUTIONS:
The experimental pellet diets were made from powdered CE-2 and MM using a mixer, a pellet maker and a dryer. The diets were kept in a refrigerator at 10°C before use. The basal CE-2 diet (crude protein: about 25%) was free of contamination by pesticides.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Basal diet CE-2
- Storage temperature of food: At 10°C
VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet CE-2
- Concentration in vehicle: 0 or 0.25% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): E2070-UZ; E2063-T8; E2034-YA and E2104-OX.
- Purity: No data available

Study 5: PREPARATION OF DOSING SOLUTIONS:
The test chemical was mixed into the basal diet powder F-2 prior to pelleting at the desired concentrations.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): F-2 basal diet powder.
- Storage temperature of food: At 4°C until use.
VEHICLE
- Justification for use and choice of vehicle (if other than water): F-2 basal diet powder
- Concentration in vehicle: 0, 0.01, 0.03 or 0.1% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Study 6: PREPARATION OF DOSING SOLUTIONS:
The test chemical was mixed into the basal diet powder F-2 prior to pelleting at the desired concentrations.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): F-2 basal diet powder.
- Storage temperature of food: At 4°C until use.
VEHICLE
- Justification for use and choice of vehicle (if other than water): F-2 basal diet powder
- Concentration in vehicle: 0, 0.12, 0.6 or 3.0% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

Study 2: - M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy vaginal plug
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility. Not available
- Further matings after two unsuccessful attempts: [no / yes (explain)] Not available
- After successful mating each pregnant female was caged (how):Not available
- Any other deviations from standard protocol:Not available

Study 3,4,5 and 6: Mating was not performed

Details on analytical verification of doses or concentrations:

Study 2,3,4,5 and 6: No Data Available

Duration of treatment / exposure:

Study 2: Male: 50-52 days
Female: 40 to 48 days.

Study 3: 2 months

Study 4: 2 months

Study 5: 18 months

Study 6: 12 weeks

Frequency of treatment:

Study 2, 3, 4,5 and 6: Daily

Details on study schedule:

Study 2,3 and 5: No data available

Study 5: - Dose selection rationale: Because of the severe histopathological changes in the testis of the lowest-dose group (0.12% per day in the diet) males in the 12-week study, doses selected for the 18-month study were 0, 0.01, 0.03 and 0.1% per day in the diet.
Male and female Wistar rats were fed 0, 100, 300 and 1000 ppm of the test substance in diet for 18 months .Various examinations were done during and after the exposure period.

Study 6: Male and female Wistar rats were fed 0,0.12, 0.6 or 3.0% per day of the test substance in diet for 12 weeks .Various examinations were done during and after the exposure period.

Doses / concentrations

Remarks:

Study 2: Doses / Concentrations:
12.5, 50, 200, 800 mg/kg/day (in 5% gum arabic)
Basis:
no data

Study 3: Doses / Concentrations:
0.06% per day (ca. 38.6-58.0 mg/kg bw/day)
Basis:
nominal in diet

Study 4: Doses / Concentrations:
0 or 0.25% per day (ca. 371-457mg/kg bw/day)
Basis:
nominal in diet

Study 5: Doses / Concentrations:
0, 0.01, 0.03 or 0.1% per day
Basis:
nominal in diet

Study 6: Doses / Concentrations:
0,1200,6000,30000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

Study 2: Control-12 female and 12 male
12.5 mg/kg/day- 12 female and 12 male
50 mg/kg/day-12 female and 12 male
200 mg/kg/day-12 female and 12 male
800 mg/kg/day-12 female and 12 male

Study 3: Control: 8 males
0.06% per day: 8 males

Study 4: Stduy 4: Control-8 male mice
0.25%-8 male mice

Study 5: Control: 30 males, 30 females
0.01% per day: 30 males, 30 females
0.03% per day: 30 males, 30 females
0.1% per day: 30 males, 30 females

Study 6:Control-10 male and 10 female
1200 ppm-10 male and 10 female
6000 ppm-10 male and 10 female
30000 ppm-10 male and 10 female

Control animals:

other: Study 2,3,4,5: Yes, concurrent; Study 6: Yes, plain diet

Details on study design:

Study 2: No data Available

Study 3: No Data Available

Study 4: Feeding Experiment:Male mice were fed 0.25% of the test substance in diet for 2 months,followed by various examinations.

Study 5: No data available

Study 6: Male and female Wistar rats were fed 0(control), 0.12, 0.6 or 3.0% of the test substance in diet for 12 weeks .Various examinations were done during and after the exposure period.

Positive control:

Study 2,3,4,5 and 6: No Data Available

Examinations

Parental animals: Observations and examinations:

Study 2: Males:
Clinical Signs: The general condition and the presence or absence of death were observed twice a day before and after administration.

Study 3: Clinical signs of toxicity were recorded daily. Body weight and food consumption were occasionally measured.
Body weights: Body weight was measured twice a week.
Food Consumption: Food consumption was measured twice a week from 14 days before the start of the mating and after the end of the mating cycle.
Necropsy and Histopathology: On the day after the final administration, the animals were sacrificed by abdominal aorta under ether anesthesia, necropsied, and the tail weight of testis, epididymis and epididymis was measured. The relative weight was also calculated by dividing each organ weight by the final body weight. Testis and epididymis heads were fixed in Bouin's solution. Prostate and seminal vesicles were fixed in 20% neutral buffered formalin. Testis and epididymis were paraffin-embedded specimens prepared according to a conventional method. HE stained tissue specimens were prepared for the control group and the testis and epididymis heads in the 800 mg / kg group, and seminal vesicles with abnormality at necropsy (6 subjects in the 800 mg / kg group), and histopathology An inspection was carried out. For testes and epididymis heads considered to be different in number of animals showing abnormalities compared with the control group in the 800 mg / kg group examination, the 12.5, 50 and 200 mg / kg group was also examined did.
Sperm paramters: The tail of the right epididymis was divided in sperm culture (0.5% bovine serum albumin plus Medium 199) warmed to 37 ° C. and used as a sperm stock solution. The sperm concentrate was used to examine sperm activity, sperm viability and sperm morphology.Sperm activity was determined by diluting the sperm diluted solution with sperm culture solution in sperm culture solution and incubating the sperm diluted solution with HTM-IVOS (Hamilton Thorne Research) after incubating for about 30 minutes (culture conditions: 37 ° C., 5% carbon dioxide, 95% The active spermatozoa ratio was obtained and the reference point moving speed, the shortest moving speed, the total moving speed and the number of crossings of the head were calculated for the active sperm.
As the viability of sperm, according to the method 2) of Kato et al., The sperm stock solution was diluted about 3 times with the sperm culture solution in the microwell plate, then 16 μM calcein acetoxy methyl ester and 8 μM ethidium homodimer-1 After incubation and staining for about 2 hours (culture conditions: 37 ° C, 5% carbon dioxide, 95% air), spermatozoa were classified as living sperm, mid-dead sperm and killed sperm under a fluorescence microscope and surviving sperm ratio and survival The sperm ratio was determined. For survival, mid-death, and death sperm determination, green fluorescent color development is observed in the head to tail area, living spermatozoa, red fluorescent coloring in the head and green fluorescent coloring in the tail are observed in the head Somewhat dead sperm, sperm on the head, red fluorescent coloring was observed in the head, but those that did not show fluorescent coloring in the tail were regarded as dead sperm.
The morphology of the sperm was prepared by mixing a sperm stock solution and a 10% eosin stain solution, and smears were prepared and observed under a microscope.
Sperm counts were calculated using HTM-IVOS after homogenizing the left epididymis tail. The number of spermatozoa per 1 g of the tail of the left epididymis was also calculated.
Females:
Clinical Signs: The general condition and the presence or absence of death were observed twice a day before and after administration.
Sex cycle: The sex cycle was observed once a day from the administration start date to the mating confirmation date. In addition, when , the estrus period was observed over 2 consecutive days it was counted as 1 time.
Body Weights: Body weights were measured twice a week during the 14 days before the mating and during the mating period, at 0, 7, 14 and 21 gestation during gestation, on 0 and 4 days of feeding during the feeding period, respectively .
Food Intake: Food consumption was measured twice a week until 14 days before the start of the mating. Also, during pregnancy, gestation was measured on 2, 9, 16 and 21 gestation, and 4 days after nursing during gestation period.
Delivery Status: Mating females were allowed to spontaneously deliver, and the presence or absence of abnormality in labor condition and confirmation of end of delivery were confirmed once a day from day 21 of pregnancy until 25th day of pregnancy. If delivery was completed at 10:00 am, that day was taken as nursing day 0. Females who did not deliver until 25th day of pregnancy were necropsied after lethality from the abdominal aorta under ether anesthesia.
Necropsy and Histopathology: Maternal animals were observed for nursing condition once a day until 4th day of nursing and autopsied after death from the abdominal aorta under ether anesthesia on the day when all newborn cases died or on nursing 4th day, necropsied, and the number of implantation traces and I counted the corpus luteum. The ovaries were weighed and fixed in 20% neutral buffered formalin. The relative weight was also calculated by dividing the ovarian weight by the final body weight. For the ovary, a paraffin-embedded specimen was prepared according to an ordinary method. HE stained tissue specimens were prepared for the control group and the ovaries of the 800 mg / kg group and histopathological examination was performed.

Study 4:Clinical signs of toxicity were recorded daily. Body weight and food consumption were occasionally measured.

Study 5 and 6: Clinical signs were recorded daily. Body weight and food consumption were monthly.

Oestrous cyclicity (parental animals):

Study 2: the estrus period was observed over 2 consecutive days

Study 3,4,5 and 6: No data available

Sperm parameters (parental animals):

Study 2: testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, viability, sperm morphology

Study 3: Testicular sperm content and daily sperm production were determined.

Study 4: Testicular sperm content and daily sperm production were determined.

Study 5 and 6: No data available

Litter observations:

Study 2: Number of pups, live birth index, body weights of both sexes, number of stillbirths.
Study 3,4,5 and 6: No data available

Postmortem examinations (parental animals):

Study 2: Yes, in males On the day after the final administration, the animals were sacrificed by abdominal aorta under ether anesthesia, necropsied, and the tail weight of testis, epididymis and epididymis was measured. The relative weight was also calculated by dividing each organ weight by the final body weight. Testis and epididymis heads were fixed in Bouin's solution. Prostate and seminal vesicles were fixed in 20% neutral buffered formalin. Testis and epididymis were paraffin-embedded specimens prepared according to a conventional method. HE stained tissue specimens were prepared for the control group and the testis and epididymis heads in the 800 mg / kg group, and seminal vesicles with abnormality at necropsy (6 subjects in the 800 mg / kg group), and histopathology An inspection was carried out. For testes and epididymis heads considered to be different in number of animals showing abnormalities compared with the control group in the 800 mg / kg group examination, the 12.5, 50 and 200 mg / kg group was also examined. In females, Maternal animals were observed for nursing condition once a day until 4th day of nursing and autopsied after death from the abdominal aorta under ether anesthesia on the day when all newborn cases died or on nursing 4th day, necropsied, and the number of implantation traces and I counted the corpus luteum. The ovaries were weighed and fixed in 20% neutral buffered formalin. The relative weight was also calculated by dividing the ovarian weight by the final body weight. For the ovary, a paraffin-embedded specimen was prepared according to an ordinary method. HE stained tissue specimens were prepared for the control group and the ovaries of the 800 mg / kg group and histopathological examination was performed.

Study 3: Hematological and serum biochemical examinations were conducted after 16 hrs starvation. All animals were studied for histological changes.

Study 4: At termination of administration, mice were killed and blood was collected. Preputial glands, testes, epididymides, prostate glands, seminal vesicles with coagulation glands, kidneys and liver were dissected out and weighed. Testis was fixed with formalin, sectioned, stained routinely with hematoxylin and eosin and observed microscopically.

Study 5: Hematological and serum biochemical examinations were conducted after 16 hrs starvation at month 6, 12 and 18. Hematological parameters examined were Red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT) and white blood cells (WBC). Serum biochemical examinations included total protein (T-PRO), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), uric acid (UA), glucose (GLU), non-esterified fatty acids (NEFA), phospholipids (PL), triglycerides (T-GLY), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 7-glutamyl transpeptidase ( 7 -GTP) , 2-hydroxybutyrate dehydrogenase (HBDH), leucine aminopeptidase (LAP) and lactate dehydrogenase (LDH) and calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), chloride (CI) and magnesium (Mg).
At autopsy, the weights of the brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary and thyroid glands were measured. The above-mentioned organs and the salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiatic nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle and bone marrow (femur and sternum) were fixed in 10% buffered formalin solution for routine histological processing and examionation.

Study 6: Hematological and serum biochemical examinations were conducted after 16 hrs starvation at week 4 and 12. Hematological parameters examined were Red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT) and white blood cells (WBC). Serum biochemical examinations included total protein (T-PRO), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), uric acid (UA), glucose (GLU), non-esterified fatty acids (NEFA), phospholipids (PL), triglycerides (T-GLY), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 7-glutamyl transpeptidase ( 7 -GTP) , 2-hydroxybutyrate dehydrogenase (HBDH), leucine aminopeptidase (LAP) and lactate dehydrogenase (LDH) and calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), chloride (CI) and magnesium (Mg).
Urinalysis was also conducted in fresh urine for pH, protein, glucose, ketone bodies, occult blood, bilirubin and urobilin in the morning at weeks 4 and 12.
At autopsy, the weights of the brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary and thyroid glands were measured. The above-mentioned organs and the salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiatic nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle and bone marrow (femur and sternum) were fixed in 10% buffered formalin solution for routine histological processing and examionation.

Postmortem examinations (offspring):

Study 2: The surviving pups was sacrificed by abdominal aorta from the abdominal aorta under necropsy under ether anesthesia on 4th day of nursing and then necropsied.

Study 3, 4, 5 and 6: No data available

Statistics:

Study 2: For statistical analysis, homodispersity was tested by the Bartlett method. In the case of equi-variance, variance analysis was performed by one-way method, and if significant, it was performed by the Dunnett method. On the other hand, when it was not recognized as equal variance, we performed analysis by the one-way method using rank order (Kruskal-Wallis test), and if significant, we used Dunnett type test method using ranking. Mating rate, conception rate and birth rate were determined by χ ^ 2 test.
In histopathological examination, the toxicological effects were suggested in the 800 mg / kg group, and findings of organs and tissues examined for other groups were ranked using comparison between groups with the control group Dunnett type test method was used. Therefore, when a significant difference was observed with the control group, the dose reactivity test was conducted using Cochran · Armitage trend test.

Study 3 and 5 and 6: All quantitative data except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheffe's multiple comparison procedures. Histopathological data were statistically analyzed with the cumulative Chi-squares test which is known to be useful for analysis of categorical data.

Study 4: Variations of body and organ weights are routinely expressedas per standard deviation. Bartlett’s test, analysis of variance (ANOVA), Kruskal–Wallis test, and Dunnett’s parametric or non-parametric test were used as tests for significance of differences. Fisher’s exact test was used for histopathologic data. The limit for significance was set at P<0.05.

Reproductive indices:

Study 2: Implantation Index, Resorption Index, Mating Index, Gestational Index
Study 3,4,5 and 6: No data available

Offspring viability indices:

Study 2: Pups viability index, Litter Index
Study 3,4,5: No data available
Study 6: Survival indices at wk 12

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: In males, transient salivation was observed in the 200 and 800 mg / kg group in general condition observation. In addition, loose stools were observed in each group including the control group, but no relation with the dose was observed.

Study 3: No effects observed

Study 4: No data available

Study 5: No effects observed

Study 6: Haemorrahage from the nasal cavity was observed from all the dead animals. Diarrhea was observed in two of the four of 3.0% of the males and one in four females from 3.0% dose group of dead animals.

Dermal irritation (if dermal study):

not specified

Description (incidence and severity):

Study 2,3,4,5: No data available

Mortality:

no mortality observed

Description (incidence):

Study 2: No Mortality was observed in either of males and female animals of treated groups.
Study 3,4,5: No data available
Study 6: Mortality was observed in males of 0.6% and 3.0% dosed group and females of 3.0% dose group from weeks 3 to 4.

Body weight and weight changes:

not specified

Description (incidence and severity):

Study 2: In males, there was no significant difference in body weight at any measurement day in each administration group compared with the control group. In females, Before the mating, there was no significant difference in body weight at any measurement day in each administration group compared with the control group.
During the gestation period, there was no significant difference in body weight at any measurement day in the 12.5, 50 and 200 mg / kg group compared with the control group. In the 800 mg / kg group, there was a significant lower value of body weight on 0 to 21 days of pregnancy than in the control group.
During the nursing period, there was no significant difference in body weight at any measurement day in the 12.5 and 50 mg / kg group compared to the control group. In the 200 and 800 mg / kg group, a significant lower value of body weight was seen on 4th day of nursing compared with the control group.

Study 3: There were no significant differences in body weights among F344 rats administered with the test chemical at a level of 0.06% per day.

Study 4: Body weight gain was not significantly depressed in male mice fed with the test chemical.

Study 5: effects observed treatment related

Study 6: In both the sexes, severe suppression was observed in 0.6 and 3.0 % dose groups.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

Study 2: In males, in the 12.5, 50 and 200 mg / kg groups, there was no significant difference in food intake on any measurement day compared to the control group. In the 800 mg / kg group, a significant low value of food intake was seen on the 3rd day of administration compared with the control group. In females, Before the mating, there was no significant difference in food consumption on any measurement day in the 12.5 and 50 mg / kg group compared with the control group. In the 200 mg / kg group, a significant lower value of food intake was observed on days 3 and 6 compared to the control group. In the 800 mg / kg group, a significant low value of food intake was seen on the 3rd day of administration compared with the control group. During the gestation period, there was no significant difference in food intake on any measurement day in the 12.5 and 50 mg / kg group compared with the control group. In the 200 and 800 mg / kg group, a significant lower value of food intake was seen on the 2nd day of pregnancy than in the control group. There was no significant difference in food intake in the 12.5 and 50 mg / kg group during the nursing period compared with the control group. In the 200 and 800 mg / kg group, a significant low value of food intake was seen on the 4th day of nursing compared with the control group.

Study 3: There were no significant differences in body weights among F344 rats administered with the test chemical at a level of 0.06% per day.

Study 4: No effects observed

Study 5: effects observed treatment related

Study 6: Decreased mean food intake was observed in males of 0.6 and 3.0% dose group and females of 3.0% dose group.

Food efficiency:

not specified

Description (incidence and severity):

Study 2,3,4,5 and 6: No data available

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

not specified

Description (incidence and severity):

Study 2,3,4,5 and 6: No data available

Ophthalmological findings:

not specified

Description (incidence and severity):

Study 2,3,4,5 and 6: No data available

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Study 2,3,4,5: No data available

Study 6: Significant decrease in the RBC was observed in 0.6% males at week 12, but not in treated females. Significant decrease in HGB at 3.0% dose group males in week 4, and 0.6% dose group males and all treated females at week 12. Although, not significant, a decrease in PLT in both the sexes of 0.3% were observed in week 12.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Study 2,3,4,5: No data available

Study 6: Significant decrease in GLU was observed in 3.0% dose groups males and 0.6 and 3.0% dose group females at week 12. PL was significantly increased in 0.6 and 3.0% males and 0.6% females at week 4 and 0.6% and 3.0% dose group females at week 12. T-CHO and F-CHO were significantly elevated elevated in both the sexes at 0.6 and 3.0% dose groups in week 4 and thereafer. CHE activity was significantly decreased in both males and females receiving 0.6 or 3.0% at week 4 and in 0.6% and 3.0% females at week 12. Gamma-GTP was significantly increased in 0.6 and 3.0% females at week 4 and 0.6% males and 0.6 and 3.0% females at week 12.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Study 2,3,4,5: No data available

Study 6: There were no changes observed in biochemical urine parameters from the test chemical treated rats.

Behaviour (functional findings):

not specified

Description (incidence and severity):

Study 2,3,4,5 and 6: No data available

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Study 2,3,4,5 and 6: No data available

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Study 2: In males, In the testes, the atrophy of seminiferous tubules was observed in 6 patients in the 200 mg / kg group and in 12 cases in the 800 mg / kg group. One case of degeneration of seminiferous tubules was observed in the 200 mg / kg group. A reduction in sperm was observed in the 200 mg / kg group in one case. Two cases of giant cells appeared in 50 mg / kg group and two cases in 200 mg / kg group. In the epididymis, nine cases of sperm decrease in 200 mg / kg group and 12 cases in 800 mg / kg group were observed. In the testes, atrophy of seminiferous tubules and spermatozoa in epididymis was significantly different from the control group in the 200 and 800 mg / kg group, and the dose correlation was also confirmed. No abnormalities were found in the seminal vesicle (at 6 months in the 800 mg / kg group) who showed atrophy at necropsy. In females, in the ovary, no abnormalities were observed in the control group and the 800 mg / kg group.

Study 3: Vacuolation of Sertoli cells, exfoliation, retention of elongated spermatids in the basilar region of a stage X, degeneration of spermatids, disappearance of basement membrane, and broken tails of elongated spermatids were, however, observed in the test chemical treated group.

Study 4: Sloughing of seminiferous tubules was found in 50% mice. Giant cells were abundantly observed in 75% mice. In addition, dilatated lumen and Leidig cells vacuolization was also observed.

Study 5: effects observed treatment related

Study 6: In male rats, testicular atrophy and appearance of the giant cells were observed in the 0.6 and 3.0% dose groups in week 4. At week 12, testicular atrophy was observed and pronounced at all the dose groups and all the male treated animals. Decreased amount of spermatogenesis was observed in all treated males in week 4 and thereafter. Interstitial edema was also observed in testes of the male rats at all dose groups at week 12. At week 4 and 12, epididymis atrophy and hypospermia at 3.0% males and atrophy of the seminal vesicles and prostate in the 0.6% and 3.0% groups were observed. In females, atrophy of the ovaries and uterus were apparent in the 0.6% and 3.0% groups. In both the sexes, thymus atrophy and bone marrow hypoplasia were noted in the 0.6 and 3.0% groups. No histopathological changes were observeds in any other organs.

Histopathological findings: neoplastic:

not specified

Description (incidence and severity):

Study 2,3,4,5 and 6: No data available

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Study 2,3,4 and 6: No data available

Study 5: Effects observed treatment related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Study 2: There was no significant difference in the number of estrus counts during the pre-mating period (14 days) compared with the control group in each administration group.

Study 3,4,5 and 6: No data available

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Study 2: In the 12.5 mg / kg group, compared with the control group, there were no significant differences in activity sperm ratio, reference point moving speed, shortest distance traveling speed, crossing number of head crossings, malformed sperm rate, survival sperm rate, surviving sperm rate, sperm count, epididymis There was no significant difference in sperm count per 1 g of tail. In the 12.5 mg / kg group, a significant increase in the total migration rate was observed compared to the control group, but it was not a change related to the dose, the other items related to sperm activity (activity sperm ratio, The reference point moving speed, the shortest moving speed, and the number of crossings of the head), it was judged that it was not due to administration. In the 50 mg / kg group, there was a significant lower value of sperm count per g of active sperm ratio, survival sperm ratio, survival sperm ratio, sperm count and epididymal tail compared with control group, significant high value of malformed sperm ratio Was observed. In the 50 mg / kg group, a significant high value of the crossing number of the head was seen compared to the control group, but because it was not a change related to the dose, it was judged that it was not due to administration. In the 200 mg / kg group, there was a significant lower value of active sperm ratio, survival sperm ratio, survival sperm ratio, sperm count and sperm count per gram of epididymal tail compared to control group. In the 200 mg / kg group, the active spermatozoa rate in 6 cases was 0%, but no significant difference was found in the calculated animal's reference point moving speed, shortest distance moving speed and total moving speed. In the 200 mg / kg group, there was a significant lower value of the crossing number of the head compared to the control group, but it was very slight difference. In the same group, seven cases where sperm morphology observation was possible showed a significant high value of malformed sperm ratio as compared with the control group. Significant lower values ​​of sperm count and sperm count per gram of epididymal body tail were observed in the 800 mg / kg group compared to the control group. In the 800 mg / kg group, the active spermatozoic rate was 0% in all animals, a significant difference was recognized as compared with the control group, and the reference point moving speed, the shortest moving speed, the total moving speed and the crossing number of the head Can not be measured. In the 800 mg / kg group, only one animal was able to observe morphology of spermatozoa, but most of it was malformation sperm. In the same group, observable spermatozoa could not be obtained, so it was not possible to calculate surviving sperm ratio and survival sperm ratio.

Study 3: Daily sperm production (DSP) and DSP/g testis were significantly decreased in the test chemical-treated rats.

Study 4: No effects observed

Study 5: No data available

Study 6: Decreased amount of spermatogenesis was observed in all treated males in week 4 and thereafter. Interstitial edema was also observed in testes of the male rats at all dose groups at week 12. At week 4 and 12, epididymis atrophy and hypospermia at 3.0% males and atrophy of the seminal vesicles and prostate in the 0.6% and 3.0% groups were observed.

Reproductive performance:

not specified

Description (incidence and severity):

Study 2: There was no significant difference in the number of estrus counts during the pre-mating period (14 days) compared with the control group in each administration group. Since mating was confirmed in all cases used for mating, the mating rates of each group were all 100%. There was no significant difference in the number of days required for copulation between the control group and each administration group. One unfertilized female was found in the 200 mg / kg group, but no significant difference was observed in the conception rate between each administration group and the control group.

Study 3,4,5 and 6: No data available

Details on results (P0)

Study 2: Male
At 50 mg/kg/day:
Giant cell formation in the testis. Decrease in sperm motility ratio and number of sperms in the cauda epididymis. Increase in abnormal sperm ratio.
At 200 mg/kg/day:
Atrophy of the testis and the epididymis. Decrease in the absolute and relative testis and epididymis weights. Atrophy of seminiferous tubules. Degeneration of seminiferous tubules. Decrease in number of sperm in the cauda epididymis. Giant cell formation.
At 800 mg/kg/day:
Transient decrease in food consumption. Atrophy of the testis, the epididymides and the seminal vesicle. Decrease in the absolute and relative testis and epididymis weights. Atrophy of seminiferous tubules in the testis. Observation of no motile sperm. Increase tendency in number of abnormal sperm. Decrease in number of sperm in the cauda epididymis.
Female
At 200 mg/kg/day:
Suppression of body weight gain during the lactation period. Lower food consumption during pre-mating, pregnancy and lactation periods.
At 800 mg/kg/day:
Suppression of body weight gain during the pregnancy and the lactation periods. Lower food consumption during pre-mating, pregnancy and lactation periods..

Study 3: No significants differences in body weight gain compared to control, no effects on the absolute organ weights, decrease in relative testicular and epididymal weights, no weights of other sex accessory organs were reduced, DSP/g testis were significant decreased, no significant change in serum testosterone level.
Histopathological findings:
Vacuolation of Sertoli cells ,exfoliation ,retention of elongated spermatids in the basilar region of stage X ,degeneration of spermatids, giant cells ,multinucleated giant cells , disappearance of basement membrane , broken tails of elongated spermatids , necrotic spermatocytes and/or spermatogonia,disappearance of germ cells , disappearance of round spermatids were observed.
ERalpha competitive binding of the test chemical:
ER alpha competitive binding of the test chemical showed that the test substance did not inhibit E2-ER alpha binding (up to 10-3 M).Serum testosterone levels were not significantly changed after treatment with the test chemical.

Study 4: No absolute sex accessory organ weights and liver and kidney weights were changed in mice fed the test substance.Sloughing of seminiferous tubules,Giant cells,Dilatated lumen,Leidig cells vacuolization.
ERalpha competitive binding of the test chemical in vitro:
the test substance did not inhibit E2 -ERalpha binding (up to 10-3M)
Serum testosterone levels were not significant changed after treatment

Study 5: Clinical signs and mortality: No remarkalbe changes in general appearance were observed in any rat. survival rates in all treated animals were comparable to those of control (see Table below).
Body weight: Significant suppression of body weight gain was only observed in 0.1% per day males from month 6 and 0.1% per day females from month 1.
Mean food intakes per rat or per kg BW in all treated groups were comparable to those of controls.Mean efficiency of feed utilization was dose-dependently decreased in both sexes.
Organ weights (testis): Significant increases or tendencies for increase in relative liver weights were observed in the 0.1% per day animals of both sexes.
Significant decreases in absolute and relative testis weights were observed in the 0.1% group throughout the study. In contrast, no change in ovary weights was observed in any of the treated females. No changes in other organs were observed in any treated groups for either sex.
Histopathology (incidence and severity):
Histopathological lesions were only observed in the testis and epididymis of males.Atrophy of testicular tubules and spermatogenic arrest and epididymis hypospermia were observed limited to the 0.1% group, throughout the study. However, no interstitial cell tumors were apparent.
In the other organs of males, no changes induced by MBMBP were observed and no changes in any organs were apparent in females.
No neoplastic lesions which could be attributed to MBMBP were observed in any organs of either sex.
Male: Atrophy of testicular tubules, spermatogenic arrest and epididymis hypospermia were observed in the 1000 ppm group.
Female: No significant effect was observed.
Haematology:In the hematological and serum biochemical analyses, several parameters demonstrated significant alteration. However, none appeared to be of biological significance, since they did not show the same tendency throughout the experimental period and/or the degrees of change were very small.
Gross pathology incidence and severity: Organ weight changes:
Male: Increase in liver weight at 300 ppm (absolute (p<0.05) and relative (p<0.01)); decrease in testis weight at 300 ppm (absolute and relative) (p<0.01).
Female: Increase in liver weight at 1000 ppm (relative) (p<0.01). no changes in ovary weights were observed in any of the treated females. no changes in other organs were observed in any treated groups for males and females.

Study 6: Clinical signs :Deaths were observed in the 0.6 and 3.0% per day in males and in 3.0% females from weeks 3 to 4. All mortalities was accompanied by hemorrhage from the nasal cavity. Diarrhea was observed in two of the four of the 3.0% per day males and in one of the four 3.0% per day females which died.
Body weight and food consumption:In both sexes, severe suppression of body weight gain was observed when treated with 0.6 and 3.0% per day. Decreases of mean food intake per rat were observed for the 0.6 and 3.0% per day males and for the 3.0% per day females.
Organ weights:Relative liver weights were dose-dependently increased in males and relative and absolute liver weights were dose-dependently increased in females. Absolute and relative testis weights were time- and dose-dependently decreased in all treated males. In females, absolute and relative ovary weights were significantly decreased at 3.0% per day at weeks 4 and 12.
Histopathology:In male rats, testicular atrophy and the appearance of giant cells were observed at 0.6 and 3.0% per day at week 4. At week 12, testicular atrophy was pronounced and observed in all-treated rats. Decrease of spermatogenesis was evident in all treated males at week 4 and thereafter. Interstitial edema in the testis was also apparent in all treated rats at week 12. At weeks 4 and 12, epididymis atrophy and hypospermia in the 3.0% per day males and atrophy of the seminal vesicles and prostate at 0.6% and 3.0% per day were observed.
In females, atrophy of the ovaries and uterus were apparent in the 0.6 and 3.0% per day groups. In both sexes, thymus atrophy and bone marrow hypoplasia were noted in the 0.6 and 3.0% per day groups.
No histopathological changes were observed in any other organs.
Hematology
Significant decrease in the RBC was observed in 0.6% per day males at week 12, but not in treated females. Significant decrease of HGB was observed in 3.0% per day males at week 4, and 0.6% per day males and all treated females at week 12. Although not significant, a decrease of PLT in both sexes of the 0.3% per day group noted at week 12.
Clinical chemistry
Significant decrease of GLU was observed in 3.0% per day males and 0.6 and 3.0% per day females at week 4 and 3.0% per day females at week 12. PL was significantly increased in 0.6 and 3.0% per day males and 0.6% per day females at week 4 and 0.6 and 3.0% per day females at week 12. T-CHO and F-'CHO were significantly elevated in both sexes of the 0.6 and 3.0% per day groups at week 4 and thereafter.
CHE activity was significantly decreased in both males and females receiving 0.6 or 3.0% per day at week 4 and in 0.6 and 3.0% per day females at week 12. 7-GTP was significantly increased in the 0.6 and 3.0% per day females at week 4 and the 0.6% per day males and 0.6 and 3.0% per day females at week 12.
There were no changes in the biochemical parameters for urine from MBMBP treated rats.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

12.5 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed organ weights,Sperm parameter.

2

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Decrease in sperm motility ratio and number of sperms in the epididymis cauda, increase in abnormal sperm ratio

3

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed on Number of pups, live birth index, body weights of both sexes, number of stillbirths.

4

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Decreased body weight gain, number of corpora lutea, number ofimplantation scars and number of pup born and lower food consumption.

5

Dose descriptor:

LOAEL

Effect level:

58 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathological changes in testis and reduces sperm production.

6

Dose descriptor:

LOAEL

Effect level:

457 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathological changes in testis

7

Dose descriptor:

NOAEL

Effect level:

4.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed.body weight; food consumption and compound intake; food efficiency; water consumption and compound intake; gross pathology; organ weights; histopathology.

8

Dose descriptor:

LOAEL

Effect level:

12.7 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathological changes in testis and effects on spermatogenesis, and absolute liver weight and body weight.

9

Dose descriptor:

NOAEL

Effect level:

15.1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed. body weight; food consumption and compound intake; food efficiency; water consumption and compound intake; gross pathology; organ weights; histopathology.

10

Dose descriptor:

LOAEL

Effect level:

88 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathological changes in the testis, decrease in testis weight, biochemical changes.

11

Dose descriptor:

NOAEL

Effect level:

104 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed except slight decrease in hemoglobin content.

12

Dose descriptor:

LOAEL

Effect level:

618 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Death, decreased body weight, increased liver weights and histopathological changes in ovary and uterus.

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Organ:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Study 2: There was no abnormality in each group in the observation of the external table of the newborn. Also, there was no abnormality in each group in the general condition of the newborn.

Study 3 and 6: No data available

Dermal irritation (if dermal study):

not specified

Description (incidence and severity):

Study 2: No data available

Study 3 and 6: No data available

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Study 2: In the 12.5 and 50 mg / kg groups, there was no significant difference in the number of surviving children and survival rate on 4th day of nursing compared to the control group. In the 200 mg / kg group, there was a significant low value of the number of surviving children on 4th day of nursing compared to the control group. In the 800 mg / kg group, although there was no significant difference compared with the control group, a low value trend of the number of surviving children on nursing 4th day was observed.

Study 3 and 6: No data available

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study 2: In the 12.5 and 50 mg / kg group, there was no significant difference in body weights of sexes on 0 and 4 days of nursing compared with the control group. In the 200 mg / kg group, there was a significant high value of sex and body weight on 0th day of nursing compared to the control group, but because there was no significant difference in the 800 mg / kg group, it was not due to administration It is judged. In the 800 mg / kg group, there was no significant difference in male body weight on 4th day of nursing compared to the control group, but there was no significant difference in female body weight on 4th day of nursing though there was no significant difference.

Study 3 and 6: No data available

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

Study 2, 3 and 6 No data available

Food efficiency:

not specified

Description (incidence and severity):

Study 2, 3 and 6: No data available

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

not specified

Description (incidence and severity):

Study 2,3 and 6: No data available

Ophthalmological findings:

not specified

Description (incidence and severity):

Study 2, 3 and 6: No data available

Haematological findings:

not specified

Description (incidence and severity):

Study 2, 3 and 6: No data available

Clinical biochemistry findings:

not specified

Description (incidence and severity):

Study 2, 3 and 6: No data available

Urinalysis findings:

not specified

Description (incidence and severity):

Study 2, 3 and 6: No data available

Sexual maturation:

not specified

Description (incidence and severity):

Study 2, 3 and 6 No data available

Organ weight findings including organ / body weight ratios:

not specified

Description (incidence and severity):

Study 2, 3 and 6 No data available

Gross pathological findings:

not specified

Description (incidence and severity):

Study 2: No abnormality was found in any of the groups.

Study 3 and 6: No data available

Histopathological findings:

not specified

Description (incidence and severity):

Study 2, 3 and 6 No data available

Other effects:

not specified

Description (incidence and severity):

Study 2, 3 and 6 No data available

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Description (incidence and severity):

Study 2, 3 and 6 No data available

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Description (incidence and severity):

Study 2 and 6: No data available

Details on results (F1)

Study 2: There was no abnormality in each group in the observation of the external table of the newborn. Also, there was no abnormality in each group in the general condition of the newborn. In the 12.5 and 50 mg / kg groups, there was no significant difference in the number of surviving children and survival rate on 4th day of nursing compared to the control group. In the 200 mg / kg group, there was a significant low value of the number of surviving children on 4th day of nursing compared to the control group. In the 800 mg / kg group, although there was no significant difference compared with the control group, a low value trend of the number of surviving children on nursing 4th day was observed. In the 12.5 and 50 mg / kg group, there was no significant difference in body weights of sexes on 0 and 4 days of nursing compared with the control group. In the 200 mg / kg group, there was a significant high value of sex and body weight on 0th day of nursing compared to the control group, but because there was no significant difference in the 800 mg / kg group, it was not due to administration It is judged. In the 800 mg / kg group, there was no significant difference in male body weight on 4th day of nursing compared to the control group, but there was no significant difference in female body weight on 4th day of nursing though there was no significant difference. No abnormality was found in any of the groups.

Study 3: No data available

Study 4: Not examined/No data

Study 6: No data available

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effect level is estimared

2

Remarks on result:

other: Not measured/tested

3

Remarks on result:

other: not

Target system / organ toxicity (F1)

1

Critical effects observed:

not specified

System:

other: Not specified

2

Critical effects observed:

not specified

System:

other: not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Applicant's summary and conclusion

Conclusions:

Based on all the observation and results, it was concluded that the NOAEL for the test chemical was considered to be 50 mg/kg bw.

Executive summary:

Reproductive Toxicity Study:

Study 2:

A combined repeated and reproductive/developmental toxicity test by oral administration to rats of the test chemical was conducted to investigate the effects on the reproductive ability of the male and foster parents and development and development of the next generation .The dose was 800 mg / kg as the highest dose, and 200, 50 and 12.5 mg / kg as below.As a control, a vehicle (5% aqueous gum arabic solution) administration group was provided. The test substance was prepared by suspending it in a 5% gum arabic solution.The test substance was converted to purity, and the dose was expressed in terms of the original weight.Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation.Also, as a result of confirming the concentration of the test substance in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test substance. 8-week-old Sprague-Dawley male and female rats [Crj: CD (SD) IGS, (SPF)] were purchased from Charles River Japan.The obtained animals were subjected to a quarantine period of 5 days and a subsequent acclimatization period of 7 days, and animals that were not abnormal in general state and body weight transition and had no abnormality by sexual cycle observation were grouped.Grouping was carried out on the administration start date so that the average body weight and variance of each group were almost equal by random sampling method after dividing body weight by stratification using a computer.The number of animals in one group was 12 male and female. The animals were kept in a room kept at room temperature 20 to 26 ° C., humidity 40 to 70%, light and dark each for 12 hours (lighting: 6 am to 6 pm), ventilation frequency 12 times / hour.During the quarantine / acclimatization period, stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.The mother animals were individually transferred to a plastic cage containing autoclaved bedding (Sunflake, Japan Charles River Co., Ltd.) on the 18th day of pregnancy to allow natural delivery and nursing.The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.), and the drinking water was freely taken in all of the tap water. The administration route was selected for oral administration.Upon administration, it was forcibly orally administered using a polypropylene disposable syringe fitted with a metal oral gastric tube.In the males, the volume of the liquid to be administered was calculated as 10 mL / kg based on the administration day or the weight on the measurement day closest to the administration day.In females, the body weight of the measurement day closest to the administration day or the administration day before the mating and during the mating period, the body weights of 0, 7, 14, and 21 days of pregnancy during gestation, the body weight of day 0 nursing during the nursing period As a standard, calculated at 10 mL / kg.The number of administrations was once a day. The age at the start of administration was 10 weeks for both males and females, the body weight ranged from 332 to 383 g for males and from 206 to 238 g for females. In males, no death or moribund cases were observed in any group.In general condition and body weight, no change due to administration was observed.Feeding amount was transient low in the 800 mg / kg group.Autopsy showed atrophy of testis and epididymis in 200 mg / kg group, atrophy of testis, epididymis and seminal vesicle in 800 mg / kg group.In the organ weight, low absolute and relative weights of testis and epididymis were observed in groups of 200 mg / kg or more.Sperm examination showed an increase in spermatozoa rate, survival sperm ratio, survival sperm ratio, sperm count, sperm count per gram of epididymal tail, and malformed sperm ratio in the 50 and 200 mg / kg group .In the 800 mg / kg group, no active spermatozoa was observed, and the increase tendency of malformed sperm, the number of sperm and the low number of sperm per 1 g of epididymal body tail were observed.Histopathological examination showed giant cell formation in the testes in the 50 mg / kg group, atrophy of the seminiferous tubules in the testis in the 200 mg / kg group, degeneration of the seminiferous tubules, spermic depletion and giant cell formation, spermatozoa in the epididymis In the 800 mg / kg group, atrophy of seminiferous tubules and spermatozoa in the epididymis were observed in the testes. No deaths or moribund cases were observed in females in either group.In general condition, no change due to administration was observed.Weight gain was suppressed in the nursing stage in the 200 mg / kg group, and increased in the pregnancy and nursing period in the 800 mg / kg group.Feeding levels were low in the 200 and 800 mg / kg groups before mating, during pregnancy and nursing.Necropsy, organ weight and histopathological examination showed no change due to administration. In sperm examination and histopathological examination, as described above, changes were observed in the group of 50 mg / kg or more. There was no change due to the administration in the number of estruses, copulation rate, number of days required for copulation, conception rate, gestation period, delivery status, and nursing condition.In the 800 mg / kg group, one female who was unable to deliver a newborn baby and one mother who died all the newborn at the nursing stage were observed.In addition, in the 800 mg / kg group, the low or low tendency of sex and body weight in sex and body weight was observed in the number of corpus luteum, number of implantation traces, number of total births, number of neonates on 0 and 4 nursing, fertility rate, There was a tendency for the number of births to be high.In the 200 mg / kg group, the number of corpus luteums, the number of implantation traces, the total number of babies born, and the low or low tendency of the number of newborns on 0 and 4 nursing were observed.There was no change due to the administration in the external table, general condition and necropsy of the newborn. As described above, the general toxicological ineffective amount of thed test chemical is 50 mg / kg in males and the sperm test results and testicular histopathological examination It was considered to be 50 mg/kg / day because it was found that 12.5 mg / kg / day was affected by the results and 200 mg / kg in females suppressed body weight gain and low food intake .In addition, reproductive developmental toxicological ineffective dose was 12.5 mg / kg / day in females because it affected sperm examination results and testicular histopathological examination results by 50 mg / kg administration in males, 200 A low tendency of the number of corpus luteum and the number of implantation traces was observed by mg / kg administration, so 50 mg / kg / day was administered at a dose of 50 mg / kg / day, and in children, 200 mg / kg administration resulted in a low number of newborns on day 0 and day 4 It was considered to be 50 mg / kg / day.

Study 3:

In a two month feeding study with male F344/Crj (Fischer) rats, the test chemical was administer in F344/Crj (Fischer) rats. The rats were housed individually in chip-bedded plastic and stainless steel suspended cages, respectively, in an air-conditioned room at a temperature of 25±1[1]C and relative humidity of 55±5%, with an 11:13 h light: dark cycle. Male rats were divided into five groups of eight each and were fed the basal diet (control) or the basal diet containing the test chemical at levels of 0.06–0.25% for 2 months. Body weight and food intake were occasionally measured. Clinical signs of toxicity were daily recorded. At termination of administration, rats were killed and the blood was collected. Preputial glands, testes, epididymides, prostate glands, seminal vesicles with coagulation glands, kidneys, and liver were dissected out and weighed. Spleen weight was also measured in the rat 0.25% study. Testis was fixed with formalin, sectioned, and stained routinely with hematoxylin and eosin, and observed microscopically. Testicular sperm content and daily sperm production (DSP) were determined. One whole testis frozen at-80˚C from each rat or mouse was reweighed and homogenized in 0.9% NaCl–Triton X-100 solution. After staining by Trypan blue, unbroken nuclei of elongated spermatids were counted on the hemocytemeter. DSP and its efficiency (DSP/g testis) were determined by the division of the number of spermatids per testis, and spermatids per g testis with 6.1 for rats and 4.8 for mice. The test chemical induced toxicity in the testes. Animals feed with 0.06% test substance showed a decreased relative testicular and epidididymal weights and histopathological changes such as vaculoation of Sertoli cells, disappearance of basement membrane and degeneration of spermatids. Moreover, the daily sperm production (DSP) was significant decreased in treated animals. In contrast, the serum testosterone levels were not significant changed in treated animals compared to control. No estrogenic activity of the test substance was noted in an in vitro ER-binding assay. Therefore, LOAEL was considered to be 0.06% per day when male F344/Du Crj (Fischer) rats were treated with test substance on daily basis in feed for 2 months.

Study 4:

In a two month feeding study, the test chemical was administered in with maleCRj: CD-1 (ICR) mice. The mice were housed individually in chip-bedded plastic and stainless steel suspended cages, respectively, in an air-conditioned room at a temperature of 25± 1 ˚C and relative humidity of 55 ± 5%, with an 11:13 h light: dark cycle. Male mice were divided into five groups of eight each and were fed the basal diet (control) or the basal diet containing the test chemical at levels of 0.06–0.25% for 2 months. Body weight and food intake were occasionally measured. Clinical signs of toxicity were daily recorded. At termination of administration, rats were killed and the blood was collected. Preputial glands, testes, epididymides, prostate glands, seminal vesicles with coagulation glands, kidneys, and liver were dissected out and weighed. Spleen weight was also measured in the rat 0.25% study. Testis was fixed with formalin, sectioned, and stained routinely with hematoxylin and eosin, and observed microscopically. Testicular sperm content and daily sperm production (DSP) were determined. One whole testis frozen at-80˚C from each mice was reweighed and homogenized in 0.9% NaCl–Triton X-100 solution. After staining by Trypan blue, unbroken nuclei of elongated spermatids were counted on the hemocytemeter. DSP and its efficiency (DSP/g testis) were determined by the division of the number of spermatids per testis, and spermatids per g testis with 6.1 for rats and 4.8 for mice. The test chemical induced no significant toxic effects in absolute organ weights such as testes, accessory organ weights, liver and kidney. However, animals feed with the test substance showed histopathological changes in giant cells and Leydig cell vacuolization, while no estrogenic activity of the test substance was noted in an in vitro ER Alpha-binding assay. Therefore based on all the observations and results, LOAEL was considered to be 0.25% per day when male CRrj: CD-1 (ICR) mice were treated with the test chemical on daily basis in feed for 2 months.

Study 5:

In a chronic feeding study, the effects of 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol were investigated in male and female Wistar rats. The animals were treated with 0, 0.01, 0.03 or 0.1% per day of test substance mixed in feed. A suppression of the body weight gain was noted at 0.1% per day, and an increase in the relative liver weight was found at 0.03 and 0.1% per day in males and at 0.1% per day in females. In males, a decrease of the absolute and relative testes weights and atrophy of testicular tubules were observed at 0.03 and 0.1% per day. In addition, a spermatogenic arrest and epididymis hypospermia were noted at 0.1% per day in males. Therefore, NOAEL was considered to be 0.01% per day for males and 0.03% per day for females, while LOAEL for the males was considered to be 0.03% per day when Wistar rats were exposed to 2,2'-methylenebis(4-methyl-6-tert-butylphenol) by oral route for 18 months. The NOAELs are 4.2 mg/kg/day (100 ppm) for male and 15.1 mg/kg/day (300 ppm) for female.LOAEL for females was assessed to be 15.1 mg/kg/day(300 ppm).

Study 6:

A subchronic feeding study was performed to assess the effect of the test chemical on the reproductive parameters of the rats. The effects of the test chemical were investigated in male and female Wistar rats. The animals were treated with 0, 0.12, 0.6 or 3.0% per day of test substance mixed in feed.

Clinical signs were recorded daily. Body weight and food consumption were weekly. Hematological and serum biochemical examinations were conducted after 16 hrs starvation at week 4 and 12. Hematological parameters examined were Red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT) and white blood cells (WBC). Serum biochemical examinations included total protein (T-PRO), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), uric acid (UA), glucose (GLU), non-esterified fatty acids (NEFA), phospholipids (PL), triglycerides (T-GLY), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 7-glutamyl transpeptidase ( 7 -GTP) , 2-hydroxybutyrate dehydrogenase (HBDH), leucine aminopeptidase (LAP) and lactate dehydrogenase (LDH) and calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), chloride (CI) and magnesium (Mg). Urinalysis was also conducted in fresh urine for pH, protein, glucose, ketone bodies, occult blood, bilirubin and urobilin in the morning at weeks 4 and 12. At autopsy, the weights of the brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary and thyroid glands were measured. The above-mentioned organs and the salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiatic nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle and bone marrow (femur and sternum) were fixed in 10% buffered formalin solution for routine histological processing and examination. All quantitative data except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheffe's multiple comparison procedures. Histopathological data were statistically analyzed with the cumulative Chi-squares test which is known to be useful for analysis of categorical data. Mortality was observed in males of 0.6% and 3.0% dosed group and females of 3.0% dose group from weeks 3 to 4. Haemorrahage from the nasal cavity was observed from all the dead animals. Diarrhea was observed in two of the four of 3.0% of the males and one in four females from 3.0% dose group of dead animals. In both the sexes, severe suppression was observed in 0.6 and 3.0 % dose groups. Decreased mean food intake was observed in males of 0.6 and 3.0% dose group and females of 3.0% dose group. Significant decrease in the RBC was observed in 0.6% males at week 12, but not in treated females. Significant decrease in HGB at 3.0% dose group males in week 4, and 0.6% dose group males and all treated females at week 12. Although, not significant, a decrease in PLT in both the sexes of 0.3% were observed in week 12. Significant decrease in GLU was observed in 3.0% dose groups males and 0.6 and 3.0% dose group females at week 12. PL was significantly increased in 0.6 and 3.0% males and 0.6% females at week 4 and 0.6% and 3.0% dose group females at week 12. T-CHO and F-CHO were significantly elevated elevated in both the sexes at 0.6 and 3.0% dose groups in week 4 and thereafer. CHE activity was significantly decreased in both males and females receiving 0.6 or 3.0% at week 4 and in 0.6% and 3.0%  females at week 12. Gamma-GTP was significantly increased in 0.6 and 3.0% females at week 4 and 0.6% males and 0.6 and 3.0% females at week 12. Relative dose-dependent increase in the liver weights in males and dose-dependent increase in the absolute liver weights in females was observed. Absolute and relative testis weights were decreased in dose and time dependent manner in all treated males. In females, absolute and relative decrease in ovary weights was observed in females of 3.0% dose groups at wek 4 and 12. In male rats, testicular atrophy and appearance of the giant cells were observed in the 0.6 and 3.0% dose groups in week 4. At week 12, testicular atrophy was observed and pronounced at all the dose groups and all the male treated animals. Decreased amount of spermatogenesis was observed in all treated males in week 4 and thereafter. Interstitial edema was also observed in testes of the male rats at all dose groups at week 12. At week 4 and 12, epididymis atrophy and hypospermia at 3.0% males and atrophy of the seminal vesicles and prostate in the 0.6% and 3.0% groups were observed. In females, atrophy of the ovaries and uterus were apparent in the 0.6% and 3.0% groups. In both the sexes, thymus atrophy and bone marrow hypoplasia were noted  in the 0.6 and 3.0% groups. No histopathological changes were observeds in any other organs. Thus, based on all the results and observations, it was concluded that, the NOAEL was 104mg/kg/day (1200 ppm) for male and  female.