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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
T41-04:Preliminary Reproduction Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
IUCLID4 Test substance: A01-03:KURARAY Co., Ltd.; Purity, 99.19%
- Name of test material (as cited in study report): 3-methoxy-3-methylbutanol (MMB); 1-Butanol, 3-methoxy-3-methyl-, ; Solfit
- Molecular formula (if other than submission substance): C6H14O2
- Molecular weight (if other than submission substance): 118.18
- Structural formula attached as image file (if other than submission substance): (H3C)2(H3CO)C(CH2)2OH
- Physical state: clear colorless liquid
- Analytical purity: 99.19% (Supplier: Kuraray Co. Ltd.)
- Impurities (identity and concentrations): 2-methoxy-4-methoxymethyl-2-buthylbutane, 0.2%; 4-t-butoxy-2-methyl-2-butanol, 0.1%
- Purity test date: 2 february 2001
- Lot/batch No.: 22517
- Received on (date): 6 february 2001
- Stability under test conditions:
- Storage condition of test material: cool dark place (4°C), sealed

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Atsugi Production site (795 Shimo-furusawa, Atsugi-shi, Kanagawa-ken
- Weight at study initiation: (P) Males: 356-433 g; Females: 225-268 g;
- Housing: 1 per stainless metallic mesh cage
- Age at study initiation: (P) 10 wks
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): ad libitum (Labo MR stock, pellet, Nosan Co.)
- Water (e.g. ad libitum): sterilized tap water
- Acclimation period: 13 days to the test environment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 10%
- Air changes (per hr): 10 per hr
- Photoperiod (hrs dark / hrs light): 12 h dark/ 12 h light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose solutions were prepared as water solutions at the concentrations for defined doses, using official purified water. A stability test was conducted for 10 and 0.2 % water solutions of this substance, confirming the stability at least for seven days under cool dark conditions (4°C). Therefore, duration of use of the dose solution was determined to be seven days after preparation, and the prepared dose solutions were divided into daily portions, tightly sealed and stored until use at a cool place (4°C) protected from lights. In addition, the first prepared dose solution was analyzed, and confirmed to be prepared at the defined concentration.

VEHICLE
- Concentration in vehicle: 0, 8, 40, 200, 1000 mg/kg bw/day
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- M/F ratio per cage:
- Length of cohabitation: The male-female pairs were made in the same group that had completed two-week administration prior to mating (afternoon on day 15 of dosing), and the paired animals were cohoused continously for at least two weeks up to confirmation of copulation.
- Proof of pregnancy: copulation was checked by formation of vaginal plug or presence/absence of sperms in the vaginal smear, and the day of confimation was considered to be day 0 of gestation
- The delivery status was observed at the same point, and the day of confirmation of completion of the delivery was considered to be day 0 of lactation. The fertility rate (%) (number of pregnant females/number of pairs with sucessful copulation*100) and gestation rate (%) (number of females with live born/number of living pregnant females*100) were calculated from the results of observation of mating and delivery.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Exposure period: 47 days for males; 42-52 days from 14 days before mating to day 4 of lactation for females
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: 47 days for males; 42-52 days for females
Frequency of treatment:
once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
8, 40, 200, 1000 mg/kgbw:/day
Basis:

No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were set based on the results obtained up to necropsy at the end of administration in a 28-day repeated dose toxicity test of 3-methoxy-3-methylbutanol, conducted a little ahead of this test.

Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Deaths/survival, appearance, behavior, etc. of the animals were checked at least twice daily in the morning and evening. In particular, the status of gestation, delivery and lactation were carefully observed.

BODY WEIGHT: Yes
- Body weight was measured on Day 1 (initial dosing day), 8, 15, 22, 29, 36, 43 and 47 of dosing for males, day 1, 8 and 15 of dosing, day 0, 7, 14 and 20 of gestation, and 0 and 4 of lactation for females. Body weight was determined also on the day of sacrifice. For food consumption, 24-hour food consumption was measured from the day of body weighing to the next day for each cage. However, the final day of measurement of food consumption was set to be day 46 of dosing for males and day 3 of lactation for females. Food consumption was not measured on day 15 of dosing during the mating period for all male/female animals and on day 22 of dosing for male/female animals that failed to copulate.

Oestrous cyclicity (parental animals):
For females, after the acclimatization and quarantine period, the phase of estrus cycle was determined by the microscopic test on prepared Giemsa-stained vaginal smears, unitl confirmation of copulation.
Postmortem examinations (parental animals):
SACRIFICE
Male animals for scheduled sacrifice were killed by exsanguinations under ether anesthesia on the next day of Day 47 of dosing, females with delivery and normal lactation on Day 5 of lactation, and animals that failed to copulate 24 days after completion of the mating period (the next day of Day 52 of dosing), and body surface, orifice mucosa and every internal organ were macroscopically observed.

GROSS NECROPSY
In addition, the liver and kidney of males and females, and testis and epididymis of males were weighed (absolute weight), and the body weight ratio (relative weight) was calculated based on the body weight on the day of sacrifice. For females, number of corpus luteum in the ovary and number of implantation sites in the uterus were examined to calculate implantation rate (%) [number of implantation sites/Number of corpus luteum×100].

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were collected and fixed with 10% neutral phosphate-buffered formalin solution (the testis and epididymis were pre-fixed with Bouin’s solution), and stored.
Brain, pituitary glands, thyroid glands, thymus, trachea, lungs, stomach, intestine, heart, liver, spleen, kidneys, adrenal glands, urinary bladder, testes, epididymis, prostate glands, seminal vesicles, ovaries, uterus, spinal code (cervical, thoracic and lumber regions), sciatic nerves, bone marrow (femur),lymph nodes (cervical lymph nodes and mesenteric lymph nodes), mammary glands and other macroscopic abnormal regions
In the histopathological examination, liver, kidneys, testes, epididymis, ovaries and macroscopic abnormal regions were examined in the control and 1000mg/kg group. In addition, for the paired animals that failed to copulate, prostate glands and seminal vesicles were examined for males and uterus and pituitary glands for females. Since the results showed no changes caused by administration of the test substance, only macroscopically abnormal regions were examined in 8, 40 and 200mg/kg dosed groups. For the examination, paraffin sections were prepared, according to the common procedure, stained with hematoxylin and eosin, and microscopically examined.
Postmortem examinations (offspring):
HISTOPATHOLOGY / ORGAN WEIGTHS
For Dead pups when died, and live pups which were sacrificed by exsanguinations under ether anesthesia on Day 4 of lactation, main thoracic and abdominal organs were macroscopically observed.
Statistics:
For observed means and frequencies, significant differences (the risk rate is 5% or less) were tested as follows between each dose group of the test substance and the control group. Parametric data (body weight, food consumption, organ weights, number of corpus luteum, number of implantation sites, gestation length and number of pups born) was tested by Bartlett’s test. As the results, when the variances were uniform, the one-way analysis of variance was conducted, and when significant differences were found, comparative test was conducted with the control group by Dunnett’s method or Scheffe’s method (when the number of samples of each group was different) . When the variances were not uniform, or in case of nonparametric data (implantation rate, live birth rate, delivery rate, viability rate of live pups, incidence of abnormal cases in external findings and visceral findings of pups born), Kruskal-Wallis rank test was performed, and if there were significant differences, Dunnett’s test or Scheffe’s test was conducted. For categorical data, Fisher’s exact test (observation of general conditions, onset rate of anomalies in necropsy and histopathological examination) or x2 test (copulation rate, fertility rate, gestation rate and sex ratio of pups) was used. For the data on pups, the mean of the litter was considered to be one sample.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased kidney and liver weights
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No changes in general conditions or any death were noted in the control or test substance dosed groups during the dosing or recovery period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no significant differences in body weight and increase in body weight between the control group and the test substance dosed groups during the dosing period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There were no significant differences between the control group and test substance dosed groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Normal return of estrus was noted from the grouping to prior to mating, in all animals, showing no significant differences in the estrus cycle of each of the test substance dosed groups from that of the control group. However, after starting of mating, the estrus cycle of one animal of the 40mg/kg group transferred from postestrus to anestrus, which continued up to the end of the mating period, resulting that copulation was not established.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Copulation rate and fertility rate:
Copulation was established in all cases except one pair of the 40mg/kg dose group, showing no significant differences in mating days until copulation. The fertility rate was 100% both in the control and test substance dosed groups.
Number of corpus luteum, number of implantation sites and implantation rate:
There were no significant differences in number of corpus luteum, number of implantation sites or implantation rate between each of the test substance dosed groups and the control group.
Gestation rate and gestation length:
The gestation rate was 100% both in the control and test substance dosed groups. For gestation length, there were no significant differences between the control and each of the test substance dosed groups.
Status of delivery and lactation:
There were no abnormalities in the status of delivery or lactation in the control and each of the test substance dosed groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Significant increase in absolute and relative kidney weights were noted in males of the 200 and 1000mg/kg groups. In addition, significant increase in relative liver and kidney weights were noted in females of the 1000mg/kg dosed group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No changes caused by administration of the test substance were noted.
As the findings sporadically noted, but not related to administration of the test substance, black spots on the lungs were noted in one female of the control group, one male of the 8mg/kg group and one female of the 1000mg/kg group, diaphragmatic nodule in one female of the control group, red spots in thymus in one male of the 8mg/kg group and small testis/epididymis (bilateral) in one male of the 1000mg/kg group. In addition, small testis/epididymis (unilateral) was also noted in one male of the pair that failed to copulate in the 40mg/kg group. There were no changes in the female of the pair that failed to copulate.

HISTOPATHOLOGY (PARENTAL ANIMALS)
(1) Organs other than reproductive ones
No changes caused by administration of the test substance were noted.
For changes considered not related to administration of the test substance, microgranuloma and focal necrosis in the liver, eosinophilic body/hyaline droplets in proximal tubular epithelium, basophilic tubules, formation of cysts, hyaline cast, cellular infiltration of lymphocytes/fibrosis of cortex, mineralization of cortico-medullary junction and hyperplasia of pelvic epithelium in the kidneys were noted in males and/or females in the control and 1000mg/kg group. Furthermore, black spots on the lungs, which were sporadically noted in necropsy, were accompanied with hemorrhage or hemorrhage with hematoidin crystals, and red spots in the thymus gland accompanied hemorrhage.
(2) Reproductive organs
No changes caused by administration of the test substance were noted.
For changes considered not related to administration of the test substance, atrophy of seminiferous tubules/hyperplasia of intestinal cells in the testis and sperm decrease/cell debris in lumen of epedidymis in the small testis/epididymis (lateral) of one male of the 1000mg/kg group, which was noted at necropsy. In the case of the 40mg/kg group that failed to copulate, atrophy of seminiferous tubules in the testis (bilateral) was noted in the paired male particularly the change in one side was severe, showing almost no sperm formation or sperms in the lumen of epidydimis. There were no changes in prostate gland or seminal vesicle of the male, or ovary, uterus or pituitary gland of the paired female.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive and development toxicity
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: general toxicity
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: general toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY and Body weight (OFFSPRING)
There were no significant differences between each of the test substance dosed groups and the control group in total number of pups born per litter, delivery rate, number of newborn, live birth rate, sex ratio, body weight on Day 0 of lactation or viability/body weight on Day 4 of lactation, and no abnormalities were noted in general conditions of newborn.

GROSS PATHOLOGY (OFFSPRING)
There were no abnormalities in external or visceral findings of the pups. For visceral variations, thymus remnant in neck and persistent left umbilical artery were noted in a few cases of each group, but there were no significant differences in their onset rates between each of the dosed groups and control group.




Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

NOAEL:40 mg/kgbw/day for repeated dose toxicity of males and
200 mg/kg bw/day for repeated dose toxicity of females, and 1000 mg/kgbw/day for reproductive performance of parents and for offspring development. 

Mortality: There was no mortality related to the test material treatment.
Clinical signs: No effects related to the test material were apparent on clinical observation. 
Body weight: No statistically significant changes.
Food consumption: No statistically significant changes.
Necropsy: No effect for males and females.
Organ weights: Absolute and relative weights of kidneys increased in males at 200 mg/kg bw/day or more and relative
weights of liver and kidneys increased in females at 1000 mg/kg bw/day. 

Males: 
Dose(mg/kg)                 0     8    40    200  1000
No.of animals               12    12    12    12    12
Kidneys   Absolute weight(g) 
Mean    3.10  3.26  3.25  3.50* 3.68

SD    0.28  0.35  0.32  0.23  0.47 
Relative weight(g%)

           Mean    0.59  0.59  0.60  0.65* 0.70**
           SD    0.04  0.05  0.06  0.04  0.04
Females:
Dose(mg/kg)                 0     8    40    200  1000
No.of animals               12    12    12    12    12
Liver   Relative weight(g%)
                    Mean    4.62  4.72  4.84  4.70  5.13**
                      SD    0.25  0.26  0.24  0.40  0.25
Kidney  Relative weight(g%)
                    Mean    0.57  0.59  0.60  0.59  0.64**
                      SD    0.05  0.04  0.03  0.03  0.05    

Histopathology: No effect for males and females.

Reproductive and developmental parameters: The parent
animals exhibited no alterations in reproductive parameters.
There were no significant differences in offspring
parameters.

Reproduction results:
Dose(mg/kgbw/day)        0     8    40   200  1000
No.of females examined  12    12    12    12    12
Estrous cycle(days)  
                     Mean    4.0   4.0   4.0   4.0   4.0
                       SD    0.0   0.1   0.1   0.0   0.1   

No.of pairs mated           12    12    12    12    12   
No.of pairs with successful
copulatation                 12    12    11    12    12
Copulation index(%)        100   100   91.7  100   100 
Pairing days until 
copulation(day)      
                     Mean   2.3   3.2   2.0   2.3   3.3      
                       SD   0.9   0.8   1.0   1.2   1.8

No.of pregnant females    12    12    11    12    12
Fertility index(%)          100   100   100   100   100
No.of corpora lutea  
                     Mean   18.7  19.5  18.2  17.8  18.3     
                       SD    2.7  2.2   1.9   2.1   2.1   

No.of implantation sites
                     Mean   17.0  18.1  16.6  16.4  16.7
                       SD    2.6   1.8   3.0   1.2   2.4

Implantation index(%)
                     Mean   91.4  93.0  90.9  92.7  91.7     
                       SD    9.0   6.2   8.9   6.5  11.5 

No.of pregnant females
with parturition             12    12    11    12    12
Gestation length(days) 
                     Mean   22.5  22.4  22.5  22.2  22.4
                       SD    0.5   0.5   0.5   0.4   0.5

No.of pregnant females
with live pups               12    12    11    12    12
Gestation index(%)          100   100   100   100   100
No.of pregnant females
with live pups on day 4      12    12    11    12    12

Litter results:
Dose(mg/kgbw/day)           0     8    40   200  1000
No.of pups born    
                   Mean    15.2  16.7  15.4  15.3  14.9     
                     SD     3.9   2.3   3.3   1.7   2.1 
Deliver index(%)   
                   Mean    89.5  92.1  92.0  93.3  89.9     
                     SD    19.8   8.6   7.4   7.1   8.1 
No.of pups on day 0 of lactaion
                   Mean    15.2  16.7  15.3  15.3  14.5     
                     SD     3.9   2.3   3.2   1.7   2.5
Live birth index(%)
                   Mean    100   100   99.5  100   97.2
                     SD      0     0   1.6     0    9.6
Sex ratio(male/female)     1.04  0.82  1.04  1.16  1.01
No.of pups alive on day 4 of lactation
                   Mean    14.9  16.3  14.9  15.0  14.1
                     SD     3.7   2.3   3.2   2.0   2.6
Viability(%)      
                   Mean    98.6  98.1  97.6  97.7  97.1
                     SD     2.6   4.4   4.9   4.5   4.5
Body weight of live pups 
 on day 0 : Male   
                   Mean     7.2   7.1   7.3   7.0   6.7
                     SD     0.6   0.8   0.8   0.5   0.6
          : Female 
                   Mean     6.9   6.7   7.0   6.4   6.5
                     SD     0.8   0.9   0.8   0.5   0.4
 on day 4 : Male   
                   Mean    11.7  11.7  11.6  11.5  11.0
                     SD     1.7   1.7   2.3   0.8   1.1
          : Female 
                   Mean    11.4  11.0  11.0  10.8  10.7
                     SD     1.8   1.6   1.9   0.7   1.1

Applicant's summary and conclusion

Conclusions:
A well reported reproductive toxicity screening study, conducted according to the current guideline for that endpoint and in accordance with GLP, identified a NOEC value of 40 mg/kg bw/day in male rats and 200 mg/kg bw/day in female rats; changes in kidney and liver weights were evident at 200 and 1000 mg/kg bw/day in male rats and 1000 mg/kg bw/day in female rats. No effects on fertility of male and female parents and on each of the indices for development/growth of pups were evident.