Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
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
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
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
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.
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: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed
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)
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
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
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.




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
Reproductive effects observed:
not specified

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

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.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the reproduction/developmental toxicity screening test [OECD TG 421], Crj:CD(SD)IGS rats (12 animals/sex/dose) were given MMB by gavage at 0 (vehicle: distilled water), 8, 40, 200 or 1000 mg/kg bw/day. Males were dosed for 47 days and females were dosed from day 14 before mating to day 4 of lactation throughout the mating and pregnancy period. Increases in absolute and relative weights of the kidney in males at 200 mg/kg bw/day and higher and relative weight of the liver and kidney in females at 1000 mg/kg bw/day were detected.

No effects of MMB were detected on reproductive parameters such as estrous cycle, number of pairs mated, number of pairs with successful copulation, copulation index, precoital interval, number of pregnant females, fertility index, number of corpora lutea, number of implantation sites, implantation index, number of pregnant females with parturition, gestation length, number of pregnant females with live pups, gestation index and number of pregnant females with live pups on day 4 of lactation. NOAELs for general toxicity were considered to be 40 mg/kg bw/day in males and 200 mg/kg bw/day in females. An NOAEL of 1000 mg/kg bw/day (maximum dose tested) was determined for reproductive and developmental toxicity in rats.


Short description of key information:
A well reported reproductive toxicity study, conducted according to the current guideline for that endpoint and in accordance with GLP is available for MMB. No effects on fertility of male and female parents were evident at the highest dose tested.

Justification for selection of Effect on fertility via oral route:
well conducted OECD 421 study

Effects on developmental toxicity

Description of key information
Two well reported studies are available for the developmental toxicty of MMB, conducted according to generally accepted scientific standards and in accordance with GLP:
The key study (Sagamihara Kanagawa, 1991) reported a maternal NOEL of 250 mg/kg bw/day and developmental NOAEL of 500 mg/kg bw/day. The second study (Atsushi Noda, 2002) identified no effects on fertility of male and female parents. No effetcs on each of the indices for development/growth of pups were evident.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: T44-03:Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use(FDA:1966)
GLP compliance:
yes
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: 72 days (birthday: 20 august 1990; female); 66 days (birthday: 18 feburary 1990; male)
- Weight at study initiation: 196 to 228 g (after day of arrival; female); 242 to 288 g (after day of arrival; male)
- Housing: 1 per stainless steel cage
- Diet (e.g. ad libitum): ad libitum ( certified rodent chow; Ralston purina)
- Water (e.g. ad libitum): ad libitum (reverse osmosis membrane)


ENVIRONMENTAL CONDITIONS
- Temperature: 70 to 78 °F
- Humidity (%): 40 to 70 %
- Air changes (per hr): 10/hr
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Solutions of the test article in deionized water were prepared daily during the study. The following concentrations of 3-methoxy-3-methyl-butan-1-ol were preparted: 0, 25, 50, 200 mg/mL

DIET PREPARATION
no details given

VEHICLE
- Concentration in vehicle: 0, 25, 50, 200 mg/mL
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): 023849
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
target concentrations: 12.5; 200 mg/mL
measured average concentrations: 12.33; 196.8 mg/mL
GC (Shimadzu, FID detector); (conducted at Lancaster Laboratories Inc.)
Details on mating procedure:
- Impregnation procedure:
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: female rats with spermatozoa observed in a vaginal lavage or a copulatory plug observed in situ were considered to be at day 0 of presumed gestation and assigned to individual housing
- All females rats that did not have a confirmed mating date, as well as all female rats that mated but were not assigned to the study, were placed in the general population of the test facility.
Duration of treatment / exposure:
day 6 through 15 of gestation
Frequency of treatment:
once a day
Duration of test:
15 days from 6 to 20 days of gestation
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: for 15 days from 6 to 20 days of gestation
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for test duration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: day 0 and on days 6 through 20 of presumed gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; day 0 and on days 6 through 20 of presumed gestation

POST-MORTEM EXAMINATIONS: Yes /
- Sacrifice on gestation day #20
- Organs examined: laparaohysterectomic examination and necropsy

OTHER:
half of the foetuses from 0 and 2000 mg/kg bw/day groups were examined for soft tissue abnormalities
half of the foetuses from all groups were examined for skeletal abnormalities
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
Statistics:
Dunnet (1955), A multiple comparison procedure for comparing several treatments with a control
Snedecor (1967), variance test for homogeneity of the binomial distribution
Siegel (1956), nonparametric statitistics for the behavioral sciences
Dunn (1964), multiple comparisons using rank sums
Kruskal-Wallis test

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No rats died during the conduct of this study
Decreased mortor activity, excess salivation, ataxia, muscle flaccidity and loss of righting reflex were observed in the 2000 mg/kgbw/day group.
Dose descriptor:
NOAEL
Effect level:
< 250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
maternal abnormalities
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
maternal abnormalities
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The 2000 mg/kgbw/day group reduced fetal body weight. No other Caesarean-sectioning or litter observations were attributable to the test material. Malformations and variations: The test material did not cause fetal malformations. The 2000 mg/kgbw/day group had significant increases in the litter and fetal incidences of variations in skeletal ossification of the ribs, sternum and pelvis.
Dose descriptor:
LOAEL
Effect level:
2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Dose descriptor:
LOAEL
Effect level:
2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in litter size and weights
Abnormalities:
not specified
Developmental effects observed:
not specified


NOEL for pregnant females: Less than 250 mg/kgbw/day
NOEL for development of fetuses: 500 mg/kgbw/day
  
Clinical signs: Decreased mortor activity, excess salivation, ataxia, muscle flaccidity and loss of righting reflex were observed in the 2000 mg/kgbw/day group.
Body weights: Decreases of body weight gains in pregnant females of 250, 500 and 2000 mg/kgbw/day.
Food consumption: The 250, 500 and 2000 mg/kgbw/day groups had significant reductions in absolute (g/day) and
relative(g/kg/day) maternal feed consumption values for the entire dosage period.
Necropsy: No gross lesions were caused by the test material.
Fetal parameters: The 2000 mg/kgbw/day group reduced fetal body weight. No other Caesarean-sectioning or litter observations were attributable to the test material.
Malformations and variations: The test material did not cause fetal malformations. The 2000 mg/kgbw/day group had
significant increases in the litter and fetal incidences of variations in skeletal ossification of the ribs, sternum and pelvis.

Conclusions:
A well reported study conducted according to generally accepted scientific standards and in accordance with GLP reported maternal toxicity (increased incidences of clinical observations, and decreases in body weight gain and food consumption) at 500 and 2000 mg/kg bw/day. The occurrence of maternal toxicity was accompanied by fetal toxicity (reduced fetal body weight and significant increases in the litter and fetal incidences of variations in skeletal ossification of the ribs, sternum and pelvis at 2000 mg/kg bw/day). No significant maternal or developmental effects were observed at 250 and 500 mg/kg bw/day, respectively. The maternal NOEL was 250 mg/kg bw/day and developmental NOAEL was 500 mg/kg bw/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a developmental toxicity study, Crj:CD(SD) female rats (25 animals/dose) were given MMB (CAS: 56539-66-3) by gavage at 0 (vehicle: deionized water), 250, 500 or 2000 mg/kg bw/day on days 6-15 of gestation. Decreased motor activity, excess salivation, ataxia, muscle flaccidity and loss of righting reflex at 2000 mg/kg bw/day and decreases in body weight gains and food consumption at 250 mg/kg bw/day and higher were observed in dams. Fetal body weights were decreased at 2000 mg/kg bw/day. No increases in embryonic/fetal deaths and fetal malformations were detected after administration of MMB. Increases in skeletal variations and delayed ossification were found at 2000 mg/kg bw/day. The NOAELs were considered to be less than 250 mg/kg bw/day for maternal toxicity and 500 mg/kg bw/day for developmental toxicity in rats.

In the reproduction/developmental toxicity screening test [OECD TG 421], Crj:CD(SD)IGS rats (12 animals/sex/dose) were given MMB by gavage at 0 (vehicle: distilled water), 8, 40, 200 or 1000 mg/kg bw/day. Males were dosed for 47 days and females were dosed from day 14 before mating to day 4 of lactation throughout the mating and pregnancy period. Increases in absolute and relative weights of the kidney in males at 200 mg/kg bw/day and higher and relative weight of the liver and kidney in females at 1000 mg/kg bw/day were detected.No effects of MMB were detected on developmental parameters such as number of pups born, delivery index, number and weight of pups on postnatal days 0 and 4, live birth index, sex ratio and viability of pups.No external or internal malformation was found in pups at any dose.


Justification for selection of Effect on developmental toxicity: via oral route:
most sensitive endpoint based on effects like increases in litter and fetal incidences of variations of skeletal-related ossification of the ribs, sternum and pelvis.

Justification for classification or non-classification

The available data do not suggest that MMB (CAS: 56539 -66 -3) should be classified for reproductive or developmental toxicity according to Regulation 1272/2008/EC.