3-Methoxy-3-methylbutyl acetate

Endpoint summary

Description of key information

The substance was administered orally by gavage (in accordance with OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Screening Test and GLP criteria) to 12 male and 12 female rats per dose. The following doses were given: 0, 25, 150 and 1000 mg/kg/day. The aim of the study was to determine the potential systemic and reproductive toxicities in parental animals and developmental toxicity of the offspring. The administration of the test substance was conducted for 42 days in males and until day 4 in females of the nursing period. The recovery groups were kept for 14 days in the vehicle control and 1000 mg/kg groups, which was 5 out of 12 males and 5 additional females. The females of the recovery groups were not mated.

The following observation were made:

Body weights and food intakes decreased in females of the 1000 mg/kg group on day 4 of the nursing period. The hematologic observations revealed decreases in red blood counts (RBC) in males and females and decreases in haemoglobin concentration and haematocrit values in female in the 1000 mg/kg groups. No abnormalities were noted in the clinical signs, functional observational battery (FOB), bloody chemistry, organ weights, necropsy and histopathology. Decreases in RBC, haemoglobin concentration and haematocrit values were not observed after 14-day recovery period.

The NOAEL of the MMB-AC in the repeated dose toxicity study is 150 mg/kg /day for both sexes due to the decreases in RBC in males and females, decreases in haemoglobin concentration and haematocrit values in females and decreased in body weights and food intakes in females on day 3 of the nursing period in the 1000 mg/kg bw.

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-07-10 to 2007-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Stability under test conditions: The infrared (IR) spectrum was measured by IR spectrophotometer before and after the dosing period in our laboratory. The test substance was stable during the dosing period.

Species:

rat

Strain:

other: Crl: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Weight at study initiation: males: 322.8 – 363.7 g; females: 199.9 – 238.7 g
- Housing: stainless steel cages with wire-mesh floor; after copulation females were placed in polycarbonate cages with wood chip bedding. 1 animal/cage, 1 female and 1 male/cage during mating period
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The test substance was repeatedly administered once daily in the morning by oral gavage using a syringe connected to a Nelaton catheter.
Dosing volume was10 mL/kg.

Vehicle:

other: Carboxymethylcellulose sodium (CMC-Na) of 1.0% with Tween 80 of 0.1% aqueous solution

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The test substance was accurately weighted and mixed with vehicle to prepare 10.0w/v% suspension. This formulation was prepared once every 7 days and stored at the dark and cold place. Two lower doses of 0.25 and 1.5 /v% were diluted from the 10.0 w/v% formulation prior to use.

- VEHICLE
- Justification for use and choice of vehicle: carboxymethylcellulse sodium (CMC-Na) of 1.0% with Tween 80 of 0.1% aqueous solution was selected as a vehicle due to good analytical precision and results, and from results of the stability test.
- Lot/batch no. (if required):
Tween 80: DPK6694, Nacalai Tesque Inc
CMC-NA: LT0117, Wako Pure Chemical Industries

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples were taken (n=1) from the middle layer of 0.25, 1.5 and 10.0 w/v% formulations immediately after preparation. These samples were pretreated and measured (n=1) with gas chromatography (GC).

Duration of treatment / exposure:

Dosing period form males was 42 days from two weeks before mating to the day before necroscopy throughout the mating period. For females it was 41 to 47 days, two weeks before mating to 4 days in the nursing period.

Frequency of treatment:

Once daily in the morning

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 14-day repeated oral dose toxicity study was performed with each 3 male and female Crj:CD(SD) rat of 7 weeks old at 4 doses of 15, 60, 250 and 1000 mg/kg/day. As a result, increases in WBC in the 1000, 250 and 15 mg/kg/day increases in GOT, GPT, ALP, LDH, CPK and neutral fat and increases or decreases in cholinesterase in all dose groups were observed; however, they were slight changes, single occurrence or no dose-related changes. Therefore, the high dose was set at 1000 mg/kg/day and lower doses of 150 and 25 mg/kg were set for the present study.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Male animals were observed twice a day, i.e., before dosing and after dosing from day 1 of administration to the day before necropsy, and once on the day of necropsy. Female animals were also observed twice a day from day 1 to day 4 of nursing period including delivery and nursing conditions, and once on day 5 of nursing period of the day of necropsy. During the recovery period, all animals were observed once a day.
Detailed Clinical Observations:
The detailed examination in all animals was performed ad blind tests before dosing and once a week thereafter. Males and females during the mating period were not examined. Mated females were observed in week 1 to 5 and after delivery. During the recovery period, the observation was performed once a week.


BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured once during the acclimation period and once at allocation to groups in all animals, and on the treatment days of 1, 3, 7, 14, 21, 28, 35, 42, 43 (necropsy) in females of the recovery groups and all males. Body weights of the other females were measured on days 1, 3, 7 and 14 before gestation, days 0, 7, 14, 17 and 20 during the gestation period and days 0 (delivery), 4 and 5 (necropsy). In the recovery period, all animals were measured on days 1, 3, 7, 14 and 15 (necropsy).

FOOD CONSUMPTION:
- Food consumption was measured on the treatment days of 1, 3, 7, 14, 21, 28, 35 and 42 in females of the recovery groups and all males. Food intakes of the females were measured on days 1, 3, 7 and 14 before gestation, on days 0, 7, 14, 17 and 20 during the gestation period and on days 0 and 4 after delivery. In the recovery period food consumption was measured on days 1, 3, 7 and 14. Mean food consumption per day was calculated from the deference between each measured day and the next measured day. All animals were fasted from the afternoon of the day before necropsy.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at completion of the dosing period and at completion of the recovery period
- Anaesthetic used for blood collection: Yes. Blood plasma samples were obtained by blood sampling from the abdominal aorta under ether anesthesia
- Animals fasted: Yes. Overnight fasting (16 to 20 hr)
- How many animals: in five males and five females whose delivery days were closed in each group, and all males and females in the recovery groups.
- Parameters checked in table [No. 2] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Serum samples were separated from blood samples collected at the same times as those for haematology examinations.
- Animals fasted: Yes. Overnight fasting (16 to 20 hr)
- How many animals: in five males and five females whose delivery days were closed in each group, and all males and females in the recovery groups.
- Parameters checked in table [No.3] were examined.


OTHER:
- Sensorimotor Function:
Each five males and five females in all groups were examined in the final week and on day 4 of the nursing period before fasting, respectively, or five females whose delivery days closed in each group were examined when the number of females was less than five in a group. Recovery groups were examined in the final week of the dosing period. In the recovery period locomotor activity were counted in females of the vehicle control and 1000 mg/kg groups since significant differences were noted in females of the 1000 mg/kg group in the dosing period.

- Organ weight:
The weights of the following organs were measured in all animals. The relative organ weight per 100 g of body weight was calculated based on the body weight at the time of necropsy:
Liver, heart, kidney, testes, epididymides, brain, spleen, thymus and adrenals.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to the detailed gross necropsy including body surface, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities, and the contents. Corpora lutea in the ovaries and implantation sites in incised uterus were counted.

HISTOPATHOLOGY: Yes
The following organs and tissues were taken in all animals:

Category Organs and Tissues
Respiratory system Trachea and lungs
Digestive system Stomach, intestine (duodenum to rectum, with Peyer´s patches) and liver
Cardiovascular system Heart
Urinary system Kidneys and urinary bladder
Reproductive system Testes, epididymides, prostate, seminal vesicle, ovaries, uterus and vagina
Nervous system Brain (cerebrum, cerebellum and pons), spinal cord and sciatic nerve
Hematopoietic and
lymphatic system Bone marrow (femur), axillar and mesenteric lymph nodes, spleen and thymus
Endocrine system Pituitary gland, thyroids (with parathyroids) and adrenals


Trachea, lungs and urinary bladder were filled with 10% neutralized buffered formalin before taken. Stomach and intestines were filled and fixed with 10% neutralized buffered formalin and the contents were wash away with water. All organs/tissues were preserved in 10% neutralized buffered formalin. However, testes and epididymides were fixed in Bouin´s solution.

Statistics:

Dunnett’s test; Kruskal-Wallis’ test; Fisher’s exact probability test

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Males: Salivation just after dosing was observed in all animals of the 1000 mg/kg group. No abnormalities in other groups.
Female: No abnormalities observed before mating period. Salivation was observed in four animals of the 1000mg/kg group. One animal in the vehicle control group showed staining on the lower abdomen continuously from delivery, and all her pups died on day 2 of the nursing period. In the 150 mg/kg group, although abnormalities were noted during the nursing period, one pup died in one animal just after delivery. Salivation was observed in one animal, and staining on the lower abdomen and around the vagina in other one animal was observed and all her pups died on day 1 of nursing period in the 1000 mg/kg group. Salivation was observed only just after dosing in three animals of the 1000 mg/kg recovery group.
During Recovery Period: No abnormalities were noted in all groups of each sex.

Salivation was observed in the 1000 mg/kg groups only after treatment, and it possibly related to the taste of the substance and was not considered to be toxicologically significant.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female died on day 23 of the gestation period in the vehicle control group. Foamy substance in the lumen of the trachea, dark reddish change in the lungs, recessed region of the mucosa in the forestomach, enlargement of the adrenals and hydrothorax were observed in the necropsy, and the cause of death was considered to be administration error.

Mortality of pups is documented in the robust study summary in the endpoint "Toxicity to reproduction"

Body weight and weight changes:

not specified

Description (incidence and severity):

During Dosing Period
Males: No abnormalities were noted.
Females: Significant lower body weights compared to the vehicle control group was noted in the 1000 mg/kg group on day 4 of the nursing period. Abnormal body weights were not noted in other groups.
During Recovery Period
No abnormalities were noted in all groups of each sex.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During Dosing Period
Males: Abnormalities were not noted in all groups
Females: Significant decreases in food consumption in the 25 mg/kg group on day 17 of the gestation period and in the 1000 mg/kg group on day 3 of the administration period before mating, on day 17 of gestation period and on day 4 of the nursing period were not compared to the vehicle control group. No abnormalities were noted in other groups.
During Recovery Period
Males: Significant increases in food consumption were noted in the 1000 mg/kg recovery group on day 3.
Females: No abnormalities were noted in the 1000 mg/kg recovery group.

Food consumption was decreased without body weight changes in females of the 1000 mg/kg group on a day 3 of the treatment and on day 17 of the gestation periods. These food consumption were not considered to be of no toxicologically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At Termination of Dosing Period
Males: RBC was significantly decreased in the 1000 mg/kg group. No abnormalities were noted in the other groups.
Females: MCHC was significantly increased in the 25 mg/kg group. Significant decreases in RBC, Hb and Ht and a reduction in PT were noted in the 1000 mg/kg group. Abnormalities were not noted in other groups.
At Termination of Recovery Period
Males: No abnormalities were noted in the 1000 mg/kg group.
Females: A significant reduction in PT was noted in the 1000 mg/kg recovery group.

PT was reduced in females of the 1000 mg/kg group and this observation was not considered to be toxicologically significant.
Decreases in RBC in males and females, decreases in Hb and Ht in females were noted in the 1000 mg/ kg groups, and they were considered to be treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period
Males: No abnormalities were noted.
Females: A significant decrease in total protein in the 150 mg/kg group, increases in IP in the 150 and 1000 mg/kg groups and an increase in total cholesterol and a decrease in chloride in the 1000 mg/kg group were noted. In the other group, abnormalities were not noted.
At Termination of Recovery Period
Males: Creartinine was significantly decreased in the 1000 mg/kg recovery group.
Females: No abnormalities were noted in the 1000 mg/kg recovery group.

Increase in IP in females of the 150 and 1000 mg/kg groups and an increase in total cholesterol and a decrease in chloride in females of the 1000 mg/kg group were noted; however, they were not considered to be treatment-related since liver and kidney weights were not affected, and any histopathological changes were not noted in these organs.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

During Dosing Period
Males: No abnormalities were noted in all groups.
Females: Locomotor activity count in 50-60 min was significantly decreased in the 1000 mg/kg recovery group in week 6. No abnormalities were noted in other groups.
During Recovery Period
Males: Not examined.
Females: Locomotor activity count in 50-60 min was significantly increased in the 1000 mg/kg recovery group in week 2.

In the sensorimotor functions, locomotor activity counts were decreased in females of the 1000 mg/kg recovery group. Theses observation were considered to be a single occurrence and to be unrelated to the treatment, since increased counts were noted in the recovery group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period
Males: Relative liver and kidney weights were significantly increased in the 1000 mg/kg group. No abnormal organ weights were noted in other groups.
Females: Absolute thymus weight was significantly decreased in the 1000 mg/kg group. No abnormalities were noted in other groups.
At Termination of Recovery Period
Males: Relative kidney weights were significantly increased in the 1000 mg/kg recovery group.
Females: Relative liver weight was significantly increased in the 1000 mg/kg recovery group.

In the organ weights, increase in relative liver and kidney weights in males and decrease in absolute thymus weight in females were noted in the 1000 mg/kg groups. These were only changes of absolute or relative weights without histopathological, changes, and they were not considered toxicologically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period
Males: Softening of the testes in two animals (nos. 13 and 23), small testes and epididymides in two animals (nos. 13 and 19) and whitish region on the capsule in the spleen in one animal (no. 16) were observed in the 25 mg/kg group. No abnormalities were noted in other groups.
Females: Cyst in the pituitary was observed in one animal (no. 109) of the vehicle control group. Small thymus was observed in three animals; of the 25 mg/kg group (no. 121), 150 mg/kg group (no. 141) and 1000 mg/kg group (no.159). Elevation of the limiting ridge in the forestomach in one animal (no. 143) and nodule in the vagina in two animals (nos. 144 and 150) were observed in the 1000 mg/kg group. In one dead animal (no. 103) of the vehicle control group, foamy substance in the lumen of the trachea, dark reddish change in the lungs, recessed region of the mucosa in the forestomach, enlargement of the adrenals and hydrothorax were observed. No abnormalities were noted in other groups.
At Termination of Recovery Period
Males: Blackish region of the mucosa in the glandular stomach was observed in one animal (no. 45) of the 1000 mg/kg recovery group.
Females: Pelvic dilatation in the kidney was observed in one animal (no. 116) of the vehicle control recovery group. Abnormal signs were not noted in the 1000 mg/kg group.

Dams which All Pups Died
No abnormalities were observed in two animals of the vehicle control group and 150 mg/kg group. Rough region of the muscosa in the forestomach, small thymus and enlargement of the adrenals were observed in one animal of the 1000 mg/kg group.
Non-pregnant Female
No abnormal signs were observed in two animals of the 25 mg/kg group and one animal of the 150 mg/kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period
Males: Mineralization in the Peyer´s patches in the jejunum in one animal microgranuloma in the liver in two animals, focal myocarditis in the heart in three animals and cyst formation in the medulla of the kidney in one animal were observed in the vehicle control group. Diffuse atrophy of the seminiferous tubules in the testis and decreased spermatozoa in the lumen, germ cell debris in the lumen and spermatic granuloma in the epididymis in one anima and capsulitis in the spleen in one animal were observed in the 25 mg/kg group. Mineralization in the Peyer´s patches in jejenum in one animal and cyst formation in the pars distalis of the pituitary gland in one animal were observed in the 1000 mg/kg group. No abnormal signs were observed in other group.

Females: Hyperplasia of the squamous epithelium in the limiting ridge of the forestomach in one animal, mineralization in the Peyer´s patches in the jejenum in one animal and cyst formation in the pars distalis of the pituitary gland in one animal were observed in the vehicle control group. Hyperplasia of the squamous epithelium in the limiting ridge of the forestomach in one animal, cyst formation in the medulla with basophilic tubules (unilateral) and mineralization in the pelvis (unilateral) of kidney in one animal, cyst formation in the vagina in two animals and atrophy of the thymus in one animal were observed in the 1000 mg/kg group. In one dead animal showed edema and thrombus in the lung, ulcer in the forestomach, focal necrosis of the hepatocytes in the liver, thrombus in the heart and kidney, atrophy of the spleen and thymus and hypertrophy of the cortex in the adrenal were observed in the vehicle control group. No abnormalities were observed in other groups.

At Termination of Recovery Period
Males: Necrosis of the fundic mucosa in the glandular stomach was observed in one animal of the 1000 mg/kg recovery group.
Females: Microgranuloma in the liver was observed in one animal and pelvic dilatation in the kidney in one animal were observed in the vehicle control recovery group. No abnormalities were observed in the 1000 mg/kg recovery group.


Dams which All Pups Died:
Hyperplasia of the squamous epithelium in the limiting ridge of the forestomach, vacuolization of the hepatocytes in the liver, vacuolization of the tubular epithelium in the kidney, atrophy of the thymus and fine cauolization of the zona fascilita in the adrenal were observed in on animal of the 150 mg/kg group. Edema in the submucosal layer, focal hyperplasia of the squamous epithelium and hyperplasia of the squamous epithelium in the limiting ridge of the forestomach, atrophy of the fundic mucosa in the glandular stomach, vacuolization of the tubular epithelium in the kidney, atrophy of the thymus and fine vacuolization of the zona fasciculate and hypertrophy of the cortex in the adrenal were observed in one animal of the 1000 mg/kg group.
Non-pregnant Female
No abnormalities were observed in two animals of the 25 mg/kg group and one animal of the 150 mg/kg group.

Mineralization in the Peyer´s pateches in the jejenum and cyst formation in the pars distalis of the pituitary gland in males and hyperplasia of the squamous epithelium in the limiting ridge of the forestomach, cyst formation in the medulla with basophilic tubules and mineralizaiton in the pelvis of the kidney, cyste formation in the vagina and atrophy of the thymus were observed in the 1000 mg/kg groups. These were single occurrence and also observed in the vehicle control groups. Therefore, they were not considered to be treatment related. The other changes did not show dose-relationships and were not considerd to be treatment-related.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Description (incidence and severity):

Males: Defecation was significantly increased in the 150 mg/kg group in week 5. All other animals showed no abnormalities.
Females: Urination was significantly decreased in the 25, 150 and 1000 mg/kg groups in week 1. No abnormalities were noted in other groups.This was not considered to be toxicologically significant since it related to the high value of the vehicle control group.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The NOAEL repeated dose toxicity was considered to be 150 mg/kg/day under conditions tested due to decreases in RBC in males and females, decreases in Hb and Ht in females, decrease in body weights and food consumption in female on day 4 of the nursing period in the 1000 mg/kg groups.
NOEL of repeated-dose toxicity was considered to be 150 mg/kg/day due to transient salivation in males and females, increased relative liver and kidney weights without histopathological changes in males, toxicologically insignificant decreases in food consumption without body weight changes, and a decrease in absolute thymus weight without histological changes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Organ:

blood

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The decrease in RBC in males and females and the decrease in haemoglobin concentration and haematocrit were observed at a dose of 1000 mg/kg bw (highest dose) and being the cut-off limit for the repeated dose classification for a subacute exposure is <=300 mg/kg bw no STOT-RE classification is derived.