Methyl stearate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance methyl laurate (CAS 111-82-0). According to the ECHA guidance document ¿Practical guide 6: How to report read-across and categories (March 2010)¿, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

: no neurobehavioural examination performed.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl laurate

EC Number:

203-911-3

EC Name:

Methyl laurate

Cas Number:

111-82-0

IUPAC Name:

methyl laurate

Details on test material:

- Name of test material (as cited in study report): Methyl dodecanoate
- CAS-No. of test material (as cited in study report): 111-82-0
- Purity: 99.2 %
- Physical state: colorless liquid
- Lot/batch No.: GD01
- Stability under test conditions: verified
- Storage condition of test material: sealed at room temperature

Test animals

Species:

rat

Strain:

other: Sprague-Dawley (Crj:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Atsugi, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: males: 354 - 386 g, femalrs: 216 - 241 g
- Housing: stainless steel cage
- Diet (e.g. ad libitum): NMF from Oriental Yeast Co., Ltd
- Water (e.g. ad libitum): tap water
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 2 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 15 times/hour
- Photoperiod (hrs dark / hrs light): 12/12 (light intensity 150 - 300 lux)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
test substance was solved in corn oil. Dosing solutions were prepared once or more a week, kept under seal, cool and dark until use and used within 7 days after preparation. 5 % (w/v) was confirmed to be stable for at least 8 days under cool and dark condition.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the concentration was conducted using a sample by random-choice from each batch. Since 98.6 - 104 % was shown as a result, it was confirmed that given amount of dodecanoic acid methyl ester was included.

Duration of treatment / exposure:

females: 14 days before mating until day 3 of lactation (41 - 55 days)
males: 45 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 250, 500 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- The animals were mated.
- Terminal kill was on day 45 for males and on day 4 of lactation for females.
- Dose selection rationale: A preliminary test was performed to determin dose. Dose group were 0, 250, 500, 750 and 1000 mg / kg for 14 consecutive days in males and females, and the general condition showed no effect relevant to test substance, deaths were also not observed. No change were observed in weight, food consumption, autopsy findings, organ weights, blood chemistry test and hematology test.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males on days 1, 8, 15, 22, 29, 36 and 43 of administration; Females on days 1, 8 and 15 before mating, on days 0, 7, 14 and 21 of gestation and on days 0 and 4 of Lactation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Time schedule: days 1-8, 8-15, 22-29, 29-36, 36-43 and 43-45 in male animals; days 1-8, 8-15 before mating, days 0-7, 7-14 and 14-21 of gestation and during days 0-4 of lactation in females.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At autopsy
- Anaesthetic used for blood collection: Yes (ether):
- Animals fasted: Yes (16 hours)
- How many animals: 12 males per dose
- Parameters checked: Hct, Hgb, RBC, MCV, MCH, MCHC, PLT, WBC, Differential leukocyte counts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At autopsy
- Animals fasted: Yes (16 hours)
- How many animals: 12 males per dose
- Parameters checked: Total protein, albumin, A/G, glucose, BUN, creatinine, total cholesterol, GOT, GPT, gamma-GTP, total bilirubin, pottasium, chloride, calcium, inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

ORGAN WEIGHTS: Yes, absolute and relative weights
- How many animals: 10- 12 animals per dose
- Organs: thymus, liver, kidneys, testes, epididymides, ovaries, lung

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Males: thymus, lungs, liver, kidney, brain, heart, spleen, adrenal gland, seminal vesicles, prostate, testis and epididymis
Females in control and highest dose group at day 4 of lactation: thymus, lung, liver, kidney, ovary, heart, spleen, adrenal, colon
Non-pregnancy females: 25 days gestation: skin, breast, lymph nodes, salivary glands, sternum, femur (including marrow), thymus, trachea, bronchi and lungs, heart, thyroid and parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, colon, liver, pancreas, spleen, kidney, adrenal gland, bladder, ovaries, uterus, vagina, eye, Harderian gland, brain, pituitary, spinal cord
Females with all dead pups: skin, breast, lymph nodes, salivary glands, sternum, femur (including marrow), thymus, trachea, bronchi, lungs, heart, thyroid and parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, colon, liver, pancreas, spleen, kidney, adrenal gland, bladder, ovaries, uterus, vagina, eye, Harderian gland, brain, pituitary and spinal cord
Newborns: major organ

HISTOPATHOLOGY: Yes
Males in control and highest dose group, successful pregnancy: brain, thymus, heart, lungs, liver, kidney, spleen, testis and adrenal gland
Females in control and highest dose group at day 4 of lactation: brain, thymus, heart, lungs, liver, kidney, spleen, adrenal and ovarian
Non-pregnancy females and not fertile males: brain, thymus, heart, lungs, liver, kidney, spleen, adrenal glands, vagina, uterus, ovary, testis, epididymis, seminal vesicles, prostate and pituitary gland
Females with all dead pups: skin, breast, lymph nodes, salivary glands, sternum, femur (including marrow), thymus, trachea, bronchi and lungs, heart, thyroid and parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenal gland, bladder, ovaries, uterus, vagina, eye, Harderian gland, brain, spinal cord and pituitary gland

Other examinations:

Estrus cycle, reproductive performance, observation of pups

Statistics:

Barlett test, One-way ANOVA, Kruskal-Wallis test, Dunnett test, Scheffe test, chi-square test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortality were not observed in either group throughout the treatment period both sexes. The anomaly of a tooth in a male of the control group (fracture of incisors) was observed in 5-7 days administered, alopecia on a neck of a male in 250 mg/kg on days 10-19 and crusting in the same male on days 20 - 21, alopecia on a neck of another male in 250 mg/kg on days 2 - 46 and trauma on the same male on days 4 - 13 and crusting on the same male on days 14 - 46. For females, any abnormal were not observed throughout treatment. But this change was regarded as non-relevant effect.

BODY WEIGHT AND WEIGHT GAIN
Difference between treatment groups and the control group throughout the treatment period was not observed in both sexes.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In males, higher mean of food consumption was observed in 500 and 250 mg/kg on day 43 - 45 compared with the control group. In females, the difference between the treatment groups and control group was not observed throughout the administration. But this change was regarded as non-relevant effect.

HAEMATOLOGY
No difference between treatment groups and control group was found.

CLINICAL CHEMISTRY
No difference between treatment groups and control group was found.

ORGAN WEIGHTS
In males 1000 mg/kg group, actual weight of the thymus was heavier compared to control group. But this change was regarded as non-relevant effect because no difference was observed in female groups.

GROSS PATHOLOGY
In fertile males, lung colored spots / areas (gray) and white spots / areas were oberved in two animals in control group, the red spots / areas in the lung in a few cases including the control group and administration group, the thymus red spot / area was observed in 2 animals in 500 mg/kg, skin colored spots / areas (gray) was observed in one animal in 250 mg/kg, respectively. In naturally delivered females, atrophy of the thymus, spots of color / area (brown) in the lungs, changing into black and atrophy of the large intestine, white spots / areas in the liver, cysts of the ovary and scarring of the kidney were found in each administration including the control group. These changes were considered to be not toxicologically relevant, since these were regarded as natural change due to frequency. Two animals in 250 mg/kg in both sexes were not successfully pregnancy but showed no abnormal findings. The two animals, whose all pups died before day 4 of lactation, were observed in control group and one case showed red eye in the retina.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the fertile male, granuloma of the liver lesion, basophilic tubules of the kidney, acidophilic bodies appearance in the kidney and vacuolate adrenal glands were observed. No abonormality was observed in thymus in 1000 mg/kg, where increase of organ weights was obsereved. In females delivered naturally, pigmented spleen, aggregated macrophages in the lungs, focal granulomas in the liver, basophilic tubules and vacuolation of the kidney were observed in each group.
In two cases in the control group, whoses pups were dead before day 4 of lactation, mammary gland hyperplasia, accumulations of macrophages, blood vessel expansion arteritis of the uterus were observed. One case had bleeding in the retina of the eye locally.
A non-reproducible male in 250 mg/kg showed sperm granulomas of the epididymis.
These changes were considered to be not toxicologically relevant, since there were no difference of the number of occuarance between each group.

OTHER FINDINGS:
Parent animal reproduction:
Reproductive performance displayed no significant changes between treatment groups and controls (estrous cycle, copulation index, fertility index, gestation length, gestation index, delivery conditions, or implantation index). In control group and 1000 mg / kg, since there was bias of the number of male and female infants, sex ratio was defference between in these groups. Since many pups died before day four of lactation in the control group, number of total live female pups were significant different between the control group and 1000 mg/kg group. But no difference was found in delivery, pregnant duration, the number of corpora lutea, number of implantation scars, and fetal birth of live or dead pups, the birth rate, implantation rate, delivery percentage and fertility rate.

Pups:
2 case of hypodermic hmorrhage, 1 case of imperforate anus and 1 case of rudiment tail were observed in control group, 1 case of nanooffspring was in 250 mg/kg, 1 case of short tail was in 500 mg/kg. Rgarding weight, no difference was observed. At autopsy, thymic remnant in the neck was observed in control group, 250 mg/kg and 1000 mg/kg, diaphragmatic hernia was observed in two dead pups in 500 mg/kg.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects (clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology;) observed in all dose groups

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test an oral NOAEL of 1000 mg/kg bw/day was found for male and female rats.