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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In or before 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
No information on whether female mice are nulliparous and non-pregnant, no data on opthalmoscopic, neurobehavorial and detailed clinical observations, limited blood chemistry analysis performed.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-chloropropane-1,2-diol
EC Number:
202-492-4
EC Name:
3-chloropropane-1,2-diol
Cas Number:
96-24-2
Molecular formula:
C3H7ClO2
IUPAC Name:
3-chloropropane-1,2-diol
Details on test material:
- Name of test material (as cited in study report): 3-MCPD
- Analytical purity: 98%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: stability in drinking water and the dose formulations of animal room samples were analyzed and the analytical results for all dose formulations were within 10% of the theoretical concentrations.
- Storage condition of test material: at 4°C protected from light
- Other: purchased from Sigma Aldrich Inc. (St. Louis, MO, USA)

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 6 weeks
- Weight at study initiation: no data
- Fasting period before study: not applicable
- Housing: female mice: 5 per cage; male mice individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 1
- Humidity (%): 55 +/- 5
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: drinking water solutions were prepared using deionized water. The dose formulations were prepared every 2 weeks and stored at 4°C in glass vessels that were protected from light.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water and the dose formulations of animal room samples were analyzed by LabFrontier Co. Ltd., Korea. The analytical results for all dose formulations were within 10% of the theoretical concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuous via the drinking water
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5, 25, 100, 200 and 400 ppm
Basis:
nominal in water
Remarks:
Doses / Concentrations:
0.94, 4.59, 18.05, 36.97 and 76.79 mg/kg bw/d
Basis:
other: average daily intake in males
Remarks:
Doses / Concentrations:
0.79, 3.94, 15.02, 30.23 and 61.34 mg/kg bw/d
Basis:
other: average daily intake in females
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: not applicable
- Section schedule rationale (if not random): no data
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, any clinical signs

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled sacrifice
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes
- How many animals: all
- Parameters examined: erythrocyte count, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelet count, differential leukocytes counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled sacrifice
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alkaline phosphatase, total protein, albumin, blood urea nitrogen, creatinine

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER:
Females: estrus cycle: for 12 consecutive days prior to the scheduled sacrifice, samples of vaginal fluid were taken and stained. The relative leukocytes, nucleated epithelial cells and large squamous epithelial cells were determined and used to determine estrus cycle stage.
Males: sperm motility: the left testis and left epididymis were isolated and weighed. The cauda epididymis was then removed and weighed. Sperm effluxing from the cauda epididymus was analysed.
Sacrifice and pathology:
A complete necropsy was performed on all animals.
Organ weights: liver, kidneys, testes, ovaries, heart, spleen
Microscopic examination: liver, kidneys, testes, ovaries, spleen, brain, clitoral gland, esophagus, eyes, femur with marrow, gallbladder, harderian glands, heart and aorta, small and large intestine, lungs and mainstem bronchi, mandibular and mesenteric lymph nodes, mammary gland with adjacent skin, muscle, nasal cavity and nasal turbinate, pancreas, parathyroid glands, pituitary gland, preputial glands, prostate, salivary glands, seminal vesicle, skin, spinal cord and sciatic nerve, stomach, epididymis, thymus, thyroid gland, trachea, urinary bladder, uterus and vagina.
Other examinations:
Not applicable.
Statistics:
Variance in the data was checked for homogeneity using Bartlett's procedure. If homogeneous the data were assessed by one-way analysis of variance. If not, the Kruskal-Wallis test was applied. When statistically significant differences were indicated, a Dunnett's multiple test was used to compare the control and treatment groups. Data comparisons were considered significant if P<0.05.
For histopathological data analysis, the incidences were compared using a Fischer's exact probability test. A P<0.05 was considered significant.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
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:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality or deterioration in the general conditions

BODY WEIGHT AND WEIGHT GAIN
Body weights were significantly suppressed (p<0.05) in the 400 ppm groups from week 12 in males and week 8 in females. Final body weights were significantly decreased at 400 ppm in both males (92.7%, p<0.05) and females (85.7%, p<0.01), and a decrease of the final body weights (but not statistically significant) was also oberserved in females at 100 ppm (95.9%) and 200 ppm (96.9%).
The body weight suppression can be explained by the sweetish taste of the test substance.

HAEMATOLOGY
No definite dose-related changes observed. There was a slight decrease (p<0.05) in MCV observed in 400 ppm males and in females given 100 and 200 ppm. Furthermore, a minimal increase in the MCHC was observed in the males given 100 and 400 ppm and in the 25 ppm females, but there was no corresponding decrease in the erythrocyte counts. The leukocyte, lymphocyte and monocyte counts were higher in 100 ppm males.
The changes are mainly within the limits of the normal biological ranges and not accompanied by other toxicological or histopathological findings.

ORGAN WEIGHTS
In the 400 ppm males, a decrease in the absolute liver weights was observed (92.2%, p<0.05) and an increase in relative kidney weights in males given 200 ppm (112.3%, p<0.01) and 400 ppm (115.1%, p<0.01).
In the 400 ppm females, an increase in the relative liver weights was observed (116.1%, p<0.05) and an increase in relative kidney weights in females given 200 ppm (118.3%, p<0.05) and 400 ppm (122.6%; p<0.01).
Liver weights changes are not considered toxicologically relevant as the changes are not consistent and in the absence of histopathological changes.
Kidney weight changes, although no significant histopathological changes occurred, are thought to be toxicologically relevant. The nephrotoxic mechanisms are thought to be due to the inhibition of glycolysis by metabolites associated with beta-chlorolactate pathway.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the testes, there was an increase in the incidence and severity of degeneration of the germinal epithelium in males given 200 ppm (4 minimal , 1 mild) and 400 ppm (4 minimal, 4 mild). Microgranulomas in the liver, interstitial nephritis, basophilic tubules and mineralization in the kidneys were detected as spontaneously occurring lesions in the males, so not related to the test substance treatment.
In females, microgranulomas in the liver, interstitial nephritis, basophilic tubules, hyaline cast, and cystic tubules in the kidneys, and pigmentation and cystic dilatation in the ovaries were also observed. Other lesions were also detected sporadically but there were no significant changes in the incidence between the control and treatment groups.
OTHER FINDINGS
The percentage of motile sperm in the 400 ppm males was significantly lower (60.2%) than those of the controls (73%).
The length of the estrus cycle in the 400 ppm females was significantly longer (6.30 days) than those of the controls (4.90 days), because the 400 ppm females spent more time in diestrus (36.5% of the cycle) than the control females (30.6% of the cycle). There were however no treatment-related histopathological changes in the female reproductive organs. Luteolytic and antiestrogenic effects may have influenced the totalestrus cycle length.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Average daily test substance intake at 5, 25, 100, 200 and 400 ppm in the drinking water:

Males: 0.94, 4.59, 18.05, 36.97 and 76.79 mg/kg bw/day, respectively

Females: 0.79, 3.94, 15.02, 30.23 and 61.34 mg/kg bw/day, respectively

Applicant's summary and conclusion

Conclusions:
A 13-week study in B6C3F1 mice showed that the test substance suppressed the body weight gains in the 400 ppm group and increased the relative kidney weights in the 200 and 400 ppm groups in both males and females. The nephrotoxic effects are thought to be due to the inhibition of glycolysis by metabolites associated with beta-chlorolactate pathway. In addition, decreased sperm motility and degeneration of the germinal epithelium was seen in male mice, which may be caused by alkylation of spermatozoa cysteine and by metabolites which have an inhibitory activity on enzymes in the spermatozoa glycolysis. In females, a delayed total estrus cycle was observed, which may be caused by luteolytic and antiestrogenic effects.

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