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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:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A fully peer reviewed GLP study carried out by a reputable organisation to recognised scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Principles of method if other than guideline:
Toxicology study carried out following a range finder study to determine appropriate doses. Exposure by drinking water with normal end points used for such a repeat dose study, including histopathology, haematology, clinical chemistry, urinalysis and reproductive system parameters.
GLP compliance:
yes
Remarks:
USA regulations 21 CFR 58
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Methoxyethanol purity: 98%
- Analytical purity: checked
- Supplied by Kodak laboratories

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Housing: 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 - 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 60-77F
- Humidity: 20-70% RH respectively
- Photoperiod: fluorescent tube, 12hrs/day.

Administration / exposure

Route of administration:
oral: drinking water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preliminary studies confirmed stability of stock solutions for 3 weeks under appropriate storage conditions and for 4 days in rodent drinking water bottles.
Duration of treatment / exposure:
90 days
Frequency of treatment:
continous
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 750, 1500, 3000, 4500 or 6000 ppm
Basis:
nominal in water
Remarks:
Doses / Concentrations:
70, 135, 297, 546, 785 mg/kg
Basis:
other: actual ingested - females
Remarks:
Doses / Concentrations:
71, 165, 324, 715, 806 mg/kg
Basis:
other: actual ingested - males
No. of animals per sex per dose:
10m/10f
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: range finder study

Examinations

Observations and examinations performed and frequency:
CAGE-SIDE OBERVATIONS: Yes
- All rats were observed twice daily throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the beginning of the study and then weekly until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded at the beginning of the study and then weekly until the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Estimates of compound consumption based on water consumption by rats as shown above in doses/concentration section. Measured by cage twice per week.

HAEMATOLOGY: Yes, Series 7000 cell counter and a Series 810 whole blood platelet analyzer (Baker Instruments). Supplemental groups of 10 rats/sex/group/time point were included for haematology and clinical chemistry observations at weeks 1 and 3. Parameters checked: HgB, HCT, RBC count, MCV, MCH, MCHC, platelets, reticulocytes, WBC total and differential count, nucleated erythrocytes, methemoglobin concentrations, bone marrow cellularity.

CLINICAL CHEMISTRY: Yes, measured with a Cobra Fara analyser (Roche Diagnostics). Parameters examined: urea nitrogen, creatinine, total protein, albumin, ALP, ALT, creatine kinase, bile acids.

URINALYSIS: Yes
- Time schedule for collection of urine: last 24 hours prior to sacrifice
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No, but no water during collection period.
- Parameters checked: volume, gravity, pH

OTHER: Evaluations of vaginal cytology and sperm morphology were done for the three highest doses tested. Results concerning these examinations are reported in more detail in the reprotoxicity section.
Sacrifice and pathology:
GROSS PATHOLOGY: Sacrifice: 70% CO2:30% O2 asphyxiation. Yes, complete autopsies were made on all study rats. Organs examined in control and high dose groups: adrenals, bone (femur) with marrow, brain (3 transverse sections), esophagus, eyes, heart/aorta, intestines, (cecum, duodenum, jejunum, ileum, colon, rectum), kidneys, larynx, liver, lung, lymph node (mesenteric, mandibular), mammary gland, nasal cavity and turbinates, ovaries, pancreas, parathyroids, pituitary, pharynx, preputial or clitoral glands, prostate, salivary gland, seminal vesicles, skin, spinal cord, spleen, stomach (fore and glandular), testes, thigh muscle, thyroid, tongue, trachea, urinary bladder, uterus, vagina, all gross lesions. In low dose group: bone marrow, epididymis, liver, spleen, testis and uterus. Organs weighed: heart, liver, kidney, lung, thymus and testes were examined.
HISTOLOGY: Tissues to be examined fixed, embedded, sectioned and stained (H&E) for microscopic examination. Tissues were examined from all control group and treated rats. Bone marrow cells collecetd from right femur for total nucleated cell counts.
OTHER: A stop-exposure group of 30 male rats was also included. 30 males were dosed at 0, 1500, 3000 and 6000ppm for 60 days. Groups of 10 were sacrificed at this time point and then 30 and 56 days after recovery.
Other examinations:
In addition, a stop exposure study group was included. 30 male rats administered 2 -methoxyethanol at given doses for 60 days. Half animals necropsied. If lesions were seen, remaining animals were kept for 56 day recovery period
Statistics:
Parametric multiple comparison methods: Organ and body weight
Non-parametric multiple comparison methods: clinical chemistry/haematology data
Jonckheere's test: for trend/dose response.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Chemical related mortality was seen reaching 100% at the highest dose. In group with 100% survival there was a decrease in water consumption for males (3000ppm) and females (1500ppm) and a significant dose related body weight decrease evident for both sexes from 1500ppm. Clinical signs of toxicity included tremors, diarrhea, emaciation, abnormal posture, pallor, tachypenia, hypoactivity and a comatose state. Both sexes showed a normochromic, normocytic, poorly regenerative (decreased reticulocyte count) anaemia and leucopenia which persisted at all time points. Bone marrow cellularity was also reduced but recovered during the study and was only evident at in the higher dose males (4500ppm) at the end of the study.

BODY WEIGHT and WATER CONSUMPTION
Reduced body weight was seen in the 1500ppm dose groups upwards as was water consumption.

HAEMATOLOGY
Normocytic anaemia (poorly regenerative), leukopenia (reduced neutrophil and lymphocyte count) and thrombocytopenia were observed associated with decreases in haemocrit and haemoglobin concentrations and reduced RBC, platelet and leucocyte count. Bone marrow cellularity reduced in higher dose groups.

CLINICAL CHEMISTRY
Changes in clinical chemistry were consistent with decreased food intake.

URINALYSIS
Decreased volume and increased specific gravity, consistent with reduced drinking water intake.

ORGAN WEIGHTS
Changes in all organ weights, except thymus and testes, were consistent with reduced body weight. For the thymus, epididymus and testes, dose related weight decreases were seen (both sexes for thymus and down to the lowest dose level.) Almost all other gross lesions were related to reduced body weight.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological changes to the testes consisted of minimal to marked degeneration of the germinal epithelium of the seminferous tubules(STs). In more severe cases atrophic STs contained only Sertoli cells and a few spermatogonia. Reduced sperm number and cell debris were also seen in the epididymus lumen. Degeneration was also seen at a minimal level in 7/10 rats at 750ppm. Chemical related fibrosis (focal thickening) of the splenic capsule was also seen in both sexes at 1500ppm and above. Other histopathological changes were thought to be secondary to general toxicity as demonstrated by marked body weight loss.


OTHER FINDINGS
Sperm motility and defects were drastically effected from 1500ppm upwards with a slight but significant reduction in concentration seen at 750ppm. In females, there was no significant changeswere no significant changes in estrous cycle length but some evidence to suggest time in estrous was reduced. In the stop exposure study, all animals at 6000ppm died. The recovery animals still showed a body weight reduction of at least 9% compared to controls even after 56 days. Histopathological examination of the testes showed some recovery from the degenerative lesions but damage remained permanent.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
71 other: mg/kg/bw
Sex:
male
Basis for effect level:
other: histopathology (testes), organ weights (thymus)
Dose descriptor:
NOAEL
Effect level:
< 71 other: mg/kg/bw
Sex:
male
Basis for effect level:
other: histopathology (testes), organ weights (thymus)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No NOAEL established since testicular degeneration in males and decreased thymus weights in males and females occurred at the lowest concentration administered.
Executive summary:

In a well conducted drinking water study, rats were exposed to methoxyethanol at concentrations ranging from 750 -6000ppm for a period of 90 days. A NOAEL was not established in the study as the lowest dose tested, equivalent to 70 -71mg/kg produced adverse changes to the male testes (degeneration). At this dose, both sexes also showed a significant reduction in thymus weight, both relative and absolute. Bone marrow cellular depletion, splenic atrophy and/or capsular fibrosis and thymic atrophy were apparent in both sexes from doses of 135 -165mg/kg upwards.

Synopsis

NOAEL (91day), rat, male/female <71mg/kg/day