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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:
1989-11-07 to 1990-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 408: no data on GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrocarbons, C10-C12, isoalkanes, <2% aromatics
EC Number:
923-037-2
Molecular formula:
None available - not a single isomer - see remarks
IUPAC Name:
Hydrocarbons, C10-C12, isoalkanes, <2% aromatics

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York, USA
- Age at study initiation: Approximately 6 to 7 weeks
- Weight at study initiation: Males, 204.3 to 246.7 grams; Females, 151.2 to 191.8 grams
- Fasting period before study: none
- Housing: Individual (except during the first two weeks of acclimation)
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002 (mash), ad libitum; Ralston Purina Company, St. Louis, Missouri USA.
- Water (e.g. ad libitum): Automatic watering system, ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-76 degrees F
- Humidity (%): 40-70% relative humidity
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: From: 1989-11-20 To: 1990-04-18

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was diluted in carrier to the appropriate concentration by the Compound Preparation Department. Fresh dosing solutions were prepared at least weekly and maintained under a nitrogen blanket until used.

VEHICLE
- Concentration in vehicle: 0.1, 0.5, or 1.0 g/kg
- Amount of vehicle (if gavage): 2 ml/kg
- Lot/batch no. (if required): Corn oil (Mazola), Best Foods, CPC. The following batch/lot numbers were used: SEPT2690B, OCT2090B, JAN0191A, FEB1591B, JUN0391A, JUN0491A, JUN1191A
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration analyses were performed every 4 weeks by the testing laboratory, with an exception for the last analysis which was performed at five weeks.
Duration of treatment / exposure:
Seven days per week
Frequency of treatment:
13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0.1, 0.5, and 1.0 g/kg
Basis:
other: gavage dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 28 days

- Two additional groups of ten males each at the Control and 0.5 g/kg dose levels were added following excessive premature mortality due to gavage accidents. The statistical t-test was performed to compare these added groups with the original male animals at these dose levels to assure they were statistically similar before combining the groups.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on Monday through Friday. Once daily on Saturdays, Sundays and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: During the week prior to dosing, at dosing initiation (Day 0), and weekly thereafter. Additionally, body weights were recorded at the scheduled sacrifice and at death for animals which succumbed prior to study termination.

FOOD CONSUMPTION
- Time schedule for examinations: Measured weekly during the test period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to dose initiation and prior to scheduled terminal sacrifice (5 to 8 days). Animals in the satellite group were examined approximately one week prior to both the main study terminal sacrifice and the recovery terminal sacrifice.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pre-dose, day 32, and terminal sacrifice (Days 92 and 93 for the main study animals; Day 120 for the satellite group)
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked: erythrocyte count, hematocrit, hemoglobin, leukocyte count (total and differential), mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, reticulocyte count.
- Note: Slides were prepared for reticulocyte count but not evaluated since RBC parameters appeared normal.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-dose, day 32, and terminal sacrifice (Days 92 and 93 for the main study animals; Day 120 for the satellite group)
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes (overnight)
- How many animals: All animals
- Parameters checked: albumin, blood urea nitrogen, calcium, cholesterol, creatinine, electrolytes (Na+, K+, Cl-), gamma glutamyl transferase, glucose, phosphorus, serum alanine transferase, serum aspartate aminotransferase, total bilirubin, total protein, triglycerides

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Included: physical examination of the external surface, all orifices, the carcass, the cranial, thoracic and abdominal cavities with their associated organs and tissues.
- The following tissues and organs were taken and preserved in 10% neutral buffered formalin for all animals: adrenals, aorta, brain (three levels), epididymides, esophagus, eyes with optic nerve, femoris muscle with sciatic nerve, femur, heart, kidneys, lacrimal gland, large intestine colon and cecum, liver, lungs, mammary glands, mesenteric lymph nodes, ovaries, oviducts, pancreas, pituitary, prostate, rectum, salivary glands, seminal vesicles, skin, small intestine duodenum, jejunum, and ileum, spinal cord cervical mid-thoracic and lumbar, spleen, sternum with marrow, stomach, testes, thymus, thyroids/parathyroids, trachea, urinary bladder, uterus corpus and cervix, and all tissues showing abnormalities.

HISTOPATHOLOGY: Yes
- Preserved tissues from the control and high dose groups, as well as from all animals that succumbed during the study, were processed, sectioned, stained (hematoxylin and eosin) and examined microscopically. Gross lesions, tissue masses, liver, lungs and kidneys from the low and mid dose group were also processed, sectioned, stained and examined microscopically.
-At the completion of the satellite recovery period, tissues from the animals in this group were examined microscopically with particular emphasis on the organs and tissues which displayed toxic effects in the other treatment groups.
Statistics:
The following parameters were statistically analyzed for significant difference:
mean hematology parameters
mean serum chemistry parameters
mean organ weights
mean organ to body weight ratios
mean body weights, by weighing period

Comparisons were limited to within sex analysis. Statistical evaluation of equality of means was done by an appropriate one-way ANOVA and a test for ordered response in the dose groups. First Bartlett's test was performed to determine if the dose group have equal variance. If so, the testing was done using parametric methods; otherwise nonparametric techniques were used.

Parametric methods: one-way ANOVA using the F distribution with Dunnett's post-test; regression analysis for linear response in the dose group; linear lack of fit

Nonparametric methods: Kruskall-Wallis test for equality of means with Dunn's Summed Rank post-test; Jonckheere's test for monotonic trend in the dose response

Bartlett's test was conducted at the 1% level of significance. All other tests were conduced at the 5% and 1% level of significance.

The statistical t-test was used to compare the additional control and 0.5 g/kg group animals to the original animals at these dose levels in order to justify their combination. In addition, the t-test was used to compare the high dose and satellite groups to ensure similar results in order to accurately evaluate the recovery effects.

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:
no effects observed
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 specified
Details on results:
CLINICAL SIGNS AND MORTALITY
- Minimal clinical in-life observations were noted in the majority of animals and were considered to be incidental and not treatment-related. These observations included a very low incidence of sores, scabs, alopecia, dyspnea, rales, soft stool, nasal discharge, ocular and dental abnormalities, urine staining, unthrifty coat, and signs of general poor health.
- Treatment-related mortality was not apparent at any dose level. All deaths occuring prior to Day 12 were considered to be the result of dosing accidents, as well as deaths on Day 45 and Day 68.

BODY WEIGHT AND WEIGHT GAIN
Overall increases in body weight were noted for all animals surviving to scheduled study termination. There were no statistically significant differences between the control and test animals' body weights nor between the high dose and the satellite animals' mean body weights.

FOOD CONSUMPTION
There were no significant differences in the food consumption values for the treated males when compared to the control animals. Dose related increases were evident in the mean food consumption values for treated females over most of the weighing periods, however, these findings are not considered to be an adverse effect.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings noted at the terminal ophthalmological examination.

HAEMATOLOGY
The changes noted in the hematology values were considered to be minimal and not biologically significant. A trend toward anemia in high dose males was suggested by a dose-related decrease in male red blood cells, hematocrit, and hemoglobin observed at both the interim and terminal analysis; however, these changes were within the range of normal biological variation. There were no significant differences noted in any hematology parameter in female animals at either the interim or terminal analysis.

CLINICAL CHEMISTRY
Many small differences were noted in in treated groups compared to controls, but the majority were considered to be within normal biological variation and thus not biologically significant. Effects that were considered to be biologically significant included a dose-related decrease in triglyceride values in male animals at both the interim and study termination analyses (mid- and high-dose groups statistically different from controls). After the recovery period, mean male triglyceride values increased nearly two-fold such that they were equivalent to the terminal value of control animals. In female animals, a dose-related decrease in aspartate animotransferase occured at study termination, reaching statistical significance in the mid- and high-dose groups. No recovery effect was noted in the satellite animals. While these findings indicate a persistent effect, there were no corresponding histopathologic changes to indicate organ damage.

ORGAN WEIGHTS
Mean liver weights and mean relative liver weights for the 500 mg/kg (15 and 21% respectively for males; 32 and 36% respectively for females) and the 1000 mg/kg (22 and 28%, respectively for males; 31 and 25% respectively for females) dose group animals were significantly greater than the control group values. Histopathologic examination of these tissues revealed slight differences in the comparison of liver cell size between all dose groups and the controls. However, these differences were not sufficient to account for the observed increases in the mean and relative liver weights for the mid- and high-dose groups. Comparison of the liver weight parameters in the satellite group and the high dose group following the 28 day recovery period indicates that these changes were reversible following cessation of dosing. Other effects not considered related to treatment included an increase in female mean kidney weight and an increase in the mid dose male relative kidney and testes weights.

GROSS PATHOLOGY
No observable effects in the majority of animals. There were single or low incidences of kidney, lymphoid, spleen, uterus, ocular, dental, adrenal, skin/fur and lung abnormalities noted intermittently among all groups. One mid-dose female was noted with a subcutaneous mass incorporating the vagina and rectum and one satellite female with a kidney and oviduct mass.

HISTOPATHOLOGY: NON-NEOPLASTIC
No microscopic changes related to treatment were observed in any animal in any dose group. All animals who died prior to study termination (except one control female) had lesions in the thoracic cavity consistent with intubation accidents. There were no significant findings in the control female that could clearly be associated with a cause of death.

Effect levels

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No treatment-related mortality or significant adverse clinical effects occurred.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for rats orally exposed to Isopar M (MRD-89-526) is greater than 1000 mg/kg body weight, based on the lack of treatment-related mortality and adverse clinical effects.
Executive summary:

A 90 -day subchronic study was conducted in rats to assess the toxicity of Isopar M (MRD-89 -256). The test mixture was administered by oral gavage at a dose of 0, 100, 500, or 1000 mg/kg, 7 days per week for a period of 13 weeks. The control animals received a carrier (corn oil) dose and a satellite group was dosed at 1000 mg/kg, 7 days/week for 13 weeks and was then observed for reversibility, persistence or delayed occurrence of toxic effects for 28 days post-treatment. Observations were made as to the nature, onset, severity, and duration of toxicological signs. No treatment-related mortalities or clinical effects were observed. Animals in all dose group exhibited an overall mean weight gain. Minimal changes were noted in the hematology and serum chemistry values, however, all were considered to be either within normal biological variation or not adverse effects. The mean absolute and relative liver weights for both the 500 and 1000 mg/kg dose groups (both sexes) were significantly greater than the corresponding control values; however, these changes were found to be reversible following the 28 day recovery period. Slight increases in mean kidney weights were also noted in the 500 and 1000 mg/kg female dose groups, however, these changes were not considered to be adverse effects. All histopathological findings were minimal and no treatment related adverse effects were noted. Based on the data recorded in this study, the NOAEL for Isopar M is greater than 1000 mg/kg.