Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1990/10/30-1991/02/27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: In agreement to OECD guideline 408.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Hydrocarbons, C10-C13, aromatics, >1% naphthalene
EC Number:
926-273-4
IUPAC Name:
Hydrocarbons, C10-C13, aromatics, >1% naphthalene

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Source: Harlan Sprague Dawley Inc.
Sex: Male (50), Female (50)
Age at study initiation: Approximately 8 weeks
Weight at study initiation: Males: 253.0 - 328.4 g; Females: 172.3 - 229.1 g
Housing: Individually
Diet (e.g. ad libitum): Purina Certified Rodent Chow 5002, ad libitum
Water (e.g. ad libitum): Automatic watering system, ad libitum
Acclimation period: 29d

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test mixture was administered by oral gavage at a dose of 0, 300, 600, or 1000 mg/ kg at a volume of 2.4 ml/kg, 7 days per week for a period of at least 13 weeks. The control animals received a carrier (corn oil) dose 2.4 ml/kg. The 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. The amount of test material administered to each animal was recalculated weekly, based on the most recent body weight.
Duration of treatment / exposure:
once daily
Frequency of treatment:
7 days/week for ~13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 300, 600, or 1000 mg/ kg
Basis:
other: oral gavage
No. of animals per sex per dose:
Males (10). Females (10) per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
The test mixture was administered by oral gavage at a dose of 300, 600, or 1000 mg/ kg at a volume of 2.4 ml/kg, 7 days per week for a period of at least 13 weeks. The control animals received a carrier (corn oil) dose 2.4 ml/kg. The 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. The amount of test material administered to each animal was recalculated weekly, based on the most recent body weight.

The animals were checked for viability twice daily on Monday through Friday, and once daily on Saturday and Sunday. Clinical observations were made daily for toxicological signs. Body weights were recorded prior to dosing and then once a week thereafter. Body weights were also recorded at the scheduled sacrifice at the death of animals that succumbed before the end of the study. Clinical laboratory studies (hematology and serum chemistry) were performed on all animals at main study termination or at the termination of the satellite/recovery study. Blood samples were collected under methoxyflurane anesthesia from the orbital sinus.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
The animals were checked for viability twice daily on Monday through Friday, and once daily on Saturday and Sunday. Clinical observations were made daily for toxicological signs. Body weights were recorded prior to dosing and then once a week thereafter. Body weights were also recorded at the scheduled sacrifice at the death of animals that succumbed before the end of the study. Clinical laboratory studies (hematology and serum chemistry) were performed on all animals at main study termination or at the termination of the satellite/recovery study. Blood samples were collected under methoxyflurane anesthesia from the orbital sinus.
Sacrifice and pathology:
The necropsy was performed on all animals. The necropsy examined the external surfaces of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. The ovaries, kidneys, adrenal, liver, and testes were weighed prior to fixation. Preserved tissue from the control and high dose group as well from all animals that were sacrificed moribund or succumbed during the study were processed, sectioned, stained (hematoxylin and eosin) and examined microscopically. Abnormalities from the low and mid doses were examined as well as the stomach, spleen, urinary bladder, and the thyroid.
Other examinations:
HEMATOLOGY
-erythrocyte count
-hematocrit
-hemoglobin
-leukocyte count (total and differential)
-mean corpuscular volume
-mean corpuscular hemoglobin
-mean corpuscular hemoglobin conc.
-platelets

SERUM CHEMISTRY
-total bilirubin
-albumin
-blood urea nitrogen
-calcium
-cholesterol
-creatinine
-electrolytes (Na, K, Cl)
-glucose
-total protein
-triglycerides
-phosphorous
-gamma glutamyl transferase
-serum aspartate aminotransferase
-serum alanine aminotransferase
Statistics:
The following parameters were statistically analyzed:
-mean hematology parameters
-mean serum chemistry parameters
-mean organ weights
-mean organ to body weights, by weighing period
-mean food consumption

Comparisons were limited to within sex analysis. Variance was assessed through the use of a one way ANOVA. Bartlett’s test (conducted at the 1% level of significance) was performed to determine if the dose groups had equal variance. If the variances were equal, the test was done using parametric methods, otherwise nonparametric techniques were used.

The parametric procedures used a one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett’s test was used to determine which treatment groups differ significantly from control. In addition, a regression analysis for linear response in the dose group was performed.

The nonparametric procedures tested for the equality of means using the Kruskal-Walls test. If significant differences were indicated, Dunn’s Summed Rank test was used to determine which treatment group differed significantly from control. Jonckheere’s test for monotonic trend in the dose response was performed.

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

The t-test was used to compare the satellite group’s main study termination and recovery termination hematology and clinical chemistry values. The t-test was also used to compare the satellite and the control groups’ organ weights. The t-test was also 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:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
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:
no effects observed
Details on results:
CLINICAL SIGNS and MORTALITY
There were a total of 10 animal deaths; four were attributed to dosing trauma or aspiration of the test material. Two animals (1 female at 300 mg/kg and a female in the satellite group) died of unknown causes. The four remaining deaths (1 male, 3 female) at the 1000 mg/kg dose appeared to be treatment-related. Animals in the 0, 300, or 600 mg/kg dose displayed little to no clinical signs. Animals in the 1000 mg/kg dose consistently displayed signs of ano-genital staining, alopecia, and emaciation. Note: The animals in the 1000mg/kg dose were initially treated with a dose of 1200 mg/kg, but displayed severe signs of hypothermia and hypoactivity and after day 3, their dose was reduced to 1000mg/kg. Sporadic clinical signs across all groups included oral discharge, red discharge from the eyes, rales, sores, scabs, soft stool, and little sign of stool.

BODY WEIGHT and WEIGHT GAIN
Statistically significant decreases in body weight for males at the 1000 mg/kg dose were noted in all intervals from day 7 through termination. There were no noted differences in any of the female groups.

FOOD CONSUMPTION
Food consumption for males at the 1000 mg/kg dose was decreased for week 1 when compared to controls. Food consumption was significantly higher for males at the 600 mg/kg dose at weeks 10-13. Food consumption was significantly higher for females at the 600 and 1000 mg/kg dose at weeks 7-10.

HAEMATOLOGY
Non-biologically relevant but statistically significant decreases were noted in:
-Hematocrit: Males at 300, 600, and 1000 mg/kg doses; Females at 600 mg/kg dose. There is no dose response for male rats. The hematocrit count was statistically affected at all does levels; however, all treated male rats had hematocrit counts that were 95% control. Hematocrit counts were only statistically affected for the female rats at 600 mg/kg (96% of control). Hematocrit counts were not different from controls in the recovery group that was initially dosed with 1000 mg/kg/day. Hematocrit levels returned to normal in the recovery group (initially dosed with 1000 mg/kg). Since only the male rats were statistically affected, no dose response was observed, and experimental values were within 10% of controls (normal physiological range), these changes are not considered to be an adverse effect.
-Hemoglobin: Males at 300, 600, and 1000 mg/kg doses: Females 600, and 1000 mg/kg dose. Male rats exposed to 300, 600, and 1000 mg/kg had decreased hemoglobin counts of 95%, 95%, and 92.5% of control, respectively. Female rat hemoglobin was 93% of control at the 600 and 1000 mg/kg doses. Hemoglobin concentrations returned to normal in the recovery group (initially dosed with 1000 mg/kg). Since no dose response was observed in either the male or female rats, and experimental values were within 10% of controls (normal physiological range), these changes are not considered to be an adverse effect.
-Mean corpuscular hemoglobin concentration (MCHC): Males at 300, 600, and 1000 mg/kg doses: Females 600, and 1000 mg/kg dose. Only male rats in the 1000 mg/kg were statistically affected, however, the MCHC level was 97.5% of control. Female rats were statistically affected at all doses, however, no dose response was observed. The values (% control) for MCHC were 96.7%, 96.9%, 96% for the 300, 600, and 1000 mg/kg treated female rats respectively. MCHC levels returned to normal in the recovery group (initially dosed with 1000 mg/kg). Since only the female rats were statistically affected, no dose response was observed, and experimental values were within 10% of controls (normal physiological range), these changes are not considered to be an adverse effect.
- Bone Marrow – There were no adverse pathology findings of the bone marrow in any animal at any dose.
- RBC– Only male rats in the 600 and 1000 mg/kg were statistically affected. The values for RBC (% control) were 95% and 91.5% for the 600 and 1000 mg/kg groups, respectively. Female rats RBC were not significantly decreased at any test dose. RBC levels returned to normal in the recovery group (initially dosed with 1000 mg/kg). Conclusion: Since only the male rats have a significant decrease in RBC counts, it is unlikely that this change is an adverse effect or is related to the splenic hemosiderosis observed in both sexes of rats. The decrease observed in the male rats was less than 10% of control (normal physiological range) and these changes are not considered to be an adverse effect.

The hematology for the recovery period revealed a recovery trend for all of the observed decreased parameters; including a significant change in the red blood cells, hemoglobin, MCHC for both males and females.

CLINICAL CHEMISTRY
Statistically significant increases were noted in mean serum chemistry values in:
-Albumin: Females at 600 and 1000 mg/kg doses
-Creatinine: Females at 1000 mg/kg doses
-Potassium: Males at 1000 mg/kg doses
-Total Protein: Females at 600 and 1000 mg/kg doses
-Cholesterol: Females at 600 and 1000 mg/kg doses
Sporadic, statistically significant increases were also noted in albumin (males) and transferase (females) and significant decreases were noted in chloride (females) and triglycerides (males).

ORGAN WEIGHT
There was a statistically significant increase in male liver weight (600 mg/kg) and in male kidney weight (300 and 600 mg/kg). Females demonstrated a significant dose related increase in both liver and kidney weight. The relative organ weights for the recovery period revealed a recovery trend in the liver and testes.

GROSS PATHOLOGY and HISTOPATHOLOY
The most frequently noted abnormalities in surviving animals were cervical lymph node redness, abnormal small/large intestine contents, small/large intestine distention, staining of the fur, alopecia, and eye damage from eye bleeding. The most frequent abnormalities for the animals which succumbed prior to study termination were: thickened liver, small spleen, enlarged adrenals, staining of fur, emaciation, abnormal stomach/small/large intestine contents, stomach distension, and stomach mucosa thinner than normal.

Treatment related changes were noted in the livers of the females of all dosage groups consisting of centrilobular hepatocellular hypertrophy. These liver changes were noted at a sporadic and lower incidence in male rats. Hepatocellular hypertrophy was not observed in the satellite group that was allowed to recover. A dose related increase amount of brown pigment (hemosiderin) in macrophages in the splenic red pulp was observed in all animals that received 600 and 1000 mg/kg doses. This hemosiderin material was still present after recovery, but was reduced. An increased incidence of minimal to moderate hyperplasia and hypertrophy of the thyroid follicular epithelial cells was noted in all male animal dosage groups and in females exposed to 600 and 1000 mg/kg. These effects were reversed in the recovery group. In response to xenobiotics, there is a greater predisposition towards proliferate lesions (e.g. hyperplasia) of follicular cells in laboratory rodents, especially males, than in the human thyroid. Capen et al. Toxicol Pathol 1989; 17:266 as cited in Werner and Ingbar’s The Thyroid. 7th ed. Braverman and Utiger. 1991. Since these effects were reversible in the recovery group and due to the rodent specific response, this effect at the lower dose is deemed to be an adaptive response.

Recovery Group:
The relative organ weight, clinical chemistry and hematology data indicated recovery during the 28-day post exposure period. The satellite group values were similar to control values.

Liver and thyroid changes seen at termination appeared to be reversible as they were not observed in the satellite group after the 28-day recovery period. The changes seen in the spleen, urinary bladder and stomach in the main study termination were at a lower incidence and/or severity in the satellite group after the 28-day recovery period also indicating reversibility. The hematology, serum chemistry and relative organ weights also indicated reversibility of the effects.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Mortality observed at the highest dose tested. Food consumption was higher at the 600 and 1000 mg/kg doses in both sexes. Clinical chemistry and haematology parameters were affected at the 600 and 1000 mg/kg doses in both sexes.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for MRD-90-884 is 300 mg/kg body weight under the test conditions of this study.
Executive summary:

A 90-day subchronic study was conducted in rats to assess the toxicity of MRD-90-884. The test mixture was administered by oral gavage at a dose of 0, 300, 600, 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. There were a total of 10 animal deaths; four deaths (1 male, 3 female) at the 1000 mg/kg dose were attributed to the treatment.   There was an overall low incidence of clinical signs at the 300 and 600 mg/kg doses.  Low food consumption and emaciation were observed in animals dosed at 1000 mg/kg.  Post mortem examinations revealed a significant increase in liver and kidney weights in male and female animals as well as increase in body weight for all animals except for males in the high dose group.  Organ weight, clinical chemistry, and hematology data from the satellite recovery group indicated recovery during the 28 day recovery period.  Based on the data recorded in this study, the NOAEL for MRD-90-884 is 300 mg/kg.