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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:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 408: GLP
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 408: GLP
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
According to EPA guideline 82-1
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley Inc.
- Age at study initiation: ca. 8 weeks
- Weight at study initiation: 238-295g (males); 180-236g (females)
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 16 days


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


IN-LIFE DATES: From: 1990-12-17 To:1991-07-13
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was mixed with corn oil to ensure a 10ml/kg dose volume at all dose levels.

Test material mixtures were administered by oral gavage at a dose volume of 10ml/kg. The control animals received carrier at a dose of 10ml/kg. The satellite group was dosed at the high dose level for the same duration as main test and allowed to recover for 28 days post-treatment.

VEHICLE
- Amount of vehicle (if gavage): 10ml/kg

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
7 days/week
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2500 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Test material mixtures were administered by oral gavage at three different doses at a dose volume of 10ml/kg. The control animals received carrier at a dose of 10ml/kg. The satellite group was dosed at the high dose level for the same duration as the main test and allowed to recover for 28 days post-treatment.

- Post-exposure recovery period in satellite groups: 28 days post-treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily monday-friday and once daily on weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing, the day of dose initiation, and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
at study initiation and during the final week of the main study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at main study termination and on satellite animals on the day of recovery sacrifice
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals:all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at main study termination and on satellite animals on the day of recovery sacrifice
- Animals fasted: Yes
- How many animals: all

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following parameters were statistically analyzed for significant differences: mean hematology parameters, serum chemistry parameters, organ weights, organ to body weight ratios, body weights, mean food consumption. Comparisons were limited to within sex analysis. Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test of ordered response in the dose groups. First, Bartlett’s test was performed to determine if the dose groups have equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.

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

For the nonparametric procedure the test of equality of means was performed using the Kruskal-Wallis test. If significant differences among the means was indicated, Dunn’s Summed Rank test was used to determine which treatment group differ significantly from control. In addition, Jonckheere’s test for monotonic trend in the dose response was performed.

The statistical t-test was used to compare the satellite group’s main study termination and recovery termination hematology and clinical chemistry values. In addition, the t-test was used to compare the satellite group's and the control group's relative 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.
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One male and 1 female died in the control group, 2 females died in the 2500 mg/kg dose group, 4 females died in the 5000 mg/kg dose group, 2 males and 3 females died in the satellite group. With the exception of one 2500 mg/kg female, all of the other 13 listed spontaneous deaths appear to be a result of dosing trauma and/or aspiration of test material (due to physical characteristics of test material and the high dosage volume).

The majority of animals in the control, low and mid dose groups displayed no observable abnormal clinical signs. Observations included but are not limited to scabs, maloccluded incisors, alopecia and staining of fur, dry/wet rales, dyspnea, nasal discharge. The type and incidence of abnormal clinical signs were similar between the high dose and satellite groups with a dramatic increase in incidence when compared to mid dose group. Clinical signs most frequently noted included swollen anus, ano-genital staining, emaciation, and alopecia. During the satellite recovery period, the incidence of abnormal signs decreased over time with an increase in the number of animals exhibiting no observable abnormalities.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant decreases from controls at the p<=0.05 level of significance were noted for mid dose males on days 77, 84, 91 and termination and for the high dose males on Day 42. A statistically significant decrease (p<=0.01) was noted for the high dose group males on Day 49 and continued through the end of the treatment period. Statistically significant decreases were noted for mid dose females (p<=0.05) on day 91 and for high dose females on days 77 and 91. At termination both mid and high dose females displayed a statistically significant decrease in body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Statistically significant increases in food consumption which were linearly related to dose were noted for males on Days 28 through 56 and Day 70 through termination. Significance levels were noted for both the mid and high dose males during these periods. These trends were also evident in the females where statistically significant increases in food consumption were noted on Days 21, 42, 49, and 63 through 95.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings.

HAEMATOLOGY
A statistically significant increase in platelets which was linearly related to dose in both the males and females was observed. In addition the male animals displayed a linear dose related increase in white blood cells. The mid dose male values were noted to differ significantly from those of controls for hematocrit and hemoglobin at the p<=0.01 level of significance and mean corpuscular volume and mean corpuscular hemoglobin at the p<=0.05 level of significance.

CLINICAL CHEMISTRY
Statistically significant increases in males (p<=0.01) for urea nitrogen and gamma glutamyl transpeptidase for the high dose males and also the mid dose males for urea nitrogen. An increase for cholesterol was noted for the mid and high dose groups of both sexes (p<=0.01). An increase in alanine aminotransferase was also noted for the mid and high dose males (p<=0.01). Glucose levels were significantly lower than the control values (p<=0.01) for both sexes in the mid and high dose and for the male low dose (P<=0.05). A statistically significant increase in bilirubin in the high dose of both sexes was observed. Other parameters showing statistically significant differences from controls included creatinine, chloride, tryglycerides.

ORGAN WEIGHTS
Liver weights were elevated in male and female rats at 2500 and 5000 mg/kg/day. Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/kg. Testes weights were elevated in male rats at 5000 mg/kg. Both the male and female relative kidney weights for all treated groups were significantly different from the control value (p<=0.01).

GROSS PATHOLOGY
Most frequently observed abnormalities include small and large intestine distension (mid and high dose groups); swollen anus (high dose groups), staining of the fur (mid and high dose groups).
Dose descriptor:
NOAEL
Effect level:
>= 5 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No treatment-related mortality or significant adverse clinical effects occurred.
Critical effects observed:
not specified
Conclusions:
The No Observed Adverse Effect Level (NOAEL) for following oral exposure to MRD-89-582 for 90-days is greater than or equal to 5000 mg/kg/day.
Executive summary:

MRD-89-582 was administered by oral gavage to rats at concentrations of 500, 2500 and 5000 mg/kg, 7 days a week for 13 weeks to assess the subchronic toxicity.  An additional group of animals, dosed at 5000 mg/kg/day, was held for 4 weeks to assess reversibility.  No treatment-related mortality was observed; however, male body weights were decreased while food consumption increased in the 2500 and 5000 mg/kg dose groups.  Liver weights were elevated in male and female rats at 2500 and 5000 mg/kg/day.  Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/kg.  Testes weights were elevated in male rats at 5000 mg/kg.  Kidney effects occurred in males at all dose levels, and are indicative of alpha-2u-globulin nephropathy.  Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats.  These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes.  These kidney effects are specific to male rats and are not considered to be of biological relevance to humans.

Dose-related changes in hematology or serum chemistry parameters were observed and were consistent with the changes seen in the liver.  Histological findings of hepatocellular hypertrophy (liver cell enlargement) were seen in livers of both sexes in all dose groups.  These findings are believed to have been a compensatory response and not an indication of toxicity.  Additionally, these liver effects were reversible and occurred only at high doses that are not typical of hydrocarbon exposures for humans.  Other treatment-related effects were mucosal thickening and other signs of irritation of the stomach and anus which appear to be the direct result of high dose intubation of a the locally irritating test substance.  These effects are believed to have been a compensatory response to local irritation and not an indication of toxicity.  All treatment-related effects were reversible within the 4-week recovery period.  Based on the results, the No Observed Adverse Effect Level (NOAEL) for the 90-day study was greater than 5000 mg/kg/day.

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
According to EPA guideline 82-1
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MRD-89-582
- Physical state: colorless liquid
- Analytical purity: assumed 100% pure
- Lot/batch No.: Batch II
- Expiration date of the lot/batch:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley Inc.
- Age at study initiation: ca. 8 weeks
- Weight at study initiation: 238-295g (males); 180-236g (females)
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 16 days


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


IN-LIFE DATES: From: 1990-12-17 To:1991-07-13

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was mixed with corn oil to ensure a 10ml/kg dose volume at all dose levels.

Test material mixtures were administered by oral gavage at a dose volume of 10ml/kg. The control animals received carrier at a dose of 10ml/kg. The satellite group was dosed at the high dose level for the same duration as main test and allowed to recover for 28 days post-treatment.

VEHICLE
- Amount of vehicle (if gavage): 10ml/kg

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2500 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Test material mixtures were administered by oral gavage at three different doses at a dose volume of 10ml/kg. The control animals received carrier at a dose of 10ml/kg. The satellite group was dosed at the high dose level for the same duration as the main test and allowed to recover for 28 days post-treatment.

- Post-exposure recovery period in satellite groups: 28 days post-treatment

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily monday-friday and once daily on weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing, the day of dose initiation, and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
at study initiation and during the final week of the main study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at main study termination and on satellite animals on the day of recovery sacrifice
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals:all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at main study termination and on satellite animals on the day of recovery sacrifice
- Animals fasted: Yes
- How many animals: all

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following parameters were statistically analyzed for significant differences: mean hematology parameters, serum chemistry parameters, organ weights, organ to body weight ratios, body weights, mean food consumption. Comparisons were limited to within sex analysis. Statistical evaluation of equality of means was done by an appropriate one way analysis of variance and a test of ordered response in the dose groups. First, Bartlett’s test was performed to determine if the dose groups have equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.

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

For the nonparametric procedure the test of equality of means was performed using the Kruskal-Wallis test. If significant differences among the means was indicated, Dunn’s Summed Rank test was used to determine which treatment group differ significantly from control. In addition, Jonckheere’s test for monotonic trend in the dose response was performed.

The statistical t-test was used to compare the satellite group’s main study termination and recovery termination hematology and clinical chemistry values. In addition, the t-test was used to compare the satellite group's and the control group's relative 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:
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One male and 1 female died in the control group, 2 females died in the 2500 mg/kg dose group, 4 females died in the 5000 mg/kg dose group, 2 males and 3 females died in the satellite group. With the exception of one 2500 mg/kg female, all of the other 13 listed spontaneous deaths appear to be a result of dosing trauma and/or aspiration of test material (due to physical characteristics of test material and the high dosage volume).

The majority of animals in the control, low and mid dose groups displayed no observable abnormal clinical signs. Observations included but are not limited to scabs, maloccluded incisors, alopecia and staining of fur, dry/wet rales, dyspnea, nasal discharge. The type and incidence of abnormal clinical signs were similar between the high dose and satellite groups with a dramatic increase in incidence when compared to mid dose group. Clinical signs most frequently noted included swollen anus, ano-genital staining, emaciation, and alopecia. During the satellite recovery period, the incidence of abnormal signs decreased over time with an increase in the number of animals exhibiting no observable abnormalities.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant decreases from controls at the p<=0.05 level of significance were noted for mid dose males on days 77, 84, 91 and termination and for the high dose males on Day 42. A statistically significant decrease (p<=0.01) was noted for the high dose group males on Day 49 and continued through the end of the treatment period. Statistically significant decreases were noted for mid dose females (p<=0.05) on day 91 and for high dose females on days 77 and 91. At termination both mid and high dose females displayed a statistically significant decrease in body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Statistically significant increases in food consumption which were linearly related to dose were noted for males on Days 28 through 56 and Day 70 through termination. Significance levels were noted for both the mid and high dose males during these periods. These trends were also evident in the females where statistically significant increases in food consumption were noted on Days 21, 42, 49, and 63 through 95.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings.

HAEMATOLOGY
A statistically significant increase in platelets which was linearly related to dose in both the males and females was observed. In addition the male animals displayed a linear dose related increase in white blood cells. The mid dose male values were noted to differ significantly from those of controls for hematocrit and hemoglobin at the p<=0.01 level of significance and mean corpuscular volume and mean corpuscular hemoglobin at the p<=0.05 level of significance.

CLINICAL CHEMISTRY
Statistically significant increases in males (p<=0.01) for urea nitrogen and gamma glutamyl transpeptidase for the high dose males and also the mid dose males for urea nitrogen. An increase for cholesterol was noted for the mid and high dose groups of both sexes (p<=0.01). An increase in alanine aminotransferase was also noted for the mid and high dose males (p<=0.01). Glucose levels were significantly lower than the control values (p<=0.01) for both sexes in the mid and high dose and for the male low dose (P<=0.05). A statistically significant increase in bilirubin in the high dose of both sexes was observed. Other parameters showing statistically significant differences from controls included creatinine, chloride, tryglycerides.

ORGAN WEIGHTS
Liver weights were elevated in male and female rats at 2500 and 5000 mg/kg/day. Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/kg. Testes weights were elevated in male rats at 5000 mg/kg. Both the male and female relative kidney weights for all treated groups were significantly different from the control value (p<=0.01).

GROSS PATHOLOGY
Most frequently observed abnormalities include small and large intestine distension (mid and high dose groups); swollen anus (high dose groups), staining of the fur (mid and high dose groups).

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 5 000 mg/kg bw/day (nominal)
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 No Observed Adverse Effect Level (NOAEL) for following oral exposure to MRD-89-582 for 90-days is greater than or equal to 5000 mg/kg/day.
Executive summary:

MRD-89-582 was administered by oral gavage to rats at concentrations of 500, 2500 and 5000 mg/kg, 7 days a week for 13 weeks to assess the subchronic toxicity.  An additional group of animals, dosed at 5000 mg/kg/day, was held for 4 weeks to assess reversibility.  No treatment-related mortality was observed; however, male body weights were decreased while food consumption increased in the 2500 and 5000 mg/kg dose groups.  Liver weights were elevated in male and female rats at 2500 and 5000 mg/kg/day.  Adrenal weights were significantly increased in male and female rats at 5000 mg/kg and in female rats at 2500 and 5000 mg/kg.  Testes weights were elevated in male rats at 5000 mg/kg.  Kidney effects occurred in males at all dose levels, and are indicative of alpha-2u-globulin nephropathy.  Alpha-2u-globulin nephropathy, also known as hyaline droplet nephropathy, results from the formation of complexes with a naturally occurring protein (alpha-2u-globulin) in the kidneys of male rats.  These complexes can accumulate in the proximal renal tubule and may produce species-specific histopathological changes.  These kidney effects are specific to male rats and are not considered to be of biological relevance to humans.

Dose-related changes in hematology or serum chemistry parameters were observed and were consistent with the changes seen in the liver.  Histological findings of hepatocellular hypertrophy (liver cell enlargement) were seen in livers of both sexes in all dose groups.  These findings are believed to have been a compensatory response and not an indication of toxicity.  Additionally, these liver effects were reversible and occurred only at high doses that are not typical of hydrocarbon exposures for humans.  Other treatment-related effects were mucosal thickening and other signs of irritation of the stomach and anus which appear to be the direct result of high dose intubation of a the locally irritating test substance.  These effects are believed to have been a compensatory response to local irritation and not an indication of toxicity.  All treatment-related effects were reversible within the 4-week recovery period.  Based on the results, the No Observed Adverse Effect Level (NOAEL) for the 90-day study was greater than 5000 mg/kg/day.