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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 28 FEB 1990 to 6 DEC 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given, comparable to standards

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
according toUS EPA 40 CFR Part 792, Federal Register 54, No. 158
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DYTEK A Amine; 1,5-Pentanediamine, 2-methyl-
- Analytical purity: 99.3%
- Lot No.:SC01049I
- Stability under test conditions: slightly unstable at concentrations >/= 3000 ppm at roomtemperature, for details see field "Any other information on results incl. tables"

Test animals

Species:
rat
Strain:
other: Crl:CD*BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. Raleigh, North Carolina, USA
- Age at study initiation: 51 days
- Weight at study initiation: approximately ranging from 133 to 196 grams
- Housing: individually housed in stainless steel, wire mesh cages
- Diet: ground Purinae Certified Rodent Chow #5002 (GPCRC), diet mixed in a Hobart hig-speed mixer with respective amount of test item, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/-2
- Humidity (%): 50 +/-10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 FEB 1990 To: 12 APR 1990

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly (and refrigerated until use)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see below, section "Any other information on results incl. tables"
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 300, 3000, 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
for feeding group 0 (control) and 10000 ppm: 10
for feeding group 300 and 3000 ppm: 5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: For the present study, 10,000 ppm was chosen as the highest dose based an the significantly depressed mean food consumption observed during the 7-day palatability study. This dose was expected to produce body weight effects but not mortality. The intermediate and low concentrations selected were expected to produce minimal compound-related toxicity and no toxicity, respectively.
- Post-exposure recovery period in satellite groups: 14 days of 5 animal each from control group and high dose group

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at each weighing

BODY WEIGHT: Yes
- Time schedule for examinations: at least twice weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined (weekly) and mean daily diet consumption estimated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study (day 28) and end of recovery period (42 days)
- Anaesthetic used for blood collection: Yes (light carbon dioxide anaesthesia)
- Animals fasted: Yes (approx. 16 h)
- How many animals: all surviving animals
- Parameters examined: erythrocyte, leukocyte, differential leukocyte, and platelet counts, hemoglobin concentration and hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study (day 28) and end of recovery period (42 days)
- Animals fasted: Yes Yes (approx. 16 h)
- How many animals: all surviving animals
- Parameters examined: blood serum was evaluated for alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transpeptidase activities and concentrations of blood urea nitrogen, total serum protein, albumin, globulin (calculated), bilirubin, creatinine, cholesterol, triglyceride, glucose, calcium, sodium, potassium, phosphate, and chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: during 16 hours before blood collection (on day 28 and 42)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: quantitative measures of volume and osmolality and semi-quantitative measures of pH, glucose, protein, bilirubin, urobilinogen, ketone and hemoglobin or occult blood. Urine colour and transparency were recorded, and sediment from each urine sample was microscopically examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes


Statistics:
Body weights, body weight gains, organ weights, and clinical laboratory measurements were analyzed by a one-way analysis of variance. When the
test for differences among test group means (F test) was significant, pairwise comparisons between test and control groups were made with Dunnett'test. Bartlett's test for homogeneity of variances was performed on the organ weight and clinical laboratory data and, if significant (alpha = 0.005), was followed by nonparametric procedures. Except for Bartlett's test, significance was judged at alpha = 0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
one male animal from the high dose group was killed in extremis
Mortality:
mortality observed, treatment-related
Description (incidence):
one male animal from the high dose group was killed in extremis
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
tansient, but significant effects on body weight gain in female rats from the high dose group
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
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
Details on results:
CLINICAL SIGNS AND MORTALITY
No adverse clinical signs of toxicity were observed during this study.
Based on the unremarkable nature of the clinical observations noted and the absence of any dose-response relationships, none of the observations were considered to be related to the dietary administration of the test item.
One male rat (Animal # 475553) from the 10000 ppm group was killed in extremis on test day 5 and submitted for pathological examination.

BODY WEIGHT AND WEIGHT GAIN
Consumption of the test material at concentrations up to 10,000 ppm for approximately 28 days had no adverse effect on mean body weights of male or female rats. Male rats fed 300 or 3000 ppm test material had slightly higher mean body weights compared to controls during the study. Male rats
from the 10,000 ppm dose group had mean body weights similar to those of the controls throughout the study.
Female rats from the 3000 ppm group had mean body weights slightly higher than controls throughout most of the study. Female rats from the 300 ppm and 10,000 ppm dose groups had slightly depressed mean body weights throughout most of the study compared to controls. No differences in mean body weights of male or female rats were statistically significant.

Overall mean body weight gains of male rats from the 3000 ppm group and female rats from the 300 and 3000 ppm groups were similar to controls. Mean body weight gains of male rats from the 300 ppm group were significantly higher than controls. Mean body weight gains of male rats from the 10,000 ppm dose group were slightly, but not significantly, depressed while mean body weight gains of female rats from the 10,000 ppm group were significantly depressed compared to controls.
During the recovery period, male rats from the 10,000 ppm dose group gained weight at a faster rate than controls. Mean body weight gains of
female rats from the 10,000 ppm dose group were similar to controls during this period.
The no-observed-adverse-effect level based on mean body weights and mean body weight gain is 10,000 ppm for male rats and 3000 ppm for female rats.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) and FOOD EFFICIENCY
Dietary concentrations of 300, 3000, or 10,000 ppm in male rats and 300 or 3000 ppm in female rats had no effect on food consumption over the 0-29 day test period. Food consumption for female rats from the 10,000 ppm group was lower (approximately 13%) compared to control rats over the course of the study. Overall mean food efficiency values of male and female rats were similar to those of rats in the control groups. The estimated mean daily intake of the test item for the 300, 3000, and 10,000 ppm dose groups, over the course of the study, was 24.3, 238.5, and 745.2 mg of test item/kg body weight/day, for male rats; in the female groups, the values were 27.5, 275.7, and 791 mg of test item/kg body weight/day,
respectively.

HAEMATOLOGY, CLINICAL CHEMISTRY and URINALYSIS
Male rats sampled at the end of the 28-day feeding period from the 300 ppm and 10,000 ppm dose groups had significantly decreased numbers of
erythrocytes and hematocrit compared to controls. Male rats from the 300 ppm and 3000 ppm dose groups had significantly higher serum concentrations of glucose and male rats from the 3000 ppm group had significantly decreased total protein compared to controls. Male rats from the 10,000 ppm dose group sampled at the end of the 14-day recovery period had significantly decreased erythrocytes, hematocrit, and serum albumin concentration.
Female rats sampled at the end of the 28-day feeding period from the 10,000 ppm dose group had significantly increased hemoglobin concentration
and total protein and female rats from the 3000 ppm dose group had significantly increased numbers of monocytes compared to controls. Female
rats from the 10,000 ppm dose group sampled at the end of the 14-day recovery period had significantly increased hemoglobin concentration, hematocrit and numbers of monocytes.
All findings in male and female rats were within the limits of expected variation and were considered biologically insignificant. The no-observed-adverse-effect level for the hematologic, clinical chemical, and urinalysis parameters is 10,000 ppm for both male and female rats.

ORGAN WEIGHTS
There were no significant compound-related differences in organ weight data in either male or female rats during this study. There was a statistically significant increase in testicular weight of the high-dose group killed at the end of the 14-day recovery period when compared to the control group. This finding was considered to be biologically insignificant since gross or microscopic lesions were not present.

GROSS PATHOLOGY
Necropsy of the male rat (Animal #475553) from the high-dose group that was killed in extremis on test day 5 revealed that the rat was anorectic and
had diarrhea. Microscopically, there were a few small ulcers in the non-glandular portion of the stomach. There was also evidence of atrophy of the thymus and hypospermia in the epididymides. The cause for the deterioration of this animal could not be determined. Since this death was an isolated event, it is unlikely to be related to the test material.

There were no compound-related changes detected either grossly or microscopically in tissues. The gross observations and microscopic changes
noted were considered incidental occurrences of spontaneous lesions common to rats of this strain and age. Based upon the final relative organ weights and gross and microscooic pathology, the no-observable-adverse-effect level for male and female rats was 10,000 ppm.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
581.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
recalculated based on the information that test item is slightly unstable at exposure conditions (i.e. RT, highest dose group = 10000 ppm; only 78 to 82%)
Sex:
male
Basis for effect level:
other: no effects, highest dose tested
Dose descriptor:
NOAEL
Effect level:
617 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
recalculated based on the information that test item is slightly unstable at exposure conditions (i.e. RT, highest dose group = 10000 ppm; only 78 to 82%)
Sex:
female
Basis for effect level:
other: effects on body weight gain only transient (due to low palatability of feed, as indicated by lower food consumption)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Stability of the test item:

Homogeneity and accuracy of concentration samples were taken at the start of the study. Stability samples were collected at diet preparation and from feed jars refrigerated for 10 days or stored at room temperature for 14 days. All samples were frozen on the day of collection for later analysis. Diet samples containing 300, 3000, or 10,000 ppm test item collected the start of the study were determined to be homogeneous and were measured be within 87% - 108% of their respective target concentrations. The 10-day refrigerated samples at all concentrations were found to be stable. Analytical measurements ranged from 89% - 102% of the target concentrations. 14-day room temperature stability samples from the 300 ppm diet were measured to be 100% - 107% of the target concentration. Analyses of the 14-day room temperature stability samples from the 3000 and 10,000 ppm diets resulted in measurements that were 70% - 78% and 78% - 82% of the nominal concentrations, respectively.

The test item is apparently slightly unstable upon exposure to the atmosphere as observed in the room temperature samples in the present study at concentrations of 3000 ppm and above.

Taking the above presented recovery rates (at 3000 ppm: 70 -78% test item recovery; at 10000 ppm: 78 -82% test item recovery)

the follwoing values for the mean daily intake after recalculationc are: - for male rats 24.3, 167 to 186, and 581.3 to 611mg of test item/kg body weight/day and - for females rats 27.5, 193 to 215, and 617 to 649 mg of test item/kg bw/day, respectively.

Applicant's summary and conclusion

Conclusions:
In this subacute toxicity test (OECD 407) male and female rats were fed diets containing 0, 300, 3000, or 10000 ppm of test material. Significant, but transient depression of body weight gain was observed in female rats from the high ppm dose group. As these animals also showed lower food consumption and bw gain was comparable to controls during the recovery period, the effects were attributed to low palatablilty of the feed and not accounted for as adverse. No other adverse body weight effects were observed. The hematologic, clinical chemical, and urinalysis parameters measured in this study were not adversely affected. There were no changes in organ weights or at the gross and microscopical evaluation of tissues. Under the conditions of this study, the no-observable-adverse-effect level (NOAEL) was 10000 ppm (i.e. 581.3 mg/kg bw/day in male rats and 617 mg/kg bw/day in female rats).
Executive summary:

Subacute toxicity was tested under the conditions set out in OECD guideline test 407. Male and female rats were fed diets containing target concentrations of 300, 3000, or 10000 ppm of test material for approximately 28 days. The estimated mean daily intake of the test item for the 300, 3000, and 10000 ppm dose groups, over the course of the study, was 24.3, 238.5, and 745.2 mg of test item/kg body weight/day, for male rats; in the female groups, the values were 27.5, 275.7, and 791 mg of test item/kg body weight/day, respectively. Taking into consideration the fact that the test item was identified to be slightly unstable under exposure conditions (i.e. room temperature) at concentrations of 3000 ppm and above and as it is not obvious from the study if the study authors considered this fact, the most conservative assumption would lead to the following values for the mean daily intake after recalculation: for male rats 24.3, 167, and 581.3 mg of test item/kg body weight/day and for females rats

27.5, 193, and 617 mg of test item/kg body weight/day, respectively (i.e. at 3000 ppm: 70 -78% test item recovery; at 10000 ppm: 78 -82% test item recovery).

Significant depression of body weight gain was observed in female rats from the 10,000 ppm dose group during the exposure period but was comparable with control group within the recovery period. Due to the fact that food consumption during the study was lower for females of this dose group, as well as the body weight itself was only slightly depressed (minus 5.3% compared to control animals at the end of the study), these effects on bw gain were not accounted for as adverse. And no other adverse body weight effects were observed. The hematologic, clinical chemical, and urinalysis parameters measured in this study were not adversely affected. There were no significant compound-related differences in organ weight data and there were no compound-related changes detected either grossly or microscopically in tissues. Under the conditions of this study, the no-observable-adverse-effect level (NOAEL) was 10000 ppm (i.e. 581.3 mg/kg bw/day in male rats and 617 mg/kg bw/day in female rats).