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Description of key information

28-day stuy (OECD 407): NOAEL = 10 mg/kg bw/day (BASF SE/RCC, 1998)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Remarks:
RCC, Research & Consulting Company, Ltd.
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals were assigned to identification numbers.
Air-conditioning with 10-15 air changes per hour and continuously monitored environment with a target range for room temperature of 22 ± 3°C, and for relative humidity between 40-70 % (values above 70 % during cleaning process possible). The animals were provided with a 12-hour light, 12-hour dark cycle. Music was played during the light period.
Groups of five rats were accomodated in Makrolon type-4 cages with standard softwood bedding.
Pelleted standard Kliba 3433, batch no. 90/97 rat maintenance diet and community tap water were available ad libitum.

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer.
Frequency of preparation: Daily, prior to each application.
Chemical analysis of dose preparations: Concentration, homogeneity and stability of the test article / vehicle mixtures were determined in samples taken during acclimatization and during week 3 of the treatment. The analyses were performed in the Analytical Laboratories of RCC Umweltchemie AG according to a method supplied by the Sponsor.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 10, 50, and 250 mg/kg bw/d
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Based on data from a 14-day dose-range fmding study (RCC selection Projeet 666786) in which the test substance was administered by gavage to 3 rats per group and sex. Treatment with the test article at 300 and 1000 mg/kg was associated with effects on food consumption and body weight in both test groups and mortality, clinical symptoms and macroscopic findings at 1000 mg/kg.
Observations and examinations performed and frequency:
Clinical signs, outside cage observations, food consumption and body weights were recorded periodically during the acclinatization, treatment and recovery periods. The animals were examined for signs of toxicity or mortality at least once a day.
Sacrifice and pathology:
At the end of the dosing period and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses and urine samples were collected for urinalysis. All animals were killed, necropsied and examined post mortem. Samples of major organs from all animals of group 1 (0 mg/kg) and group 4 (250 mg/kg), as well as kidneys, mandibular lymph nodes, spleen, testes, thymus and thyroid glands from the animals of the intermediate dose groups 2 (10 mg/kg) and 3 (50 mg/kg) and gross lesions from all animals were processed as hematoxylin and eosin stained slides and examined by light microscopy.
Other examinations:
At pretest and at weeks 4 and 6 a modified Irwin screen test was performed on all rats per group and sex.
Statistics:
The following statistical methods were used to analyze the body weights, organ weights and all ratios as well as clinical laboratory data:
If the variables could be assumed to foilow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The Fisher's exact test was applied to the macroscopic findings.
Details on results:
All animals survived the scheduled study period.
The only treatment-related clinical signs, observed during the daily clinical observations and 1 or the weekly outside cage observations, were slight sedation and ruffled fur, which were observed in all group 4 (250 mg/kg) animals, predominantly during weeks 2 and 3 of the dosing period. No clinical signs were noted during the recovery period. Incidental clinical observations included soft feces, increased salivation and wounds / scars / fissures on tail / ears in occasional animals.
For the group 4 (250 mg/kg) animals, food consumption was reduced throughout the 4-week dosing period. On cessation of dosing, food consumption increased and was slightly higher than that of the controls in weeks 1 (females only) and 2 (both sexes) of the recovery period. Relative food consumption was lower than that of the control group in weeks 1 and 2 of the dosing period and was higher than that of the control group in weeks 1 and 2 of the recovery period. Both food consumption and relative food consumption of the group 2 (10 mg/kg) and 3 (50 mg/kg) animals were similar to that of the control group throughout the 4-week dosing period.
For the group 4 (250 mg/kg) animals, body weight gain was retarded throughout the 4-week dosing period. For both sexes, mean body weights were significantly lower than controls from Day 8 of the dosing period. During the 2-week recovery period, there was an increase in weight gain for both sexes so that mean terminal body weight of the females, but not the males, was the same as the control value. For the group 3 (50 mg/kg) animals, there was a slight retardation in body weight gain during the 4-week dosing period, but none of the lower mean body weight values attained statistical
significance. Body weights and body weight gain of the group 2 (10 mg/kg) animals were similar to those of the control group throughout the study.
At pretest and at weeks 4 and 6, a modified Irwin screen test was performed on all rats. This involved the observation of individual animals in the home cage, in the arena, in the hand and on return to the home cage, and the measurement of hind- and forelimb grip strength and locomotor activity. During the assessments performed at both 4 and 6 weeks, all observations recorded (decreased / increased pupil diameter, vocalization, decreased exploratory activity) were considered to be incidental and not related to treatment with the test article. In all groups, values for grip strength increased over the duration of the study, as the animals increased in weight and developed from ca. 7 weeks to 13 weeks of age. The significantly lower values for fore- and hindlimb grip strength at 6 weeks (recovery period) in group 4 (250 mg/kg) were considered to be incidental as the values at the end of the 4-week dosing period were similar to those of the control group and the value for forelimb grip strength was also significantly lower than controls at pretest. The significantly increased value for forelimb grip strength after 4 weeks in group 2 (10 mg/kg) was similarly considered to be incidental. There was a large inter-individual variability in the values obtained for the animals from all groups.
Quantitative assessment of locomotor activity over a period of 60 minutes was measured using an Activity Monitor in week 4 of the study. For the group 4 (250 mg/kg) females, overall activity was significantly lower than controls and for the group 2 (10 mg/kg) females, activity was significantly increased at 50 minutes. Both of these findings were considered to be incidental and to have occurred because of the large inter-individual variability in this type of measurement.
After 6 weeks, there was evidence of slight anemia in both males and females at 250 mg/kg (RBC, HB and WBC reduced). At the end of the 4-week dosing period, there were indications of this effect in the slightly lower, but not statistically significant (p>0.05) values for total leukocyte count, but the erythrocyte values were not affected. Increased hemosiderin deposits in the spleen at histological examination correlate with these hematological findings. The slight increase in reticulocytes (botih sexes) and in nucleated erythrocytes and platelets (males) at 250 mg/kg at 6 weeks indicate a compensatory effect by the hemopoietic organs, showing that the repair mechanism had started. The decreased Iymphocyte count at both 4 and 6 weeks correlates with the lymphoid atrophy in the spleen and thymus observed at histological examination and is considered to have contributed to the decrease in the leukocyte values mentioned above. There were no changes in the hematological parameters in groups 2 (10 mg/kg) or 3 (50 mg/kg), which were considered to be related to treatment with the test substance.
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female
Critical effects observed:
not specified
Conclusions:
Oral administration of the test substance to Wistar rats at doses of 10, 50 and 250 mg/kg body weight/day for 28 days was associated with clinical signs, effects on food consumption, body weight, clinical laboratory parameters, organ weights and macroscopic and microscopic findings at 250 mg/kg/day and, to a lesser extent, at 50 mg/kg/day. Target organs were identified as spleen, thymus (both sexes), kidneys, testes and thyroids (males). There was evidence of recovery in some, but not all parameters at the end of the recovery period.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline study with Klimisch score 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Supporting study (14- day range finding):

In a range- finding study, the test substance was administered to Wistar rats by repeated oral gavage, for a period of 5 days. The study was comprised of two groups, each containing three male and three female rats. The oral administration of NN'-DIMETHYLPROPYLENE UREA to Wistar rats at doses of 300 mg/kg/day, for 14 days resulted in no mortality, no clinical signs and no macroscopic findings, whereas food consumption, body weights and relative food consumption were affected in the animais of this group when compared to controls. The high-dose animals (1000 mg/kg) showed effects related to test article treatment in mortality, clinical signs, food consumption, body weights, relative food consumption and macroscopic findings. On the basis of the results obtained in this 14-day range-finding study the following dose levels for the 28-day study were proposed: 250, 50, 10 mg/kg bw/day.

Key study (28 - day):

Based on the findings in the 14 -days dose-finding study, the test item, was administered once daily by oral route for 4 weeks to Wistar rats, at the dose-levels of 0 (group 1) 10 (group 2), 50 (group 3) and 250 (group 4) mg/kg/day according to OECD guideline 407 and GLP.

All animals survived the scheduled study period.

The only treatment-related clinical signs, observed during the daily clinical observations or the weekly outside cage observations, were slight sedation and ruffled fur, which were observed in all group 4 (250 mg/kg) animals, predominantly during weeks 2 and 3 of the dosing period. No clinical signs were noted during the recovery period. Incidental clinical observations included soft feces, increased salivation and wounds / scars / fissures on tail / ears in occasional animals.

For the group 4 (250 mg/kg) animals, food consumption was reduced throughout the 4-week dosing period. On cessation of dosing, food consumption increased and was slightly higher than that of the controls in weeks 1 (females only) and 2 (both sexes) of the recovery period. Relative food consumption was lower than that of the control group in weeks 1 and 2 of the dosing period and was higher than that of the control group in weeks 1 and 2 of the recovery period. Both food consumption and relative food consumption of the group 2 (10 mg/kg) and 3 (50 mg/kg) animals were similar to that of the control group throughout the 4-week dosing period.

For the group 4 (250 mg/kg) animals, body weight gain was retarded throughout the 4-week dosing period. For both sexes, mean body weights were significantly lower than controls from day 8 of the dosing period. During the 2-week recovery period, there was an increase in weight gain for both sexes so that mean terminal body weight of the females, but not the males, was the same as the control value. For the group 3 (50 mg/kg) animals, there was a slight retardation in body weight gain during the 4-week dosing period, but none of the lower mean body weight values attained statistical significance. Body weights and body weight gain of the group 2 (10 mg/kg) animals were similar to those of the control group throughout the study.

At pretest and at weeks 4 and 6, a modified Irwin screen test was performed on all rats. This involved the observation of individual animals in the home cage, in the arena, in the hand and on return to the home cage, and the measurement of hind- and forelimb grip strength and locomotor activity. During the assessments performed at both 4 and 6 weeks, all observations recorded (decreased / increased pupil diameter, vocalization, decreased exploratory activity) were considered to be incidental and not related to treatment with the test article. In all groups, values for grip strength increased over the duration of the study, as the animals increased in weight and developed from ca. 7 weeks to 13 weeks of age. The significantly lower values for fore- and hindlimb grip strength at 6 weeks (recovery period) in group 4 (250 mg/kg) were considered to be incidental as the values at the end of the 4-week dosing period were similar to those of the control group and the value for forelimb grip strength was also significantly lower than controls at pretest. The significantly increased value for forelimb grip strength after 4 weeks in group 2 (10 mg/kg) was similarly considered to be incidental. There was a large inter-individual variability in the values obtained for the animals from all groups.

Quantitative assessment of locomotor activity over a period of 60 minutes was measured using an Activity Monitor in week 4 of the study. For the group 4 (250 mg/kg) females, overall activity was significantly lower than controls and for the group 2 (10 mg/kg) females, activity was significantly increased at 50 minutes. Both of these findings were considered to be incidental and to have occurred because of the large inter-individual variability in this type of measurement.

After 6 weeks, there was evidence of slight anemia in both males and females at 250 mg/kg (RBC, HB and WBC reduced). At the end of the 4-week dosing period, there were indications of this effect in the slightly lower, but not statistically significant (p>0.05) values for total leukocyte count, but the erythrocyte values were not affected. Increased hemosiderin deposits in the spleen at histological examination correlate with these hematological findings. The slight increase in reticulocytes (both sexes) and in nucleated erythrocytes and platelets (males) at 250 mg/kg at 6 weeks indicate a compensatory effect by the hemopoietic organs, showing that the repair mechanism had started. The decreased Iymphocyte count at both 4 and 6 weeks correlates with the lymphoid atrophy in the spleen and thymus observed at histological examination and is considered to have contributed to the decrease in the leukocyte values mentioned above. There were no changes in the hematological parameters in groups 2 (10 mg/kg) or 3 (50 mg/kg), which were considered to be related to treatment with the test substance.

Increased cholesterol and triglyceride values in both sexes at 250 mg/kg at the end of the 4-week dosing period indicated a marginal change in lipid metabolism. The significantly lower activities for the liver enzymes, ALAT (both sexes), ASAT and ALP (males) at 250 mg/kg after 4 weeks were considered to be incidental as there was no histological evidence of an effect on the liver. Lower values for total protein (both sexes) at 250 mg/kg after 4 weeks could be associated with the reduced food consumption in this group during the dosing period. However, for all the clinical chemistry parameters, mean values were within the historical control range, even when the difference from the concurrent control value was statistically

significant, indicating the narrow range of values obtained in this study. There were no changes in the clinlical chemistry parameters in groups 2 (10 mg/kg) or 3 (50 mg/kg) which were considered to be related to treatment with the test article.

As a result of the retardation in body weight gain in the group 4 (250 mg/kg) animals, there was a significant reduction in the absolute weights of the following organs - heart, thymus, spleen, testes and epididymides (males) and brain, thymus and spleen (females). After the 2-week recovery period, the only organ weights that were significantly lower than their respective control values (absolute values, organ/body weight and organ/brain weight ratios) were testes and epididymides. Thymus and spleen weights showed considerable recovery in both sexes. One slight anomaly was the significantly increased thymus weight for the group 4 (250 mg/kg) females at 6 weeks (all values). This occurred because 2/5 control females showed advanced thymic involution, indicatecl by low thymus weights and moderate lymphocytolysis. In groups 2 (10 mg/kg) and 3 (50 mg/kg), the only statistically significant difference from the control value which was considered to be related to treatment with NN'-DIMETHYLPROPYLENE UREA was the lower absolute thymus weight for the group 3 (50 mg/kg) females.

The following macroscopic findings were considered to be related to treatment with the test article:

- Testes - reduced in size, all five group 4 (250 mg/kg) rats at 4 weeks and after 2 weeks recovery.

- Spleen - reduced in size, one male and three females of group 4 (250 mg/lcg) at 4 weeks.

- Thymus - reduced in size, three males and four females of group 4 (250 mg/kg) at 4 weeks.

All other macroscopic findings were unremarkable and did not distinguish treated animals from the controls.

Alterations in the kidneys of male rats consisted of increased incidences and/or severity grades of hyaline droplet and granular cast formation, tubular degeneration/regeneration and tubular atrophy. Hyaline droplets were seen in all male rats at the end of the main study at minimal or slight degree in group 1 (control) and 2 (10 mg/kg/day), at minimal to moderate degree in group 3 (50 mg/kg/day) and at severe degree in group 4 (250 mg/kg/day). This was accompanied by minimal granular cast formation in one group 3 and two group 4 rats. Tubular degeneration was noted in one group 3 animal - minimal, and three from group 4 - minimal or slight. Minimal tubular regeneration was seen in one group 1 and two group 3 rats, and at a minimal or moderate degree in all five group 4 animals. Tubular atrophy of minimal degree was recorded in two group 1 and four group 3, and at minimal or slight degree in four group 4 animais. These findings were consistent with inducible male rat alpha-2-micro-globulin nephropathy syndrome.

Following the recovery period, hyaline droplets were seen at minimal or slight degree in three rats from both groups 1 and 4. Tubular degeneration and tubular regeneration were both recorded at a minimal degree in two cases from group 4. Tubular atrophy was present at a minimal degree in one group 1 and at a minimal or slight degree in all five group 4 animals. In the testes, at the end ofthe main study, there was a moderate degree of seminiferous tubular atrophy in one group 3 and a massive degree in all group 4 animais. This was the histological correlate to the gross findings noted at necropsy. A minimal degree of this finding was also recorded in one case in group 1. Following the recovery period, tubular atrophy was present at severe or massive degree in all group 4 animals. However, some individual testes had a moderate degree of this finding and demonstrated a degree of re-epithelialization.

At the end of the main treatment period, there was a minimal or slight degree of thymic cortical atrophy in four males of group 4 and slight to moderate degree in all five group 4 females. These were the microscopic correlates to the necropsy findings. Minimal thymic cortical atrophy was also present in two group 3 females. In addition, there was a slight increase in the degree of lymphocytolysis (single cell necrosis of lymphocytes, referred to in the past by the descriptive term "starry sky appearance") in both sexes of group 4, compared to controls. Following the recovery period, both of these findings had reverted to incidence and severity values similar to those of the control group. An additional finding was present only in males from treated groups and consisted of a slight degree of follicular hypertrophy in the thyroid glands of two group 2, one group 3 and all five group 4 male rats at the end of the main study. After the recovery period a minimal or slight grade of this finding was present in two animals of group 4. This finding was considered to indicate a slight hyperfunctional state. As all group 4 males were affected at the end of the main treatment period, a test article related effect cannot be ruled out at this dosage. However, the incidences in groups 2 and 3 showed no dose-relationship and were considered to be incidental.

On the basis of the results obtained in this study, 10 mg/kg body weight/day of N,N'-DIMETHYLPROPYLENE UREA is considered to be the no-observed-adverse-effect-level (NOAEL) for both sexes.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study with the longest duration (28 days) and lowest NOAEL was chosen (key study).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In accordance with column 2 of REACH Annex VIII, the study does not need to be conducted as one other route is provided.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
In accordance with column 2 of REACH Annex VIII, the study does not need to be conducted as one other route is provided.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
In accordance with column 2 of REACH Annex VIII, the study does not need to be conducted as one other route is provided.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
In accordance with column 2 of REACH Annex VIII, the study does not need to be conducted as one other route is provided.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: spleen; cardiovascular / hematological: thymus; glandular: thyroids; urogenital: kidneys; urogenital: testes

Justification for classification or non-classification

Based on a subacute repeated oral toxicity study, the test item was not classified and labelled according to Directive 67/548/EEC (DSD) and to Regulation /EC) No 1272/2008 (CLP).