Carbamic acid,N,N'-[1,3-phenylenebis[methyleneiminocarbonylimino(methyl-3,1-phenylene)]]bis-di-C11-14-isoalkylesters, C13-rich

Repeated dose toxicity: oral

Administrative data

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

3 blood samples from the female vehicle control group were misidentified at PI 1 and labelled with identical ID’s. Since the blood data from all 3samples originated all from a single group the deviation is rated minor and does not affect the study result

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

bis(11-methyldodecyl) ((((1,3-phenylenebis(methylene)bis(azanediyl))bis(carbonyl) bis(azanediyl))bis(4-methyl-3,1-phenylene) dicarbamate

Cas Number:

865536-03-4

Molecular formula:

C52H80N6O6

IUPAC Name:

bis(11-methyldodecyl) ((((1,3-phenylenebis(methylene)bis(azanediyl))bis(carbonyl) bis(azanediyl))bis(4-methyl-3,1-phenylene) dicarbamate

Constituent 2

Reference substance name:

bis(11-methyldodecyl)(4-methyl-1,3-phenylene)dicarbamate

Molecular formula:

C35H62N2O4

IUPAC Name:

bis(11-methyldodecyl)(4-methyl-1,3-phenylene)dicarbamate

Constituent 3

Reference substance name:

11-methyldodecyl (3-(3-(3-(aminomethyl)benzyl)ureido)-4-methylphenyl)carbamate

Molecular formula:

C30H46N4O3

IUPAC Name:

11-methyldodecyl (3-(3-(3-(aminomethyl)benzyl)ureido)-4-methylphenyl)carbamate

Constituent 4

Reference substance name:

1,1'-(4-methyl-1,3-phenylene)bis(3-(3-(aminomethyl)benzyl)urea)

Molecular formula:

C25H30N6O2

IUPAC Name:

1,1'-(4-methyl-1,3-phenylene)bis(3-(3-(aminomethyl)benzyl)urea)

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar-Unilever

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing conditions Clean conventional housing: aeration with approx. 10 air changes per hour, room climate 22 ± 3°C at 30-70% relative humidity, artificial lighting 12 h light/12 h dark Caging Eight groups of five animals each in open makrolon cages type 2000P (TechniPlast) Bedding Lignocel hygienic animal bedding (J. Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg) Diet Maintenance diet rat/mouse, pellets, No. 1324 (Altromin GmbH & Co. KG, 32791 Lage), ad lib. Water Autoclaved community tap water, ad lib.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item ADDUKT TI 65-MXDA was administered daily by oral gavage at dose levels of 63, 250 and 1000 mg/kg BM/day to 5 male and 5 female Wistar rats each over a period of 28 days. Another 5 male and 5 female rats received the same total volume of corn oil as vehicle control. Of these solutions, 8 ml/kg BM was administered daily to each animal.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

63 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

The test item ADDUKT TI 65-MXDA was administered daily by oral gavage at dose levels of 63, 250 and 1000 mg/kg BM/day to 5 male and 5 female Wistar rats each over a period of 28 days. Another 5 male and 5 female rats received the same total volume of corn oil as vehicle control. Of these solutions, 8 ml/kg BM was administered daily to each animal. During the in-life phase, viability, general and detailed clinical signs, food and water consumption, body mass, grip strength and reactivity to sensory stimuli (limb placing test) were recorded. At the end of the in-life phase, blood samples from all animals were collected to provide data on haematology and serum biochemistry. All animals were sacrificed immediately after bleeding and examined by gross necropsy. Weights of selected organs (see table 5) were recorded, and tissues and organs were preserved. All samples were processed for histopathological examination. The latter was conducted on samples from the high dose groups and the vehicle groups exclusively.

Examinations

Observations and examinations performed and frequency:

The following parameters were observed and documented during the in life period:
- Viability/fatalities Daily
- General clinical signs/behaviour Once workdays
- Detailed clinical signs Once weekly (including once before beginning of
application)
- Grip strength, limb placing test Once before application and once during the last
exposure week
- Body mass Once weekly (including once before application
start)
- Group food consumption Once weekly (including once before application
start)
- Group water consumption Twice weekly (including once before application
start)
3.2.1 Viability, general clinical signs and behaviour
Cage-side observations to detect signs of illness, reactions to treatment, moribund animals,
or fatalities were performed at least once daily throughout the study, up to the day of
necropsy. Data were recorded on checklists. In case of any findings, the individual animal
was examined by use of an extra „health status“ form sheet.
On day 29, approx. 600 - 1000 μl blood was taken from the retroorbital vein plexus of each
animal immediately before they were dispatched. In order to avoid coagulation, 90 μl of each
individual blood sample was mixed with 10 μl 10x HEPES-EDTA solution directly after
bleeding. 30 μl of the remaining blood was used to directly determine the blood clotting time
and potential, the rest of the blood was prepared as serum.

Sacrifice and pathology:

The in life phase was terminated on day 29. Animals were sacrificed by asphyxiation in a CO2 atmosphere. A full macroscopic examination was performed and all occurring lesions were noted. Individual animal data were recorded on checklists. The mass of the organsdenoted by „W“ in table 5 was recorded; tissues denoted by „P“ were preserved in the appropriate fixativesHistological preparation was performed of the organs and tissues denoted by „P“ in table 5. The selected tissues were embedded in paraffin wax, sectioned, and stained with hemalaum and eosin. The stained sections were transferred to the Principal Investigator 2 for histopathological examination. The results were returned in a pathology expert report to the hands of the Study Director.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

4.1.1 Viability, general clinical signs and behaviour
No abnormalities concerning general clinical signs or behaviour were observed.

4.1.3 Detailed clinical signs
In the course of this study, no signs of illness (e.g. changes in skin, secretions), autonomic
activity (e.g. lacrimation, piloerection), stereotypies or behavioural reactions (e.g. selfmutilation)
in response to the treatment or otherwise was observed.

Mortality:

no mortality observed

Description (incidence):

4.1.2 Fatalities
No animal of the test item groups or the vehicle groups died during the in-life phase of this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

4.1.4 Body mass
The body mass increase of the male and female animals from the vehicle control groups was within normal range for rats of this strain and age. Body mass gain of rats subjected to the test item was similar to that of their vehicle control groups (figure 1), and showed no significant alterations.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of all animals was comparable and within normal range for rats of this strain and age. No marked differences occurred between all animal groups.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

The water consumption of all animals was within normal range for rats of this strain and age. Water consumption of the male rats showed a mild tendency for a dose dependent decrease of water consumption with an increasing test item dosage.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of haematology and serum biochemistry showed normal results in the vast majority of parameters monitored. A mild but significant effect in male test animals occurred in the medium dose group, where kreatinin was lowered. Since this effect was not noted in the other dose groups of both sexes, this result is considered to be an artefact due to the relative small sample size. Blood clotting times were found to be significantly lowered in the high, medium and low dose group, but that finding was due to a relatively high mean blood clotting time in the control group, which most likely was an artefact caused by a relatively small sample size. In the female test animals, the few significant changes occurred in the high dose group exclusively. The most prominent alterations in comparison to their control group were a significantly lowered level of glutamic-oxaloacetic transaminase (GOT) and the mean corpuscular haemoglobin (MCH) of erythrocytes. Mildly but significantly lowered calcium and raised lymphocyte levels were also observed. Significance of the only remaining alteration (mean corpuscular volume, MCV) was most likely due to a statistical artefact caused by unusually low data dispersal. Few significant changes were observed and none of the observed average data points were overall extremely out of range for rats of this strain and age.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Other biochemical parameters and the blood cell counts showed no significant differences among the groups.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No abnormalities concerning general clinical signs or behaviour were observed.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At macroscopic examination, no pathological findings were noted for the test animals. In order to allow a comparison between organ mass, data were standardised against the body mass of the individual animals. The analysis showed normal results for most of the organs investigated. In comparison to their control group, the relative organ mass of right kidneys of the male test animals of the all three dose groups was slightly but significantly increased. The relative organ mass of both kidneys of the female test animals was mildly but significantly increased in the high dose group as well as in the low dose group, however, for the latter only the data of the left kidney reached the level of statistical significance. In addition, mild but significant changes of organ mass when compared to their control group occurred in the male high dose group, where the left adrenal gland was slightly reduced, and in the medium dose group of the females, where brain mass was slightly but significantly raised. Taking into consideration that no other groups showed this effect or even a supporting non-significant tendency, an artefact caused by a relatively small sample size was the most obvious explanation. Other than the observations above, no significant changes were observed and none of the observed average data points were overall extremely out of range for rats of this strain and age.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Inflammatory lesions occurred in various organs such as liver, lungs, trachea, kidney and colon in both control and/or test item treated animals. The inflammatory reaction was associated with minimal to mild lymphoid hyperplasia in the spleen, lymph nodes and the gut-associated lymphoid tissue in the intestine. A minimal reduction of lymphoid tissue (involution) was noted in the cortex and medulla of the thymus in the male and female rats of all groups. Coincidental findings in a small number of control and/or test item-treated animals are:
Adrenal: Vacuolisation of cortical cells
Cerebrum: Hydrocephalus;
Epididymides: Spermatic cyst;
Kidney: Basophilic tubular cells, cysts;
Larynx: Glandular ectasia;
Liver: Microgranuloma;
Lungs: Focal interstitial pneumonia, foamy macrophages;
Trachea: Tracheal gland dilatation;
Urinary bladder: Proteinaceous content in male rats, dilatation;
Uterus: Hydrometra.
The histomorphological examination of rat organs from the 28-day toxicity study by oral administration of animals treated with ADDUKT TI 65-MXDA did not reveal morphological lesions that were considered to be related to the test item. There was no morphological difference between the vehicle control group and the groups subjected to the high dose of the test item.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Grip strength and Limb placing test
Motor activity and reactivity to visual and proprioceptive stimuli were not altered by the administration of the test item. At the end of the observation period, no differences in grip strength were observed in relation to the vehicle control group, and none of the animals showed significant differences to the vehicle group during the limb placing test.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

haematopoietic

Any other information on results incl. tables

.

Applicant's summary and conclusion

Conclusions:

Detailed clinical signs, body mass, food and water consumption, motoric activity, and reactivity to sensory stimuli showed no abnormalities. The most noticeable among the very few observable effects in the animal groups treated with the test item were haematological changes in females of the high dose group. Compared to their vehicle control group, they showed significantly lowered levels of glutamic-oxaloacetic transaminase (GOT) and MCH. In addition, kidneys were slightly enlarged, but significance for both organs was only attained in female rats of the high dose group. A large number of medicinal and chemical agents with different chemical structures and/or therapeutic activities produce kidney enlargement when given in high doses to species used in toxicity studies. GOT often respond to kidney damage. Findings of this nature are in favour of an adaptive response rather than a direct toxic effect of the test item. These findings, not present in the control animals, are considered to be related to the treatment with the test item. Although some additional significant changes were observed, none of the average data points were overall extremely out of range for rats of this strain and age. The histomorphological examination of the selected rat organs did not reveal morphological lesions considered to be related to the test item. The inflammatory lesions in different organs are considered to be coincidental findings or spontaneous organ changes and are therefore not test item related. No morphological differences were noted between the groups. The involution of the thymus in some rats corresponded in type, incidence and severity to the age of the animals. All the findings recorded in this study are commonly encountered in rats of this strain and age. Type, incidence, and severity of the lesions recorded were not increased in the treated animals as compared to the control animals. A daily oral administration of the test item ADDUKT TI 65-MXDA to Wistar rats at a dose level of 63, 250 and 1000 mg/kg body mass over a time period of 28 days resulted in few and only mild systemic and local effects in test animals treated with the high dose (1000 mg /kg) but did not produce any pathological evidence of a local or systemic toxicity of the test item.

Executive summary:

Aim of the study:

The aim of this study was to assess data on the subchronic toxicity of the chemical

substance ADDUKT TI 65-MXDA, suspended in corn oil.

Experimental model:

The test item ADDUKT TI 65-MXDA was administered daily by oral gavage at dose levels of

63, 250 and 1000 mg/kg BM/day to 5 male and 5 female Wistar rats each over a period of 28

days. Another 5 male and 5 female rats received the same total volume of corn oil as vehicle

control. Of these solutions, 8 ml/kg BM were administered daily to each animal.

Assessed parameters:

During the in-life phase, viability, general and detailed clinical signs, food and water

consumption, body mass, grip strength and reactivity to sensory stimuli (limb placing test)

were recorded.

At the end of the in-life phase, blood samples from all animals were collected to provide data

on haematology and serum biochemistry. All animals were sacrificed immediately after

bleeding and examined by gross necropsy. Weights of selected organs (see Table 5) were

recorded, and tissues and organs were preserved. All samples were processed for

histopathological examination. The latter was conducted on samples from the high dose

groups and the vehicle groups exclusively.

Results:

Detailed clinical signs, body mass, food and water consumption, motoric activity, and

reactivity to sensory stimuli showed no abnormalities.

The most noticeable among the very few observable effects in the animal groups treated with

the test item were haematological changes in females of the high dose group. Compared to

their vehicle control group, they showed significantly lowered levels of glutamic-oxaloacetic

transaminase (GOT) and MCH. In addition, kidneys were slightly enlarged, but significance

for both organs was only attained in female rats of the high dose group. A large number of

medicinal and chemical agents with different chemical structures and/or therapeutic activities

produce kidney enlargement when given in high doses to species used in toxicity studies.

GOT often respond to kidney damage. Findings of this nature are in favour of an adaptive

response rather than a direct toxic effect of the test item.

These findings, not present in the control animals, are considered to be related to the

treatment with the test item. Although some additional significant changes were observed,

none of the average data points were overall extremely out of range for rats of this strain and

age.

The histomorphological examination of the selected rat organs did not reveal morphological

lesions considered to be related to the test item. The inflammatory lesions in different organs

are considered to be coincidental findings or spontaneous organ changes and are therefore

not test item related. No morphological differences were noted between the groups. The

involution of the thymus in some rats corresponded in type, incidence and severity to the age

of the animals. All the findings recorded in this study are commonly encountered in rats of

this strain and age. Type, incidence, and severity of the lesions recorded were not increased

in the treated animals as compared to the control animals.

Conclusion:

A daily oral administration of the test item ADDUKT TI 65-MXDA to Wistar rats at a dose

level of 63, 250 and 1000 mg/kg body mass over a time period of 28 days resulted in few and

only mild systemic and local effects in test animals treated with the high dose (1000 mg /kg)

but did not produce any pathological evidence of a local or systemic toxicity of the test item.