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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 04 - June 25 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: Combined repeated dose Toxicity study with the Reproduction/ developmental Toxicity screening Test. EPA 712-C-00-368, July 2000.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine and triethylenetetramine
EC Number:
273-601-0
EC Name:
Fatty acids, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine and triethylenetetramine
Cas Number:
68990-47-6
Molecular formula:
The substance is a UVCB substance. One of the most likely and the smallest molecule arising from the reaction process is assumed to be: C18H33O. C18H31O. 2C4H12N3 . C4H2O2
IUPAC Name:
Reaction product of 2,5-Furandione with reaction products of tall-oil fatty acids, diethylenetriamine, triethylenetetramine and tetraethylenepentamine
Details on test material:
Name: Fatty acids, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine and triethylenetetramine
CAS No.: 68990-47-6
Batch no.: TEE3316/22
Expiry Date: November 2011
Physical state at RT: solid
Colour: dark
Purity: Date of analysis 11 February 2010 97% (w/w)
Storage Conditions: at room temperature, protected from light
Solubility in Water: very low
Safety Precautions: Standard Safety procedures were sufficient to assure personnel health and safety.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Young healthy male and nulliparous, non-pregnant female rats [strain: Wistar Crl:WI] (Full-Barrier), were used in this study. The animals were derived from a controlled full barrier-maintained breeding system (SPF) (Source: Charles River, 97633 Sulzfeld, Germany).
According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
At the beginning of the study, the age of the animals was 8-9 weeks. The range of the body weight was:
Females: 154 to 183 g, (mean: 167.93 g, ±20% = 33.59 g)
Males: 213 to 233 g, (mean: 222.30 g, ±20% = 44.46 g)
Sex:
male/female
Details on test animals or test system and environmental conditions:
After an adequate acclimatisation period (at least five days) the animals were barrier maintained (full barrier) in air-conditioned rooms under the following conditions:
- Temperature: 22 ±3 °C
- Relative humidity: 55 ±10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice.
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed at the test facility
80 animals (10 females and 10 males /group) were included in the study.
The animals were randomly assigned (using Microsoft Excel template) to the dose/control groups, each animal was assigned a unique identification number and caged individually. The animals were acclimatised for at least five days before the first dose administration.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
Name: Corn oil
Justification for use and choice of vehicle (if other than water): The vehicle was chosen as suggested by sponsor and the test item‟s solubility.
Batch No.: 11KBC6753
Storage conditions: at room temperature (RT),
Safety precautions: Routine hygienic procedures were sufficient to assure personnel health and safety
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of the concentration measurements revealed recoveries between 87.5% and 109.8% in HD group, between 89.2% and 107.9% in MD group and between 95.6% and 109.8% in LD group. Homogeneous distribution of the test substance in the preparation was approved.
Duration of treatment / exposure:
The animals will be dosed with the test item on 7 days per week for a period of approximately 54 days. The test substance will be administered daily during 14 days pre mating and 14 days mating in both male and in female, during gestation period and up to post natal day 3 in females. Males will be dosed after the mating period until the minimum total dosing period of 28 days has been completed.
Frequency of treatment:
daily; The animals were dosed with the test item on 7 days per week basis.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
80 animals (10 non pregnant nulliparous females and 10 adult males /group) were included in the study.
Control animals:
yes, concurrent vehicle
Positive control:
none

Examinations

Observations and examinations performed and frequency:
Animals were examined for the daily clinical signs, mortality, weekly detailed clinical observations, pre-treatment and at termination for functional observations. Body weight and food consumption was measured weekly except food consumption was not measured during the mating period (female) and mating/post mating period (male). Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.
Sacrifice and pathology:
Males and females were sacrificed on treatment day 29 - 30, post natal day 4 respectively and subjected to necropsy. Non-Pregnant females were sacrificed on their respective day 26 after the evidence of mating.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
due to gavaging error
Mortality:
mortality observed, treatment-related
Description (incidence):
due to gavaging error
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In males, the body weight and body weight changes remain unaffected during the treatment period in all treatment groups when compared with the controls.
In females, no treatment related effect was observed for body weight and body weight changes in any of the treatment groups when compared with the controls.
However, statistically significant reduction in weight development was observed in MD group during premation days 7-14 when compared with controls. But, this cannot be attributed to treatment due to lack of dose response pattern.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In both sexes (males and females), the food consumption statistically remained unaffected during the treatment period when compared to corresponding controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Statistically, no significant deviation was recorded in any of the hematological parameters between the treatment groups and the corresponding control values. However, some individual values were below or above the historical control range and were considered incidental.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Statistically no significant deviation was observed for the clinical biochemistry parameters evaluated. The individual and mean values were within the range except for CHOL, TP, ALBB and K. The deviation from the historical control range were considered incidental.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urinalysis revealed no test item related effect in any of the treatment groups compared to corresponding control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No relevant differences were observed concerning functional and behavioural examination in males and females.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In males, statistically no significant differences in the absolute and relative organ weights of the treatment groups was observed when compared with the controls.
In females, except for relative brain weight (LD and HD groups), statistically no significant differences was observed for any of the organs (absolute and relative organ weights) of the treatment groups when compared with the controls. The findings of brain relative weight in LD and HD group may not be considered treatment related due lack of dose response and consistent findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic findings observed in control and treatment groups were as follows:
Control: Males: animal no. 44 = small extra lobe of liver; animal no. 47 = lung covered with white spots; animal no. 49 = yellow spots on left epididymides; animal no. 50 = bloody axillary lymph node.
Females-animal no. 1 = discoloured lung with white spots; animal no. 10 = liver with extra little yellowish part attached to it.
Low Dose: Males: animal no. 53 = discoloured lung with white spots; animals no. 54 = lung with white spots; animal no. 59 = white spots on epididymides (caput), discoloured jejunum and ileum, discoloured lung with greyish and hard left lobe.
Females: animal no. 15 = discoloured lung; animal no.18 =cyst on right ovary, discoloured (greyish white) and hard lung lobes; animal no. 19 = discoloured (greyish) and hard lung.
Mid dose: Males: animal no. 61 = bloody lung with test item, animal no. 66 = discoloured lung with greyish and hard lobes; animal no. 69 = yellowish spots on epididymides.
Females: animal no. 22 = discoloured (greyish) and hard lung; animal no. 23 = bloody lung with residues of test item, animal found dead prematurely; animal no. 26 = discoloured lung (dark), focus greyish white and hard; animal no. 27 = enlarged adrenals
High dose: Males: animal no. 71 = epididymides with yellowish spots, discoloured lung with greyish and hard left lobe; animal no. 72 = liver with extra lobe; animal no. 73 = discoloured (dark) and hard lung; animal no. 77 = discoloured (dark) and hard lung; animal 78 = epididymides with yellow spots.
Females: animal no. 31= discoloured (grey) and hard lung; animal no. 33= hard and discoloured (dark) lung, focus greyish white; animal no. 36 = hard and discoloured lung with greyish white focus.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the female and male reproductive organs, no histopathological lesions considered to be test item-related were noted. In most surviving females, the reproductive organs showed physiological postpartum morphology. The number of large corpora lutea in the ovaries was similar in all study groups.
There was one non-pregnant female each in control, LD and MD groups at terminal sacrifice. The reproductive organ histomorphology of these animals indicate normal sexual cycling activity.
In one male of MD group, a minimal number of unilateral atrophic tubules in the testis were observed. In the epididymis, spermatic granuloma was observed in few treated males and in one control. There were also infiltrates with inflammatory cells in the prostate gland. These findings were considered incidental.
Test item-related histopathological lung changes like multifocal subacute bronchopneumonia, characterized by peribronchial foci of prominent fibrosis, with re-epithelialization, infiltration with mononuclear cells, histiocytes and occasional multinucleated cells was observed in a small proportion of treated males and females of all dose groups, without dose relationship. In some animals, the lesions contained foci of necrosis and in some the pulmonary changes were not well organized and contained larger numbers of mixed cells. A mild amount of intrahistiocytic black material was seen in the lung of individual male of LD and HD groups.
All other histopathological changes seen in this study were considered incidental in origin.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
<= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Based on the limited histopathological evaluation of the two decedents treated at 600 mg/kg/day, no cause of death could be determined for female animal No. 23. Histopathological lung findings in male No. 61 (intraalveolar eosinophilic material, perivascular edema/hemorrhage and diffuse congestion) suggest gavaging error as its cause of death.

Reproductive organs:

In the female and male reproductive organs, no histopathological lesions considered to be test item-related were noted. Histologically, female reproductive organs showed physiological postpartum morphology in most surviving animals. The number of large corpora lutea in the ovaries was essentially similar in all study groups. Based on clinical observations, each one female treated at 0, 300 or 600 mg/kg/day was found not to be pregnant at terminal sacrifice, and reproductive organ histomorphology of these animals indicate normal sexual cycling activity.

In the male reproductive organs, a minimal number of unilateral atrophic tubules in the testis were seen in one male treated at 600 mg/kg/day without dose relationship and were considered to be incidental. In the epididymis, spermatic granuloma was observed in a small number of treated males and in one control, corroborating the macroscopic finding of spots on the epididymis, and was considered a spontaneous lesion. There were also incidental infiltrates with inflammatory cells in the prostate gland.

Other organs:

In the other organs evaluated in this study, test item-related histopathological changes were seen in the lung. Multifocal subacute bronchopneumonia, characterized by peribronchial foci of prominent fibrosis, with re-epithelialization, infiltration with mononuclear cells, histiocytes and occasional multinucleated cells, was observed in a small proportion of treated males and females of all dose groups, without dose relationship. In some animals the lesions contained also foci of necrosis, and in some animals the pulmonary changes were less well organized and contained larger numbers of mixed cells. The mechanism of this lesion is not clear.

A mild amount of intrahistiocytic black material was seen in the lung of each one male treated at 300 or 1000 mg/kg/day.

All other histopathological changes seen in this study were considered to be incidental in origin and/or within the range of expected changes for rats of this age and strain kept under laboratory conditions.

Applicant's summary and conclusion

Conclusions:
Test item-related histopathological changes were restricted to the lung. Multifocal subacute bronchopneumonia, characterized by peribronchial foci of prominent fibrosis, with re-epithelialization, infiltration with mononuclear cells, histiocytes and occasional multinucleated cells, was observed in a small proportion of treated males and females of all dose groups, without dose relationship. In addition, a mild amount of intrahistiocytic black material was seen in the lung of each one male treated at 300 or 1000 mg/kg/day.
As a conclusion, based on the pathological evaluation, a No-Observed-Effect-Level (NOEL) could not be determined in this study.
Executive summary:

Macroscopic observations recorded at necropsy and histological slides were evaluated from male and female Wistar rats of a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with daily oral administration by gavage of Fatty acids, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine and triethylenetetramine (test item) at dose levels of 0 (vehicle control), 300, 600 or 1000 mg/kg/day. The dosing period was daily during 14 days pre-mating, during the mating period, then during the gestation period and up to post-natal day 3 in females. Dosing of the males was continued after the mating period until completion of a minimum total dosing period of 28 days. Two rats treated at 600 mg/kg/day were found dead during the early treatment period. Based on the limited pathological evaluation of these decedents, no cause of death could be determined for the female, and gavaging error was considered to be the probable cause of death for the male. In the female and male reproductive organs, no histopathological lesions considered to be test item-related were noted. Based on clinical observations, three females had been recorded not to be pregnant during the study and showed normal sexual cycling activity in histopathology. Test item-related histopathological changes were restricted to the lung. Multifocal subacute bronchopneumonia, characterized by peribronchial foci of prominent fibrosis, with reepithelialization, infiltration with mononuclear cells, histiocytes and occasional multinucleated cells, was observed in a small proportion of treated males and females of all dose groups, without dose relationship. In addition, a mild amount of intrahistiocytic black material was seen in the lung of each one male treated at 300 or 1000 mg/kg/day. As a conclusion, based on the pathological evaluation, a No-Observed-Effect-Level (NOEL) could not be determined in this study.