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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2012 to 07 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, July 2000.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear slightly yellow very viscous liquid
Details on test material:
- Substance type: Clear slightly yellow very viscous liquid
- Purity: 100% Mono constituent substance
- Storage condition of test material: In refrigerator (2-8°C) in the dark

Test animals

Species:
rat
Strain:
other: Wistar (Han)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 169 grams; females: 135 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 09 November 2012 to 07 December 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity/density of the test substance (1.220, average of range 1.215-1.225 g/cm3) and specific gravity of the vehicle. No correction was made for the purity of the test substance.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe and on information provided by the sponsor.

DOSE VOLUME:
5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method (project 501424). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, 450 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a dose range finding study with m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline (project 501433). Conclusion dose range finding study Project 501433: Based on the results of this range finding study, dose levels for the main study were 50, 150 and 450 mg/kg body weight. No clear peak effect of occurrence of clinical signs was observed in the range finding study. Therefore, clinical observations in the main study were conducted immediately after dosing, and functional observation tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
Positive control:
Not required.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately (0-15 minutes) after dosing (as no peak effect of clinical signs occurred in the range finding study). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION :
- Time schedule for examinations: Weekly.

WATER CONSUMPTION AND COMPOUND INTAKE: no

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations:
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.
Sacrifice and pathology:
GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Based on subjective appraisal.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY AND CLINICAL SIGNS
No mortality occurred during the study period. At 450 mg/kg, lethargy, hunched posture, piloerection, pale faeces, lean appearance and ptosis were noted among all animals during the study period. At 150 mg/kg hunched posture (with or without lethargy) was observed in all animals on two treatment days. Salivation seen after dosing among animals of the 50, 150 and 450 mg/kg dose group during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Other findings that were noted in these animals consisted of rales, chromodacryorrhoea and scabs on the shoulder. These findings were observed in individual animals only and were considered to be within the normal range of biological variation for rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN
All animals at 450 mg/kg showed lower body weight gain compared to control animals throughout the treatment period. Slight body weight loss (up to -4%) was noted in three males at 450 mg/kg during the first week, which slightly recovered thereafter. Body weight loss (up to -10%) was also noted in four females at 450 mg/kg in Week 4 when compared to Week 3. Mean body weights at the end of the treatment period were approximately 40 and 20% lower than controls in males and females respectively. No treatment-related findings on body weights and body weight gain were noted at 50 and 150 mg/kg. The slight lower body weight gain observed in males treated at 150 mg/kg (achieving a level of statistical significance for weight gain) was slight and within the normal range of variation. Therefore this finding was considered to be of no toxicological relevance.

FOOD CONSUMPTION
At 450 mg/kg, food consumption before or after correction for body weight was lower than controls in males and females throughout the treatment period. No toxicologically significant changes in food consumption before or after correction for body weight were noted at 50 and 150 mg/kg.

HAEMATOLOGY
Treatment with 50 mg/kg resulted in the following (statistically significant) changes in haematology parameters, compared to controls:
- Higher relative neutrophil count in females
Treatment with 150 mg/kg resulted in the following (statistically significant) changes in haematology parameters, compared to controls:
- Higher relative neutrophil count in males and females
- Lower relative lymphocyte count in males and in females (not statistically significant for females)
Treatment with 450 mg/kg resulted in the following (statistically significant) changes in haematology parameters, compared to controls:
- Higher relative neutrophil count in males and females
- Lower relative lymphocyte count in males and females (not statistically significant for females)
- Higher relative monocyte count in females
- Lower red blood cell count in males and females
- Higher reticulocyte count in males and females
- Higher red blood cell distribution width (RDW) in males
- Lower haemoglobin level in males and females
- Lower haematocrit in males and females (not statistically significant for females)
- Lower mean corpuscular haemoglobin concentration (MCHC) in males (not statistically significant) and females
- Higher platelet count in males and females
- Shortened activated partial thromboplastin time (APTT) in males (not statistically significant) and females
No treatment related changes in haematology parameters were noted in males treated at 50 mg/kg.

CLINICAL CHEMISTRY
Treatment with 150 mg/kg resulted in the following (statistically significant) changes in clinical biochemistry parameters, compared to controls:
- Higher bilirubin level in males (not statistically significant) and females
- Higher cholesterol level in females
- Higher bile acid level in females (not statistically significant)
- Higher potassium level in males and females
Treatment with 450 mg/kg resulted in the following (statistically significant) changes in clinical biochemistry parameters, compared to controls:
- Lower albumin level in males and females
- Higher bilirubin level in males and females
- Lower creatinine level in males and females
- Lower glucose level in males and females (not statistically significant for females)
- Higher cholesterol level in males and females
- Higher bile acid level in males and females (not statistically significant for females)
- Lower sodium level in males
- Lower chloride level in males and females
- Higher potassium level in females (not statistically significant)
No treatment related changes in clinical biochemistry parameters were noted in animals treated at 50 mg/kg.
All other (statistically significant) changes in clinical biochemistry parameters between treated and control animals were considered to be of no toxicological significance as these changes occurred in the absence of a treatment-related distribution and/or were within the normal range of variation.

NEUROBEHAVIOUR
A lower motor activity was recorded for males and females at 450 mg/kg, as shown by a statistically significant lower number of total movements and ambulations (ambulation counts not statistically significant in males). This difference appeared essentially confined to the habituation phase (i.e. the first 10 minutes of the measurement). The other groups showed a normal motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

ORGAN WEIGHTS
Treatment with 50 mg/kg resulted in the following organ weight changes, when compared to controls:
- Lower prostate weights and a trend towards lower seminal vesicle weights.
Treatment with 150 mg/kg resulted in the following organ weight changes, when compared to controls:
- Lower prostate weights, lower prostate to body weight ratio, lower seminal vesicle weights and a trend towards lower seminal vesicle weights to body weight ratio.
- Lower uterus weight, lower uterus to body weight ratio (not statistically significant)
Treatment with 450 mg/kg resulted in the following changes in organ weight which were considered to be related to treatment:
- Lower terminal body weight in males and females.
- Higher liver weights in females and higher liver to body weight ratio in males and females.
- Higher adrenal gland weights and higher adrenal gland to body weight ratio in males and females (not statistically significant in females regarding the ratio)
- Lower thymus weight and thymus to body weight ratio in males and females
- Lower prostate weight and prostate to body weight ratio, seminal vesicles weight and seminal vesicles weight to body weight ratio.
- Lower uterus weight and uterus to body weight ratio.
Higher liver and kidney to body weight ratios were noted in males at 150 mg/kg. Lower thymus weight and thymus to body weight ratio was observed in females at 150 mg/kg. In the lack of any relevant corroborative findings at the microscopic examinations, these organ weight changes were considered to be without toxicological significance. The higher brain, heart, thyroid, kidney, testes and epididymides weight at 450 mg/kg was considered to be related to the lower terminal body weight and occurred in the absence of microscopic correlates. In addition, lower spleen weight in both sexes occurred without any corroborative findings. Therefore, these findings were considered not to be toxicologically relevant.

GROSS PATHOLOGY
Treatment with 50 mg/kg resulted in the following macroscopic findings:
- Pancreatic lymph node, discolouration reddish was noted in 1/5 female.
Treatment with 150 mg/kg resulted in the following macroscopic findings
- Stomach, forestomach irregular surface was noted in 5/5 male and 3/5 female.
- Stomach, forestomach focus/foci, yellowish/tan, reddish/dark red was noted in 1/5 male.
- Duodenum, wall thickened/dilation was noted in 3/5 male and 3/5 female.
- Duodenum, contents gelatinous was noted in 3/5 male and 2/5 female.
- Jejunum, wall thickened/dilation was noted in 2/5 male and 2/5 female.
- Jejunum, contents gelatinous was noted in 2/5 male and 1/5 female. There was no microscopic correlate to this finding.
- Mesenteric lymph node, discolouration reddish/dark red/purple was noted in 4/5 male and 2/5 female.
- Pancreatic lymph node, enlarged was noted in 2/5 male and 1/5 female.
- Pancreatic lymph node, discolouration reddish was noted in 1/5 male and 2/5 female.
- Uterus, reduced in size was noted in 1/5 female.
Treatment with 450 mg/kg resulted in the following macroscopic findings
- Emaciated was noted in 4/5 male and 5/5 female.
- Thymus, reduced in size was noted in 5/5 male and 4/5 female.
- Stomach, forestomach irregular surface was noted in 4/5 male and 5/5 female.
- Stomach, forestomach focus/foci, yellowish/tan, reddish/dark red was noted in 3/5 female.
- Duodenum, wall thickened/dilation was noted in 4/5 male and 4/5 female.
- Duodenum, contents gelatinous was noted in 3/5 male and 1/5 female.
- Jejunum, wall thickened/dilation was noted in 4/5 male and 4/5 female.
- Jejunum, contents gelatinous was noted in 4/5 male and 2/5 female. There was no microscopic correlate to this finding.
- Mesenteric lymph node, discolouration reddish/dark red/purple was noted in 5/5 male and 4/5 female.
- Pancreatic lymph node, enlarged was noted in 3/5 male and 4/5 female.
- Renal lymph node, enlarged was noted in 1/5 male and 1/5 female.
- Adrenal glands, discolouration pale was noted in 4/5 male and 4/5 female.
- Kidneys, discolouration greenish was noted in 5/5 male and 5/5 female.
- Uterus, reduced in size was noted in 5/5 female.
- Ovaries, reduced in size was noted in 3/5 female.
- Prostate gland, reduced in size was noted in 5/5 male.
- Seminal vesicles, reduced in size was noted in 5/5 male.
All other macroscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.

HISTOPATHOLOGY
Treatment resulted in the following microscopic findings in males or females starting at 50 mg/kg:
- Duodenum, hemorrhage was present in 4/5 male (4 minimal) and 3/5 female (3 minimal) rats at 50 mg/kg, in 3/5 male (3 minimal) and 4/5 female (1 minimal, 3 slight) rats at 150 mg/kg and in 5/5 male (2 minimal, 3 slight) and 4/5 female (2 minimal, 2 slight) rats treated at 450 mg/kg.
- Duodenum, hypertrophy with villous stunting was present in 5/5 male (4 minimal, 1 slight) and 5/5 female (2 minimal, 3 slight) rats treated at 50 mg/kg, in 5/5 male (5 slight) and 5/5 female (1 minimal, 3 slight, 1 moderate) rats treated at 150 mg/kg and in 5/5 male (1 slight, 4 moderate) and 5/5 female (4 slight, 1 moderate) rats treated at 450 mg/kg. This finding corresponded with the macroscopic finding wall thickened.
- Peyer’s patches, lymphoid atrophy was present in 1/5 female (1 slight) treated at 50 mg/kg, in 1/5 male (1 slight) and 1/5 female (1 moderate) rats treated at 150 and in 3/5 male (2 minimal, 1 slight) and 4/5 female (1 minimal, 3 slight) rats at 450 mg/kg.
- Mesenteric lymph node, erythrophagocytosis was present in 1/5 male (slight) and 1/5 female (minimal) rats treated at 50 mg/kg, in 5/5 male (3 slight, 2 moderate) and 3/5 female (3 slight) rats at 150 mg/kg and in 5/5 male (5 moderate) and 5/5 female (3 slight, 2 moderate) rats at 450 mg/kg. This finding correlated with the macroscopic finding discolouration reddish/dark red/purple.
Treatment resulted in the following microscopic findings in males or females starting at 150 mg/kg:
- Stomach, ulcer forestomach was present in 2/5 male rat (1 slight, 1 moderate) treated at 150 mg/kg and in 5/5 male (1 moderate, 4 marked) and 5/5 female (5 marked) rats at 450 mg/kg. This finding corresponded with the macroscopic finding forestomach discoloured focus/foci.
- Stomach, hyperplasia forestomach was present at increased incidence and severity in 5/5 male (3 slight, 2 moderate) and 5/5 female (3 minimal, 1 slight, 1 moderate) rats treated at 150 mg/kg and in 5/5 male (2 slight, 3 moderate) and 5/5 female (4 moderate, 1 marked) rats at 450 mg/kg. This finding was present at a background incidence and severity in 1/5 male (minimal) 50 mg/kg treated. This finding corresponded with the macroscopic finding forestomach irregular surface.
- Jejunum, hypertrophy with villous stunting was present in 2/5 male (1 minimal, 1 slight) and 1/5 female (1 minimal) rats at 150 mg/kg and in 5/5 male (3 slight, 2 moderate) and 5/5 female (1 minimal, 4 slight) rats at 450 mg/kg. This finding corresponded with the macroscopic finding wall thickened.
- Mesenteric lymph node, lymphoid atrophy was present in 3/5 male (3 minimal) and 2/5 female (2 minimal) rats treated at 150 mg/kg and in 5/5 male (1 minimal, 2 slight, 2 moderate) and 4/5 female (2 minimal, 2 slight) rats at 450 mg/kg.
- Mesenteric lymph node, increased mast cells were present in 5/5 male (3 slight, 2 moderate) and 3/5 female (3 slight) rats treated at 150 mg/kg and in 1/5 male (1 slight) and 2/5 female (2 slight) rats at 450 mg/kg.
- Uterus, cervix and vagina, atrophy was present in 2/5 female rats treated at 150 and 5/5 female rats at 450 mg/kg.
- Liver, centrilobular hypertrophy was present in 1/5 female (minimal) at 150 mg/kg and in 5/5 female (1 minimal, 4 slight) rats.
Treatment resulted in the following microscopic findings in males or females at 450 mg/kg:
- Thymus, lymphoid atrophy was present in 5/5 male ( 1 minimal, 3 slight, 1 marked) and 5/5 female (1 minimal, 2 slight, 2 moderate) rats. This finding corresponded with the macroscopic finding reduced in size.
- Jejunum, hemorrhage was present in 3/5 male (2 minimal, 1 slight) and 3/5 female (3 slight) rats.
- Spleen, lymphoid atrophy was present in 5/5 male (1 minimal, 4 slight) and 1/5 female (minimal) rats.
- Adrenal glands, vacuolation zona fasciculata was present at increased incidence and severity in 4/5 male (2 minimal, 2 slight) and 4/5 female (1 minimal, 3 slight) rats. This finding was present at a background incidence and severity in 1/5 male (minimal) at 50 mg/kg and 1/5 male (minimal) at 150 mg/kg. This finding corresponded with the macroscopic finding discoloured.
- Femur including joint, decreased bone, secondary spongiosa was present in 5/5 male (2 slight, 3 moderate) and 5/5 female (1 minimal, 3 slight, 1 moderate) rats. This was likely related to the emaciation.
- Prostate gland, small with normal histology was present in 5/5 male rats. This finding corresponded with the macroscopic finding reduced in size.
- Seminal vesicles, reduced contents was bilaterally present in 5/5 male (5 slight) rats and, as background finding, it was unilaterally present in 1/5 (moderate) control Group rat. This finding corresponded with the macroscopic finding reduced in size.
- Coagulating glands, reduced contents was present in 5/5 male (5 slight) rats.
In addition, there were microscopic findings in organs that were only sampled in animals which showed macroscopic findings at 50, 150 and 450 mg/kg:
- Pancreatic lymph node, erythrophagocytosis was present in 1/5 female at 50 mg/kg (1 lymph node sampled), in 2/5 female and 1/5 male at 150 mg/kg (3 and 2 lymph nodes sampled respectively) treated rats. This correlated with the macroscopic finding discolouration reddish.
- Pancreatic lymph node, lymphoid hyperplasia was present 2/5 male and 3/5 female at 150 mg/kg (2 and 3 lymph nodes sampled respectively) and in 3/5 male and 4/5 female at 450 mg/kg (3 and 4 lymph nodes sampled respectively) treated rats. This correlated with the macroscopic finding enlarged.
- Renal lymph node, lymphoid hyperplasia was present 1/5 male and 1/5 female at 450 mg/kg (both 1 lymph node sampled) treated rats. This correlated with the macroscopic finding enlarged. All other microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analysis of dose preparations:

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Applicant's summary and conclusion

Conclusions:
In a 28-day oral repeated dose toxicity study with rats, the systemic NOAEL was determined to be 50 mg/kg bw/day based on atrophy of the female reproductive organs (uterus, cervix and vagina) observed at 150 mg/kg/day. Based on the local effects in the gastrointestinal tract (duodenum, Peyer’s patches and lymph nodes), likely to be secondary effects due to the irritation potential of the test substance, starting at the lowest dose level of 50 mg/kg/day, a local No Observed Adverse Effect Level (NOAEL) could not be established in this study.
Executive summary:

The Repeated dose 28-day oral toxicity study with m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

by daily gavage in the rat according to theOECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.

Based on the results of a 5- to10-day range finding study (Project 501433), the dose levels for this 28-day oral gavage study were selected to be 0, 50, 150 and 450 mg/kg.

The test substance, formulated in propylene glycol, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

Evaluated parameters

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

RESULTS

Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 6 hours. No mortality occurred in the study.

At 50 mg/kg, treatment resulted in changes in the gastro-intestinal tract and changes in organ weights of the male reproductive system.

The local gastro-intestinal tract findings were noted consisting of hemorrhage and hypertrophy with villous stunting in the duodenum in males and females. In addition, lymphoid atrophy in the Peyer’s patches and erythrophagocytosis and/or discolouration in the mesenteric and pancreatic lymph nodes was noted.

At 150 mg/kg, treatment resulted in clinical signs, changes in the gastro-intestinal tract, changes in blood parameters, changes in the reproductive organs of females, changes in organ weights of the male reproductive system and slight changes in the adrenal glands.

During the in-life phase, hunched posture (with or without lethargy) was observed in all animals on two treatment days.

Local effects in the gastro-intestinal tract consisted of hemorrhage in the duodenum, hypertrophy with villous stunting, gelatinous content and thickened wall in the duodenum and in the jejunum. In the stomach effects included ulcer, hyperplasia, foci and irregular surface of the forestomach. In addition, lymphoid atrophy in the Peyer’s patches, increased mast cells in the mesenteric lymph node, lymphoid hyperplasia of the pancreatic lymph node and erythrophagocytosis and/or discolouration in the mesenteric and pancreatic lymph nodes were noted. Centrilobular hypertrophy in the liver was noted in one female, although not correlating to higher liver weight or effects at the macroscopic examinations. Changes in the female reproductive organs consisted of atrophy of the uterus (with lower uterus weight), cervix and vagina. Effects on blood parameters included higher bilirubin, potassium, cholesterol and bile acid levels in males and/or females.

At 450 mg/kg, treatment resulted in poor health status of the animals during the study period, in changes in the gastro-intestinal tract, changes in blood parameters, changes in the male and female reproductive organs and in changes in liver and renal system.

Reduced body weight gain and food consumption, reduced motor activity and several symptoms indicative of ill health including lethargy, hunched posture, piloerection, pale faeces, lean appearance and ptosis were noted among all animals. All animals survived the scheduled study duration. Local effects in the gastro-intestinal tract consisted of hemorrhage and hypertrophy with villous stunting, gelatinous content and thickened wall in the duodenum and in the jejunum. In the stomach effects included ulcer, hyperplasia, foci and irregular surface of the forestomach. In addition, lymphoid atrophy in the Peyer’s patches, increased mast cells in the mesenteric lymph node, lymphoid hyperplasia of the pancreatic lymph node and erythrophagocytosis and/or discolouration in the mesenteric and pancreatic lymph nodes were noted.

Changes in the reproductive organs consisted of atrophy in the uterus, cervix and vagina (macroscopically seen as reduced size and/or weight of ovaries and uterus), a small prostate gland (with normal histology), and reduced contents of the seminal vesicles and coagulating glands were noted. Macroscopically, a reduced size and weight of the prostate and seminal vesicles was observed.

Other microscopic findings consisted of centrilobular hypertrophy in the liver (correlating to higher liver weight), discoloration of kidney, vacuolation of the zona fasciculata of the adrenal glands (with macroscopic discolouration, enlargement and increase in weight), and lymphoid hyperplasia of the renal lymph nodes (macroscopically supported by enlargement).

Effects on blood parameters consisted of changes in red blood cell parameters, platelet counts and activated partial thromboplastin time, and higher bilirubin, cholesterol, potassium and bile acid levels, and lower albumin, creatinine, glucose, sodium and chloride levels in one or both sexes.

The lower prostate and seminal vesicle weight observed at 50 and 150 mg/kg was slight in nature and had no microscopic correlate. As such they were considered not adverse at these dose levels.

The shift in type of white blood cells (i.e. higher neutrophil counts with lower lymphocyte counts observed in males at 150 and 450 mg/kg and females at 50, 150 and 450 mg/kg) was considered to be related to the lymphoid atrophy in the thymus and spleen as recorded at 450 mg/kg. This lymphoid atrophy was considered to be a secondary non-specific response to stress associated with treatment. Therefore, the change in white blood cells may be considered not to be of primary toxicological significance.

CONCLUSION

Based on the local effects in the gastrointestinal tract (duodenum, Peyer’s patches and lymph nodes), likely to be secondary effects due to the irritation potential of the test substance, starting at the lowest dose level of 50 mg/kg/day, a local No Observed Adverse Effect Level (NOAEL) could not be established in this study.

The systemic NOAEL is considered to be 50 mg/kg/day, essentially based on atrophy of the female reproductive organs (uterus, cervix and vagina) observed at 150 mg/kg/day.