Reaction mass of 2-ethyl-2-[[3-(2-methylaziridin-1-yl)propionyl]methyl]propane-1,3-diyl bis(2-methylaziridine-1-propionate) and 2,2-bis({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butyl 3-[2,2-bis({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butoxy]propanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 April 2012 - 03 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study has been performed according to OECD guidelines and GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

03 October 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

No. L142, May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

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 (incl. QA statement)

Remarks:

issued 19 May 2011

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-({3-[2,2-bis({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butoxy]-3-oxopropoxy}methyl)-2-({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butyl 3-(2-methylaziridin-1-yl)propanoate; 2-ethyl-6-(2-methylaziridin-1-yl)-2-({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)-4-oxohexyl 3-(2-methylaziridin-1-yl)propanoate

EC Number:

939-180-9

Molecular formula:

Not relevant - Multiconstituent substance

IUPAC Name:

2-({3-[2,2-bis({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butoxy]-3-oxopropoxy}methyl)-2-({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)butyl 3-(2-methylaziridin-1-yl)propanoate; 2-ethyl-6-(2-methylaziridin-1-yl)-2-({[3-(2-methylaziridin-1-yl)propanoyl]oxy}methyl)-4-oxohexyl 3-(2-methylaziridin-1-yl)propanoate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Crosslinker CX-100
- Substance type: Slightly viscous clear yellowish liquid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation:
- Fasting period before study: No
- Housing: Group housing of 5 animals per sex in Macrolon cages. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): appr. 15
- Photoperiod (hrs dark / hrs light): 12/12
Temporary deviations from the daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

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 density of the test substance. No correction was made for the purity of the test substance.
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg 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 treatment phase, according to a validated method (project 498882). 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 was also determined (highest and lowest concentration).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg/ 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 selection was based on Dose Range Finding Study, that was conducted before start of Main study. Three females per group were exposed to 500 or 1000 mg/ kg bw/ day for 5 days. No mortality occurred in any of the groups. At 500 mg/kg bw/ day, salivation was noted on one day. At 1000 mg/kg bw/ day, salivation and labored respiration on one day only and/or at a very low incidence were noted. This was considered to be background finding. No unexpected changes in body weight gain occurred, food consumption was normal and no abnormalities were noted at macroscopic examination for both groups. Liver and kidney weights were considered to be normal for all animals.
Based on the results of this range finding study, dose levels for the Main study were chosen to be 100, 300 and 1000 mg/kg body weight.
No clinical signs (indicative of toxicity) were observed. Therefore, clinical observations in the main study were conducted immediately after dosing.
- Positive control: No, not required.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE:
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (maximum 20 hours)
- How many animals: all
- Parameters as described in the test guideline were meassured.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just before necropsy
- Animals fasted: Yes (maximum 20 hours)
- How many animals: all
- Parameters as described in the test guideline were meassured.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all
- Battery of functions tested: Hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
- Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, according to test guideline (including organ weights)
HISTOPATHOLOGY: Yes, according to test guideline

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 when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 4) to determine intergroup differences followed by the Wilcoxon test 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. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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:

CLINICAL SIGNS AND MORTALITY
All animals at 1000 mg/kg were sacrificed for ethical reasons or were found death between Day 7 and 18. Three males at 300 mg/kg were sacrificed for ethical reasons on Day 25. No mortality occurred in controls and animals treated at 100 mg/kg.
Among all animals at 300 and 1000 mg/kg the following clinical signs were noted during the observation period: lethargy, hunched posture, abnormal gait, uncoordinated movements, rales, laboured respiration, piloerection, lean appearance, swelling of abdomen, chromodacryorrhoea, and/or diarrhea.
Salivation is noted in all test substance treated groups from Day 4 and onwards (most probably related to taste of the test substance). One female at 100 mg/kg showed hunched posture and rales on Day18 only. One control male showed a wound on Day 14 and scabs during the observation period thereafter.

BODY WEIGHT AND WEIGHT GAIN
Lower body weight gain and/or body weight loss was noted in animals at 300 and 1000 mg/kg. Body weights and body weight gain at 100 mg/kg was considered to be within the range considered normal for rats of this age and strain.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption before or after allowance for body weight was lower in males at 300 and 1000 mg/kg and females at 1000 mg/kg. No toxicologically significant changes in food consumption before or after correction for body weight were noted in animals at 100 mg/kg and females at 300 mg/kg.

HAEMATOLOGY
The following (statistically significant) changes in haematology parameters distinguished treated females from control animals:
- Lower red blood cell counts at 300 mg/kg and slightly lower at 100 mg/kg
- Higher reticulocytes at 300 mg/kg
- Higher red blood cell distribution width at 300 mg/kg
- Lower haemaglobin and heamatocrit levels at 300 mg/kg
- Higher mean corpuscular volume at 300 mg/kg and slightly lower at 100 mg/kg
- Lower activated partial thromboplastin time at 300 mg/kg
Other observed changes were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
The following statistically significant changes in clinical biochemistry parameters distinguished treated animals at 300 mg/kg from control animals:
- Higher total bilirubin in males
- Higher urea level in males and females
- Higher bile acid level in males (not statistically significant in females)
- Higher inorganic phosphate in females.
No changes were noted in animals at 100 mg/kg.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. Motor activity was similar between treated animals at 100 mg/kg and control groups. Lower motor activity was noted in the males and females at 300 mg/kg although not statistically significant. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
The following changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment:
- Lower thymus weight (absolute and relative) in males and females at 300 mg/kg
- Higher relative kidney weight in males and females at 300 mg/kg
- Slightly higher liver weight in females at 300 mg/kg
Changes in brain, heart, adrenals, seminal vesicles and testes were considered to be affected due to lower terminal body weight compared to control animals.
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations at 100 and 300 mg/kg in the surviving animals. The animals at 300 and 1000 mg/kg, which did not survive up to the end of the study period showed the following incidental macroscopic findings: beginning of autolyse, gastrointestinal tract and body cavities distended with gas, emaciated appearance, hardened heart, esophagus containing fluid, discolouration of blood vessels, enlarged lungs, foci in the stomach and/or caecum, discolouration of the stomach, thymus and/or mandibular lymph nodes, reduced size of the spleen, preputial glands, testes, epididymides, seminal vesicles, prostate and/or thymus.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were treatment-related microscopic findings in the relevant groups, main findings were present in kidneys, and minor findings were present in seminal vesicles, prostate gland and female thymus.

In kidneys the following findings were noted:
- Diffuse basophilia, medullary of the kidneys (4/5 females at 100 mg/kg and 5/5 females at 300 mg/kg) up to a slight degree.
- Papillary degeneration/necrosis (2/5 females at 300 mg/kg) up to a slight degree.
- Basophilic tubules (moderate) combined with slight cast formation and minimal necrosis of tubular epithelium (1/5 males at 100 mg/kg; not at 300 mg/kg bw).
Minimal reduced contents in seminal vesicles (3/5) and minimal atrophy of prostate gland (1/5) were noted in males at 100 mg/kg, this was not seen at 300 mg/kg bw.
Slight lymphoid atrophy of the thymus was noted for one female at 300 mg/kg (1/5).

All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Microscopic examination revealed effects in the kidney of males and females at 100 mg/kg/day, and slight hematological effects..

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The concentrations analysed in the formulations of the different exposure groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the formulation of the control group. The formulations of the highest and the lowest dose 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 an oral OECD407 study with rats, a LOAEL of 100 mg/kg bw/day was determined, based on microscopic changes in males and females at this concentration. Additionally minimal reduced contents in seminal vesicles in three of the five males and minimal atrophy of prostate gland in one male at 100 mg/kg/ day was found.

Executive summary:

An oral OECD 407 repeated dose toxicity study was conducted according to OECD guidelines and GLP principles. CX-100 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. All animals at 1000 mg/kg were sacrificed for ethical reasons or were found death during the study period. Three males at 300 mg/kg/day were sacrificed for ethical reasons on Day 25. Only minor clinical signs were noted at 100 mg/kg (incidental hunched posture and rales). No toxicologically significant changes were noted at this dose level in functional observations, body weight, food consumption, macroscopic examination and organ weights. Changes in hematology investigations at this lowest dose level included slightly lower red blood cell counts, slightly higher red blood cell distribution width and higher mean corpuscular volume. During microscopic examination treatment-related microscopic findings were noted in males at 100 mg/kg /d and females at 100 and 300 mg/kg/d, main findings were present in kidneys: minimal to slight diffuse basophilia, medullary of the kidneys (4/5 females at 100 mg/kg and 5/5 females at 300 mg/kg), minimal to slight papillary degeneration/necrosis (2/5 females at 300 mg/kg), moderate basophilic tubules combined with slight cast formation and minimal necrosis of tubular epithelium (1/5 males at 100 mg/kg). Additionally minor findings were found in these animals: minimal reduced contents in seminal vesicles three males and minimal atrophy of prostate gland in one male at 100 mg/kg. Slight lymphoid atrophy of the thymus was noted one female at 300 mg/kg.

From the results presented in this report and mainly based on the dose-related microscopic findings in the kidneys the LOAEL was determined to be 100 mg/kg bw/d.