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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 7, 2004 - October 5, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
(1995)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
(1996)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050 (2000)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
453-140-3
EC Name:
-
Cas Number:
667465-46-5
Molecular formula:
C20H38N6O13 (not dissociated) C20H44N6O13 (dissociated in water)
IUPAC Name:
3,6,9-tris(2-{[(2-hydroxyethyl)amino]oxy}-2-oxoethyl)-3,6,9-triazaundecan-1,11-dioic acid
Constituent 2
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Water
Test material form:
liquid
Details on test material:
- Name of test material: DHX2
- CAS no.: 667465-46-5
- 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:
- 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
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages
- Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany))
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 - 23.6
- Humidity (%): 38 - 98
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: September 7, 2004 - October 4, 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Milli-U)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
- It was determined that the test substance was stable in the vehicle for at least 5 hours at room temperature
- Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level
- The solutions were stored at ambient temperature until use
- The solutions were placed on a magnetic stirrer during dosing

VEHICLE
- Justification for use and choice of vehicle: based on performed trial formulations

DOSE VOLUME: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight. No adjustment was made for the density of the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Samples of formulations prepared after the in-life phase (preparations equal to formulations in the in-life phase), were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours was also determined (highest and lowest concentration). The analytical method used was based on the results of a separate project for the development and validation of the analytical method for the determination of the substance in water: See IUCLID section 8.
- Test substance formulations in Milli-U water were noted as stable for at least 5 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 96-101% (cationic part) and 96-103% (anionic part) of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrationsopen allclose all
Dose / conc.:
50.1 mg/kg bw/day (actual dose received)
Remarks:
adjusted for the density of the test substance
Dose / conc.:
151 mg/kg bw/day (actual dose received)
Remarks:
adjusted for the density of the test substance
Dose / conc.:
1 025 mg/kg bw/day (actual dose received)
Remarks:
adjusted for the density of the test substance
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected on the basis of a 5-day dose range finding study in 3 rats/sex. In the pilot, no treatment-related effects were noted at 151 and 1025 mg/kg bw/day (adjusted by the assessor for the density of the test substance)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 1, 8, 15, 22 and 28

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (weekly)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: According to OECD 407 (1995)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: all
- Parameters checked: According to OECD 407 (1995)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all 40 animals
- Battery of functions tested:
*Hearing ability, pupillary reflex, static righting reflex and grip strength
*Motor activity (recording period: 12 hours during overnight for individual animals, using computerised monitoring system)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Dose groups that were examined: all 40 animals
- Tissues/organs checked: According to OECD 407 (1995)

ORGAN WEIGHTS:
Organs checked according to OECD 407 (1995)

HISTOPATHOLOGY: Yes
- All tissues and organs from all animals of the control and the highest dose group
- All gross lesions of all animals
Statistics:
The following statistical methods were used to analyse 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 Student's t-test was applied for motor activity data.
- 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

All tests were 2-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, 2 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, non-treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity or behavioural changes that were considered to be related to treatment. Alopecia, diarrhoea and missing upper incisors were occasionally observed. These findings are not uncommon in rats of this age and strain housed under the conditions of this study. At the incidences observed, these signs were not considered to be related to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights of treated animals remained in the same range as controls
A significantly reduced body weight gain was observed on day 8 for males dosed with 151 mg/kg bw/day (-37.9% compared to control). As this was no longer present from day 15 onwards, this finding was considered toxicologically not relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
A statistically significant difference was only found in white blood cell counts between control females and females receiving 151 mg/kg bw/day (decrease of 39%). This was considered to be incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant deviations from control mean were observed at 1025 mg/kg bw/day:
- Increased inorganic phosphate levels (+14.5%) and decreased total protein (-5.5%) / albumin (-4.8%) levels in males
- Decreased glucose (-11.8%) and alkaline phosphatase (-22.3%) levels in females
The following statistically significant deviations from control mean were observed at 151 mg/kg bw/day:
- Decreased calcium levels in males (-3.9%)
- Decreased glucose levels in females (-16%)
The following statistically significant deviations from control mean were observed at 50.1 mg/kg bw/day:
- Decreased calcium (-2.8%) and total protein levels (-4.3%) in males
- Decreased glucose (-13.5%) and inorganic phosphate levels (-14.6%) in females
As all of these deviations were within the range of normal biological variation and/or there was no dose-response relationship for any of the deviations, it is concluded that the observed deviations were not considered to be related to treatment.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals
The variation in motor activity did not indicate a relation with treatment (no statistically significant deviations from control)
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant changes were noted regarding organ weights and organ:body weight ratios. Higher absolute (+24.6%) and higher relative (+26.3%) weights of the adrenal gland were recorded for males at 1025 mg/kg bw/day. In the absence of a microscopic correlate, these deviations were not considered to be related to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant alterations were observed. Incidental findings included pelvic dilation of the kidneys, thymic foci, enlarged mandibular lymph nodes, discoloration of the clitoral gland and alopecia. These findings are occasionally seen among rats of this age in this type of study. In the absence of a dose-response relationship, they were not considered to be treatment-related.
- Watery fluid in the uterus, as seen in 2 females at 50.1 mg/kg bw/day and 1 female at 151 mg/kg bw/day, is a normal finding related to the oestrous cycle.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no microscopic findings recorded which could be attributed to treatment. All 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.
Details on the histopathological examinations are attached below.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 025 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects up to the highest dose level tested.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a 28-day oral repeated dose toxicity study with rats, the NOAEL was determined to be ≥ 1025 mg/kg bw/day.
Executive summary:

In accordance with OECD 407 (1995), EU Method B.7 (1996), EPA OPPTS 870.3050 (2000) and according to GLP principles, a 28 -day oral repeated dose toxicity study was performed. Rats (5/sex) were given 0, 50.1, 151 and 1025 mg/kg bw/day by gavage (adjusted by the assessor for the density of the test substance, 1.14 g/mL). As it was concluded that there were no treatment-related effects, a NOAEL for the substance of ≥ 1025 mg/kg bw/day follows from the results of this study.