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Administrative data

Description of key information

Key, M-428958-01-1, U.S. EPA test guideline, rat, 28 days,
NOAEL (systemic): 100 ppm (corresponding to 5.78 mg/kg bw/day)
LOAEL (systemic): 300 ppm (corresponding to 25.7 mg/kg bw/day)
NOAEL (immunotoxicity): 1000 ppm (corresponding to 80.7 mg/kg bw/day)

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
immunotoxicity: short-term oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Oct - 29 Nov 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.7800
Version / remarks:
adopted 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Weight at study initiation: 163 - 199 g
- Fasting period before study: not applicable
- Housing: individually in suspended, stainless steel, wire-mesh cages
- Diet: Certified rodent powdered and irradiated diet A04CP1-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: tap water, filtered and softened, ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY: The feed and water was analyzed regularly and no contaminations were found.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Oct To: 29 Nov 2011
Route of administration:
oral: feed
Vehicle:
other: acetone and corn oil
Mass median aerodynamic diameter (MMAD):
other: not applicable
Remarks on MMAD:
not applicable
Details on exposure:
TEST ITEM
DIET PREPARATION
- Rate of preparation of diet (frequency): once for each concentration
- Mixing appropriate amounts with: The appropriate amount of the test item was dissolved in acetone and corn oil and this mixture was added to the diet. Acetone was evaporated and the final concentration of corn oil in diet was 1% (all groups, including control).
- Storage temperature of food: when not in use, the diet was stored at -20°C

CYCLOPHOSPHAMIDE
PREPARATION OF DOSING SOLUTIONS:
- Lot/Batch Nr: 120M1253
- Appearance: white powder
- Purity: 100.6% (analysis by HPLC)
- Storage: 2 - 8 °C
- Date of Analysis: 03 Jan 2011
- Route of exposure: gavage
-Time schedule: daily
- Concentration: 3.5 mg/kg bw/day
- Volume: 5 mL/kg body weight

Preparation:
- Rate of preparation (frequency): twice
Procedure: The dosing formulation of cyclophosphamide was prepared by suspending the
substance in sterilized water to produce the required dosing concentration.
Storage: +5°C in the dark when not in use
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity of the test substance in the diet was verified for the lowest and highest administered concentration. Stability of the test item in the diet was analyzed previously to this study at concentrations of 20 and 2000 ppm and verified for the duration of the present study.

The stability and homogeneity of the positive control was proven in previous studies at concentrations of 0.1 and 1 g/L for a time period which covers the period of storage and usage for the current study.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
100 ppm (nominal)
Remarks:
corresponding to 5.78 mg/kg bw/day
Dose / conc.:
300 ppm (nominal)
Remarks:
corresponding to 25.7 mg/kg bw/day
Dose / conc.:
1 000 ppm (nominal)
Remarks:
corresponding to 80.7 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Gender selection rational: The selection of the gender for the immunotoxicity study is based on the fact that the female Wistar rat has been used extensively in toxicity evaluation studies confirming the sensitivity of this gender.
- Species selection rationale: The selection of the species for the immunotoxicity study is based on the fact that the Wistar rat has been used extensively in toxicity evaluation studies and is a recommended rodent model for immunotoxicity studies.
- Dose selection rationale: The dose levels of 0, 100, 300 and 1000 ppm were set based on the general systemic toxicity data as available from previous rat studies conducted with this test item.

Sheep Red Blood Cell (SRBC) ADMINISTRATION
SRBC
- Supplier: BioMérieux
- Reference Number: 72141
- Storage: 2 - 8 °C
-Time schedule: Day 26
- Anesthesia: YES if necessary (Isoflurane (Baxter, Maurepas, France))
- number of cells injected: 2.8 * 10^8 SRBC/animal
- Procedure: On the day of injection, Sheep Red Blood Cells were washed in PBS (Phosphate
Buffered Saline), counted using a cell counting instrument (Siemens Advia 120) and diluted in PBS in order to obtain a 5 x 10^8 cells/mL preparation. SRBC preparation was kept on ice until use. All animals of all groups were injected (tail vein, 05 mL/animal) with SRBC preparation.
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily on weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 4 days after SRBC immunization (terminal sacrifice)
- Animals fasted: No
- Animals anesthetized: Yes (inhalation of Isoflurane)
- How many animals: all survived animals
- Parameters examined: SRBC-specific IgM

HEMATOLOGY: No

URINALYSIS: No
Sacrifice and pathology:
Surviving animals were sacrificed by exsanguination while under deep anesthesia (Isoflurane inhalation).

GROSS PATHOLOGY: Yes
necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abn
ormalities were recorded but not sampled.
Organ weight was recorded for: Spleen, thymus

HISTOPATHOLOGY: No
Cell viabilities:
No data
Humoral immunity examinations:
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: Rat Anti-Sheep Red Blood Cell IgM ELISA kits from Life Diagnostics (West Chester, PA 19380 – USA)
- Dose groups: all
- No. of animals: 10/dose group (all animals)
Specific cell-mediated immunity:
No data
Non-specific cell-mediated immunity:
No data
Other functional activity assays:
None
Other examinations:
None
Positive control:
Yes, one group of animals received 3.5 mg/kg bw/day cyclophosphamide (immunosuppressive agent) in sterilized water by gavage

Cyclophosphamide: batch number 120M1253: a white powder, 100.6% purity
Statistics:
Mean and standard deviation were calculated for each group and time period.

Body weight change, terminal body weight, absolute and relative organ weight parameters
To analyze the differences between the control and the positive group cyclophosphamide, an F-test was performed. If this showed significant differences, a 2-sided modified t-test followed. If no significance was found, a 2-sided T-test was performed.

To compare control and test substance groups, group variances were analyzed with the Bartlett test followed with an Analysis of Variance (ANOVA) and a 2-sided Dunnett's test (if needed) when variance was equal and a Kruskal-Wallis and 2-sided Dunn test (if needed) when variance differed.

Body weight and average food consumption/day parameters
The comparison between the control and positive group was similar to the above described, only that the data was transformed using the log transformation if the F test showed a significant difference. Then, the procedure was repeated on the transformed data.

The comparison between the control and test substance groups was also similar to the above described, only that the data was transformed using the log transformation if the Bartlett test showed a significant difference. Then, the procedure was repeated on the transformed data and followed as described above.

If one or more group variance (s) equaled 0, means were compared using non-parametric procedures.

Immunological parameter
The control and positive group were compared using the 2-sided Mann-Whitney-test.
The control and test substance group were compared with the Kruskal-Wallis-test. If a significant difference was found, the 2-sided Dunn Test was used.

Significance in all of the tests was defined as p ≤ 0.05.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related signs observed in the treatment and positive control group.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: mean body weight reduced throughout the study (7-9%) compared to controls, mean body weight gain was reduced (-32% at termination) compared to control animals

Summarized data can be found in Attachment 1.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: reduced mean food consumption compared to controls (-33% during the first week of the study and 9% lower in Week 2 and 3, only the first week was statistically significant)

Summarized data can be found in Attachment 2.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
no effects observed
Description (incidence and severity):
For the test substance, no statistically significant differences were noticed as compared to the concurrent vehicle control group.

Cyclophosphamide
Anti-SRBC IgM mean concentration was statistically significantly lower as compared to the solvent control (88%, p =<0.01), proving the sensitivity of the assay to identify an immunosuppressive agent.

Data is summarized in Attachment 3.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weight
- 1000 ppm: reduced mean terminal body weight compared to controls (-10%)

Cyclophosphamide
no effects observed

Organ weight
- 300 ppm: increased mean spleen to body weight ratio (+16%)
- 1000 ppm: increased mean absolute (+18%) and relative (+31%) spleen weight compared to controls

Cyclophosphamide
Mean absolute and relative spleen (-24% and -20%) and thymus (-21% and -17%) weights were decreased compared to controls.

Summarized data can be found in Attachment 4.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no differences between solvent control and treatment groups. In the positive control group, atrophic/small spleen and/or thymus were noted (8/10 and 3/10 animals, respectively)
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Cell viabilities:
not examined
Description (incidence and severity):
not applicable
Humoral immunity examinations:
not examined
Description (incidence and severity):
not applicable
Specific cell-mediated immunity:
not examined
Description (incidence and severity):
not applicable
Non-specific cell-mediated immunity:
not examined
Description (incidence and severity):
not applicable
Other functional activity assays:
not examined
Description (incidence and severity):
not applicable
Other findings:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
other: corresponding to 5.78 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 25.7 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
Immunotoxicity
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
other: corresponding to 80.7 mg/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
The study assessed the effects of the test item on the immunologic system in rats after dietary administration for 28 days. The study is considered valid and in compliance with GLP and the guideline US-EPA-OPPTS Series 870, Health Effects Testing Guidelines, N°870.6300.
Under the conditions of the study, the dietary NOAEL for systemic toxicity was set at 100 ppm (5.78 mg/kg bw/day). This was based on reduced body weight and food consumption, and organ weight effects seen at 300 and 1000 ppm. Increased absolute and relative spleen weights as reported for 300 and 1000 ppm, were considered to be due to systemic toxicity rather than immunotoxicity. The NOAEL for immunotoxicity was set at 80.7 mg/kg bw (1000 ppm, the highest administered dose).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80.7 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
U.S. EPA test guideline and GLP study, fulfilling the criteria of suitability as key study.

Effect on immunotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Please refer to the information given in the endpoint summary. 

Additional information

The potential immunotoxicity of present substance was assessed in female Wistar rats under subacute test conditions (feed, 28 days), according to an U.S. EPA test guideline, under GLP conditions. This study is considered suitable as key study (M-428958-01-1).


Groups of 10 Wistar rats/per dose were exposed daily via the diet to 100, 300 and 1000 ppm test item, corresponding to 5.78, 25.7 and 80.7 mg/kg bw/day (mean ratios) of the test substance for at least 28 days according to US-EPA-OPPTS Series 870, Health Effects Testing Guidelines, N°870.6300 and in compliance to GLP. One group of rats received 3.5 mg/kg bw Cyclophosphamide as a positive control for immunosuppression. Cyclophosphamide was administered solubilized in deionized water via gavage.


Both the concentration and homogeneity of the test substance in the diet and the positive control in the vehicle were analyzed and considered valid. Clinical signs were recorded daily and body weight and food consumption were measured weekly. Detailed physical examination was performed weekly throughout the study. On Day 26, rats were injected in the tail vein with 0.5 mL preparation of Sheep Red Blood Cells (SRBC), containing 5 x 10^8 cells/mL. 4 days after SRBC immunization, blood was taken from all animals (non-fasted) and analyzed for anti SRBC- IgM using an ELISA kit. When animals were sacrificed, the gross necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded but not sampled. Organ weight was recorded for spleen and thymus.


No mortality was observed in the study. At 1000 ppm of the test substance, systemic toxicity was evident, observed as reduced body weight (7-9%), reduced body weight change (-32%) and reduced food consumption (-33% in Week 1, -9% in Week 2 and 3) compared to the control group. Animals of this test group also showed increased mean absolute (+18%) and relative (+31%) spleen weight compared to controls. At 300 ppm, animals had an increased mean spleen to body weight ratio (+16%). However, these findings were not specifically attributed to immunotoxicity but rather related to the systemic toxicity.


 


The SRBC response was unchanged in treatment groups compared to control groups. In the positive control group, no treatment-related clinical signs or influence on food consumption or body weight occurred. The concentration of anti-SRBC IgM was 88% lower than the controls. Spleen and thymus were smaller and lighter than in control animals. With these results, the positive control cyclophosphamide confirmed the sensitivity of the test system.


 


Under the conditions of the study, the NOAEL for immunotoxicity was set at 80.7 mg/kg bw (1000 ppm, the highest administered dose), whereas the overall NOAEL with respect to systemic toxicity was set at 100 ppm (5.78 mg/kg bw/day).

Justification for classification or non-classification

No immunotoxic potential was evidenced with respect to present substance; thus, no classification is required.