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Description of key information

Repeated dose toxicity (according to OECD 407, GLP): NOAEL ≥ 1000 mg/kg bw/d

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Dec 1990 - 16 Jan 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also in accordance with GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Crl: CD SD BR VAF/Plus
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd., Margate, Kent, England
- Age at study initiation: 28 ± 1 days
- Weight at study initiation: range of 66 - 80 g
- Housing: in groups of 5
- Diet: Biosure LAD 1 Diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 50 -60
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Dec 1990 To: 16 Jan 1991
Route of administration:
oral: gavage
Vehicle:
methylcellulose
Remarks:
1% aqueous methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was ground in a mortar with a small amount of the vehicle (1% methylcellulose) until a smooth paste was formed. The formulation was then gradually made up to volume and mixed using a high shear homogeniser. A series of formulations were prepared freshly each day. Prior to dosing the test substance formulations were mixed by inversion (x20) and subsequently using a magnetic stirrer for a period of at least 10 minutes before dosing commenced. Dosing was completed within one hour of the commencement of stirring.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test substance in a representative sub-sample was quantified by high performance liquid chromatography using ultra-violet detection, to determine concentration and chemical and physical stability. For calibration a standard solution was prepared by dissolving an accurately weighed quantity of test substance and preparing serial dilutions. The results were within 5% of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Remarks:
Doses / Concentrations:
0.1, 1, 10% (w/v)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10, 100, 1000 mg/kg bw
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 selected based on a 7-day preliminary oral toxicity study
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing and subsequently at weekly intervals

FOOD CONSUMPTION:
- Food consumption for each cage: measured at weekly intervals

WATER CONSUMPTION: Yes
- Time schedule for examinations: first 14 days by visual appraisal, since treatment-related effect was suspected, water consumption was measured by gravimetric analysis from week3 on

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.2] were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table3)
HISTOPATHOLOGY: Yes (see table4)
Statistics:
All statistical analysis were carried out seperatedly for males and females. Bodyweight data were analysed using weight gains.
The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:
1) If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of values different from the mode was analysed by appropriate methods. Otherwise:
2) Bartlett's test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level)heterogeneity was found, a logarithmic transformation was tried to see if a more variance structure could be obtained.
3) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
4) Analysis of variance were followed by student's t-test and Williams test for a dose-related response, although only the one thought more appropriate for the response pattern observed reported. The Kruskal-Wallis analysis were followed by the non-parametric equivalents of the t test and Williams test (Shirleys test).
Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, analysis initially involved a correlation analysis between organ weights and final body weight. For organs where a correlation at the 10 % level of significance was established, analysis of organ weight data was performed using adjusted organ weights by analysis of covariance with final bodyweight as covariate. Where a correlation between organ weight and bodyweight was not established the organ weight analysis was carried out using routine analysis of variance on unadjusted organ weights.
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical findings were only observed after withdrawal of blood (slight pallor of extremities, slight to moderate swelling of the right eye in three animals).
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No changes in body weight gain were observed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No apparent differences were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
A slight disturbance of water consumption at 100 and 1000 mg/kg/day, but this was considered toxicologically not relevant.
100 mg/kg bw/day females: 24% increase, 1000 mg/kg bw/day females: 35% increase, 100 + 1000 mg/kg bw/day males: slight decrease (20%).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A decreased lymphocyte and total white blood cell count was observed at 1000 mg/kg/day in 1 female and where therefore considered to be of minor importance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment related effects were found.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
An increase in relative liver weights and adrenal weights were found for females. Increase of liver weights were associated with hepatic enzyme induction as an adaptive response. No histological findings were observed in the adrenal glands, therefore the weight change was considered toxicologically not relevant.

1000 mg/kg bw/day - female: adjusted adrenal and liver weights significantly increased, males: adjusted liver weights slightly increased
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Only one female showed minimal centrilobular hepatocyte enlargement, which is usually associated with increased hepatic activity and can be considered to be an adaptive response.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to and including highest dose tested
Critical effects observed:
no
Conclusions:
The NOAEL for the oral repeated dose toxicity of the test material is considered to be 1000 mg/kg/day (highest dose tested).
Executive summary:

The oral repeated dose toxicity of the test material was evaluated according to the OECD guideline 407 and following GLP principles. It was formulated daily as 0.1, 1.0 and 10% (w/v) suspensions in 1% aqueous methylcellulose and administered by intragastric intubation to groups of ten rats (five males and five females) at dosage levels of 10, 100 and 1000 mg/kg/day, for 28 days. Control rats received the vehicle alone.

No treatment-related observations were made for bodyweight changes, food consumption, clinical signs and blood biochemistry.

The following treatment-related observations were recorded, but all are considered as not toxicologically relevant. A dose-dependent increase in water consumtion was recorded for female rats receiving 100 and 1000 mg/kg/day. The trend among male rats was for a slight decrease in consumption among all treated rats in comparison with controls. A reduced lymphocyte and total white blood cell counts were recorded for one female rat at 1000 mg/kg/day. Significantly higher adjusted adrenal and liver weights were recorded at termination for female rates at 1000 mg/kg. Minimal centrilobular hepatocyte enlargement was seen in the leiver of one female at 1000 mg/kg/day.

Therefore, the NOAEL for the oral repeated dose toxicity of the test material is considered to be 1000 mg/kg/day (highest dose tested). No classification or labeling needs to be applied.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information
requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A 28-day oral repeated dose toxicity study was performed according to OECD 407 in Crl: CD SD BR VAF/Plus rats, using 4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS 107934-68-9) (Edwards, 1991). 5 animals per sex and dose were administered 10, 100 and 1000 mg/kg bw/day by gavage, 7 days/week, over a period of 28 days. No mortality was observed and clinical findings were only observed after withdrawal of blood (slight pallor of extremities, slight to moderate swelling of the right eye in three animals). No apparent differences were observed for the food consumption, whereas a slight disturbance of water consumption at 100 and 1000 mg/kg/day was observed, but this was considered toxicologically not relevant. A decreased lymphocyte and total white blood cell count was observed at 1000 mg/kg/day in 1 female and but was considered not relevant. No treatment related effects were found for clinical chemical parameters. An increase in relative liver weights was observed in females, and one female showed minimal centrilobular hepatocyte enlargement, which is usually associated with increased hepatic activity and can be considered to be an adaptive response of hepatic enzyme induction. An increase in relative adrenal weights was also found for females, but no associated histological change was observed. Based on the lack of toxicologically relevant effects up to and including the highest dose level, the NOAEL is considered to be ≥ 1000 mg/kg bw/day.

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The reliable GLP compliant OECD Guideline study was chosen.

Justification for classification or non-classification

The available data on toxicity after repeated exposure of 4,4´-(9H-fluoren-9 -ylidene)bis(2 -chloroaniline) do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.