Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
subacute inhalation toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16.02.76 - 22.03.76
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978
Report Date:
1978

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Test of the inhalation toxicity followed the method of KIMMERLE (H. NIESSEN, H. TIETZ, G. HECHT and G. KIMMERLE, Arch. toxicol. 20, 44, 1963)
Version / remarks:
Test has been performed before publication of the OECD Guidelines for the Testing of Chemicals
Principles of method if other than guideline:
- Principle of test: repeated daily inhalation exposure to the test substance.
- Short description of test conditions: 10 male and 10 female rats were exposed 18 times 6 hours per working day; the undiluted product (3 % aqueous K-HDO) was atomised by a continuous infusion apparatus with a constant amount of 12 ml per hour and with 600 litres pressurized air (8 atü (atü = technical atmosphere above reference level)) / hour. The animals of the control group were exposed in an equal apparatus by which 12 ml demineralised water per hour were atomized with 600 litres pressurized air (8 atü) / hour. The total test period was 4 weeks (28 days)
- Parameters analysed: body weight, clinical symptoms, clinical chemistry and haematology, pathology
GLP compliance:
no
Remarks:
GLP was not mandatory at the time the study was performed

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test item: (N-Cyclohexyl-diazeniumdioxy)-potassium
IUPAC name: Cyclohexylhydroxydiazene 1-oxide, potassium salt
Chemical name: Cyclohexylhydroxydiazene 1-oxide, potassium salt; synonyma: (N-Cyclohexyl-diazeniumdioxy)-potassium, K-HDO, K-NCH, Xyligen K powder, Xyligen K
Molecular formula: C6 H11 K N2 O2
Molecular mass: 182.27
Specific details on test material used for the study:
Nomination of test substance: Reu-E 3403 (Xyligen-K)
Formulation: 3 % aqueous solution
Chemical name: N-cyclohexyl-diazeniumdioxy-Kalium (K-HDO)
Substance number: XXV / 437
Appearance: honey-yellow liquid
Purity: technical

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF-breed, Company WIGA, Sulzfeld, Germany
- Age at study initiation: 35 days
- Weight at study initiation: male rats: 90 (83-100) g / female rats: 85 (77- 91) g
- Housing: during the exposure-free time 3 or 2 rats were kept in one V2A wire cage (bottom area ca. 750 cm²) in a conventional heated and aerated room.
- Diet (e.g. ad libitum): Altromin-R of company ALTROGGE Lage/L ad libitum
- Water (e.g. ad libitum): tap water ad libitum during exposure-free time
- Acclimation period: animals were exposed in an inhalation chamber during a pre-streaming period of 5 days for 6 hours per day to a fresh-air stream of 600 litres per hour

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation chamber
- Source of air: pressurized air
- System of generating particulates/aerosols: permanent infusion apparatus
- Pressure in air chamber: 8 atü / per hour
- Air flow rate: 600 litres pressurized air per hour
- Test concentration: 20 µl Xyligen K per litre air
- Volume of test substance applied to the infusion apparatus: 12 ml


Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analysis performed
Duration of treatment / exposure:
18 times 6 hour per day; total test period of 4 weeks
Frequency of treatment:
daily during working days
Doses / concentrations
Dose / conc.:
20 other: µl/L
Remarks:
The undiluted product (= 3 % aqueous Xyligen K) was constantly applied to the apparatus in a volume of 12 ml/ hour and atomized with 600 litres pressurized air / hour
No. of animals per sex per dose:
10 animals per sex and dose
Control animals:
yes, concurrent vehicle
Details on study design:
The test of the inhalation toxicity was carried out according to the method of Kimmerle (H. Niessen, H. Tietz, G. Hecht, and G. Kimmerle, Arch. toxicol. 20, 44, (1963).
After a pre-inhalation period of 5 days, during which the animals were exposed in the inhalation chambers at a time for 6 hours to a fresh air stream of 600 litres per hour, the daily (working-day) 6-hour exposure to an aqueous 3% solution of Xyligen K of totally 18 times exposures started.
The undiluted test substance (Xyligen K as 3 % aqueous solution) was administered in constant amounts of 12 ml/h (via infusion apparatus type UNITA I from B. Braun, Melsungen) and sprayed with 600 litres compressed air (8 atü)/hour into the inhalation chamber. The exposure period was 6-hours/working day.
The animals of the control group were exposed to demineralised water under the same test conditions.
A dosage of 12 ml aqueous Xyligen K solution in 600 litres air per hour amounts to a nominal concentration of 20-µg/l air or, on acceptance of a density of 1 g/ml, a value of 20 mg/l air.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Behaviour and appearance were controlled daily

BODY WEIGHT: Yes
- Time schedule for examinations: 2 times per week (Monday and Thursday)

MORTALITY: Yes
- Time schedule for examinations: daily

FOOD EFFICIENCY:
- Body weight gain has been determined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 7 days before start of substance application (blood sampling 0) as well as 14 and 28 days after inhalation start (blood sampling 1 and 2) blood was collected from all animals from the retroorbital venous plexus. Blood collection occurred each between 7:00 and 11:00 a.m.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 7 days before start of substance application (blood sampling 0) as well as 14 and 28 days after inhalation start (blood sampling 1 and 2) blood was collected from all animals from the retroorbital venous plexus. Blood collection occurred each between 7:00 and 11:00 a.m.
- How many animals: all animals
- Parameters examined:
+Chemogram: sodium, potassium, carbon dioxide, chloride, calcium, inorganic phosphate, glucose, urea, total protein, total lipids, total bilirubin, creatinine
+Enzymogram: Glutamate-pyruvate-transaminase, alkaline phosphatase
+Haematogram and differential blood haemogram: Haemoglobin, erythrocytes, haematocrit, haemoglobin content of the single erythrocytes (HbE), mean cellular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), thrombocytes, leukocytes, differential blood haemogram

URINALYSIS: Yes
- Time schedule for collection of urine: 10 and 24 days after inhalation start (urine collection 1 and 2) urine was collected from all animals over night
- Parameters examined: pH-measurement, protein, glucose, urobilinogen, sediment-microscopy

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

SECTION:
- After termination of the test period, the animals were sacrificed after a fasting-period of 16 hours. Killing occurred after anaesthetising with CO2 via decapitation and bleeding. Afterwards the animals were weighed, dissected, and macroscopically evaluated. Subsequently the organs were weighed.

BODY AND ORGAN WEIGHTS:
- Parameters tested: bloodless body weight, organ weights of: heart, liver, kidneys, spleen, testis, thyroid gland, adrenal gland, lung. From this the relative organ weights (organ weight/100 g body weight) have been calculated

HISTOPATHOLOGY: Yes
The organs preserved in 4% buffered, neutral formalin solution were in part or in toto histologically processed.
Examined organs: cerebellum, cerebrum, pituitary, thyroid gland, adrenal glands, uterus, ovaries, epididymis, testis, urinary bladder, kidney, spleen, pancreas, liver, submandibular glands; colon, jejunum, ileum, duodenum, stomach, oesophagus, lung, trachea, heart. The organs of all animals of the Xyligen K test group were microscopically examined.
Methods used: hematoxylin – eosin method (all organs), Lillie's Oil Red O Method (lung, liver, kidney), PAS reaction
Other examinations:
No other examinations performed
Statistics:
Mean values, standard deviation and standard error have been calculated. T-test according to Williams (body weight, organ weights). chi2 test (urinalysis)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All control and test animals showed no toxication symptoms during the test period and were in a good physical status.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female of totally 20 test substance exposed animals died intercurrent during the 11th exposition without specific clinical symptoms.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain of the test animals was unimpaired.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Comparison of the body weight gain of the male and female rats showed no significant difference between the control group and the Xyligen K test group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Description (incidence and severity):
Erythrocytes, haematocrit, MCV, differential blood count: unaffected
Haemoglobin: increased after blood donation 2 (females)
HbE, MCHC: decreased after blood donation 2 (males)
Leukocytes: decreased solely after blood donation 1 (males)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
– Sodium, potassium, CO2, urea, total protein were not influenced
– Chloride: solely increased after blood donation 1 (males)
– Calcium: solely decreased after blood donation 1 (males)
– Anorg. phosphyte: increased after blood donation 2 (males)
– Glucose: increased solely after blood donation 1 (females)
– Total lipids: decreased after blood donation 2 (males)
– Bilirubin: increased solely after blood donation 1 (males and females)
– Creatinine: increased (females) and decreased (males), respectively after blood donation 2

Enzymes:
- Glutamate-pyruvate-transaminase: unaffected
- Alkaline phosphatase: increased after blood donation 2 (males)
Description (incidence and severity):
PH, protein, glucose, urobilinogen: unaffected
Sediment: after urine donation 2, increase of round epithelia (males) and increase of leucocytes (females), respectively
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
See haematological findings
Description (incidence and severity):
Absolute organ weights:
– Liver weight of the male animals of test group 1 was reduced (S ≥ 99 %).

Relative organ weights:
– reduced relative liver weights of the male animals of test group 1 (S ≥ 95 %)
– increased relative weights of adrenal glands and thyroid glands of the male animals (S ≥ 95 %)
Description (incidence and severity):
Gross and histopathology:
The animals of the control- and the test group showed, caused by death, changes of the heart and the lungs, thereunder erythrodiapedesis, blood aspiration, and dystelectasis as well as rat-specific lymphocytic focal-like predominant peribronchial lungs- and liver infiltrates. In addition, hydrometra was seen several times on female animals. Several female animals of both test groups showed a slight extra-marrow haemopoiesis.
The male animals of the substance-treated group showed a slight increase of the foam cells in the lung. The female animals showed a slight fatty metamorphosis of liver. Three female rats had focal-like liver necrosis. The intercurrent after 15 days perished female rat of the test substance-treated group showed hyperaemia of the lung, liver, adrenal glands, and the hypophysis as well as tubolonephrosis and a slight vacuolisation of the adrenal glands (Zona fasciculata).
Valuation of the findings:
Histological focal like liver necrosis was primarily seen on 3 female animals as well as a slight increase of foam cells in the lungs of active-exposed male animals. In addition, a slight decrease of the absolute and the relative liver weights were seen on male animals of the active exposed dose group. A coherency with the application of the active cannot be ruled out, even though at least the histological liver changes are single findings.
Neuropathological findings:
not examined
Description (incidence and severity):
see above
Description (incidence and severity):
see above
Other effects:
no effects observed
Description (incidence and severity):
No signs of intolerance or intoxication after an exposure period of 4 weeks (6 /day) observed.

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks on result:
other: see remarks
Remarks:
a detailed description of the results is given in the summary below

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No clinical signs of intoxication or incompatibility observed during and after eposure.
Executive summary:

Xyligen K has been tested in a subacute inhalation test with rats as 3 % aqueous solution in a nominal concentration of 20 µl/l air (equivalent to 0.6 mg Xyligen K/l air). For control, rats have been exposed under identical test conditions to the solvent (demineralised water).

The test period was 4 weeks with working-daily 6-hours exposure.

No clinical signs of intoxication or incompatibility have been seen during and after the single expositions. Body weight gain of the animals was not affected.

One female of totally 20 test substance exposed animals died intercurrent during the 11th exposition without specific clinical symptoms.

Regarding the clinical chemistry and the haematology only male animals showed a decrease of the blood total lipid values as well as an increase of the activity of the alkaline phosphatase. The urine sediment of the male animals showed an increase of the round epithelia. The urine sediment of the female animals showed an increase of the leucocytes. A clear test substance induced change cannot be stated because of the lack of adequate clinical and additional clinical-chemistry findings, respectively.

Regarding gross-pathology a significant decrease of the relative and absolute liver weights of the substance treated male animals has been seen.

The histopathological examination showed focal-like liver necrosis for 3 substance treated female animals as well as a slight increase of the foam cells in the lungs of male animals.

A coherency with the application of the active ingredient cannot be ruled out although, at least the liver changes, were single findings.