Diamminediisocyanatozinc

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

until 1989-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

OECD: Guidelines for Testing of Chemicals, Section 4, Health Effects Testing Guideline 408, 12 May 1981

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

EEC: Annex V to Directive 87/302 EEC, 18 November 1987

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA, TSCA & FIFRA guidelines

Version / remarks:

US.EPA (TSCA): 40 CFR Part 772, Federal Register, 9 May 1979
US.EPA (FIFRA): 40 CFR Part 158, Federal Register, November 1982

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diamminediisocyanatozinc

EC Number:

401-610-3

EC Name:

Diamminediisocyanatozinc

Cas Number:

122012-52-6

Molecular formula:

C2H6N4O2Zn

IUPAC Name:

copper(2+) bis(carbonylazanide) diamine

Test material form:

solid: particulate/powder

Remarks:

white

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR strain (VAF plus)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, England.
- Females (if applicable) nulliparous and non-pregnant: not stated, but presumed due to compliance with OECD TG 408
- Age at study initiation: not stated
- Weight at study initiation: At the start of treatment males were in the weight range 123-172g and females weighed 109-139g.
- Fasting period before study: no, only in periods of deprivation associated with laboratory investigations
- Housing: Before the animals arrived the room, and cages were scrubbed with a solution of Soraplex S25HD (Th. Goldschmidt Limited, Eastcote, England), allowed to dry and then treated with, a 1% solution of Tegodor 73, (Th. Goldschmidt) after which the cages were rinsed in clean water and allowed to dry.
The animals were housed in groups of 5, by sex, in grid bottomed polypropylene cages (pattern RC1, North Kent Plastics Limited, Dartford, England) measuring 56 x 38 x 18cm) suspended over cardboard lined excreta trays.
- Diet (e.g. ad libitum): SQC Rat and Mouse Maintenance Diet No. 1, Expanded, (Special Diets Services Limited, Witham, England), ad libitum
- Water (e.g. ad libitum): Mains water was provided in polypropylene bottles, ad libitum
With the exception of periods of deprivation associated with laboratory investigations, both were freely available throughout the study.
- Acclimation period: All animals were found to be healthy on arrival. The animals were acclimatised for 11 days before the start of treatment. Towards the end of acclimatisation they were re-examined and confirmed to be suitable for experimental use.

DETAILS OF FOOD AND WATER QUALITY:
The supplier's certificates of analysis for the batches of diet used in this study and a representative certiricate of analysis for the drinking water are available.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The roora was air conditioned and recorded temperature was in the range 17-24°C.
- Humidity (%): range of 26-69%
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800 hours) and 12 hours dark.

IN-LIFE DATES: From: 1988-03-04 To: 102 days later

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

A constant dose volume of 5ml/kg bodyweight was used. Individual doses were adjusted according to the most recent bodyweight.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
All formulations were prepared freshly each day.
The high dose formulation was prepared by gradually adding a known volume of vehicle to a weighed amount of test article and grinding in a pestle and mortar. The resultant suspension was then homogenised further using a Silverson mixer-
The intermediate and low dose formulations were prepared separately by dilutions of known volumes of the high dose formulation with further vehicle followed by mixing in a Silverson mixer.

VEHICLE
- Amount of vehicle (if gavage): 5ml/kg bw
The vehicle used was corn oil and came from a consignment of 16 x 2.5 litres, identified as lot number C-8267, for laboratory use only, ex Sigma, provided by the sponsor.

Analytical verification of doses or concentrations:

yes

Remarks:

The sponsor indicated that the formulations would be stable under the conditions of use.

Details on analytical verification of doses or concentrations:

The sponsor indicated that the formulations would be stable under the conditions of use.
As a check on the accuracy of preparation and to confirm homogeneity, 4 samples of each concentration, including the control formulation, prepared on 2 occasions in week one, and on one day in each of weeks 2, 3, 4, 7, 10 and 13 were returned to the sponsor for analysis.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once daily, 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

medium dose

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

10/sex/dose for all dose groups plus 10/sex/dose for the recovery groups (control & high dose)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on previous studies
- Rationale for animal assignment (if not random):
Five days before the start of treatment animals were allocated randomly to treatment groups using a stratified bodyweight procedure.
- Fasting period before blood sampling for clinical biochemistry: Food was withheld during periods of deprivation associated with laboratory investigations.
- Rationale for selecting satellite groups: to investigate the reversibility of any findings
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality checks were performed twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for changes in condition or behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal wa s weighed at the start of treatment, twice week] y for the first 4 weeks of treatment and then weekly thereafter.

FOOD CONSUMPTION:
The food consumed by each cage of animals was recorded weekly throughout the treatment period and group mean weekly intakes were calculated.

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: Not specified

WATER CONSUMPTION: Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All animals were examined before the start of treatment. In week 12 the eyes of all surviving animals were examined.
On both occasions the examinations were performed using either an indirect or a direct ophthalmoscope after previous instillation of a mydriatic agent (Homatropine hydrobroraide 2% w/v).
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken during weeks 4 and 13 of treatment
- Anaesthetic used for blood collection: Yes, Samples were taken by puncture of the retro-orbital sinus under light ether anaesthesia.
- Animals fasted: Yes, overnight
- How many animals: all animals scheduled to be killed after 13 weeks treatment
- Parameters examined:
Packed cell volume (PCV) Coulter accessory unit *
Haemoglobin concentration (Hb) Cyanmethaemoglobin in Isoton II **
Erythrocyte count (RBC) Direct count *
Total leucocyte count (WBC) Direct count *
Leucocyte differential count Direct visual count using May Grunwald Giemsa stain with leucocytes classified as follows:
-Neutrophils (Neut)
-Lymphocytes (Lymph)
-Monocytes (Mono)
-Eosinophils (Eosin)
Platelet count (Plate) Direct count following sedimentation*
Reticulocyte count (Retics)+ Direct count
Mean cell volume (MCV) Coulter accessory unit*
Mean cell haemoglobin (MCH) Calculation Hb x 10 / RBC
Mean cell haemoglobin concentration (MCHC) Calculation Hb x 100 / PCV

+ smear prepared but not evaluated
* Coulter counter model ZF
**SP-6 Spectrophotometer
Blood samples were taken into EDTA anticoagulant.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken during weeks 4 and 13 of treatment
- Anaesthetic used for blood collection: Yes, Samples were taken by puncture of the retro-orbital sinus under light ether anaesthesia.
- Animals fasted: Yes, overnight
- How many animals: all animals scheduled to be killed after 13 weeks treatment
- Parameters examined:
Blood urea nitrogen (BUN) Kinetic substrate at 30°C +
Glucose (Glu) Endpoint at 25°C +
Alkaline phosphatase (A. Phos) Kinetic enzyme at 30°C +
Alanine aminotransferase (ALT) Kinetic enzyme at 30°C +
Aspartate aminotransferase (AST) Kinetic enzyme at 30°C +
Total protein (TP) Endpoint at 25°C +
Albumin (Alb) Endpoint at 25°C +
Albumin/Globulin ratio (A/G ratio) Calculation
Sodium (Na) IL 243 Flame photometer
Potassium (K) IL 243 Flame photometer
Calcium (Ca) Endpoint at 25°C +
Chloride (CI) Endpoint at 25°C +
Total bilirubin (Bili) Endpoint at 25°C +
Inorganic phosphorus (I.Phos) Kinetic substrate 25°C +
Creatinine (Creat) Kinetic substrate at 30°C +
Cholesterol (Chol) Endpoint at 30°C +
Creatine phosphokinase (CPK) Kinetic enzyme at 30°C +
Gamma glutamyl transferase (GGT) Kinetic enzyme at 30°C +

+ Gilford Impact 400 discrete batch analyser.
Blood samples were taken into lithium heparin anticoagulant

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight, under food and water deprivation during week 12 of treatment from all animals scheduled to be killed after 13 weeks treatment, and during the final week of the reversibility period (males only), to clarify previous findings. - Parameters examined:
Specific gravity (SG) Atago Uricon optical hydrometer
Volume, pH, Glucose, Protein, Ketones, Bilirubin (Bili), Urobilinogen, Blood Ames N-Multistix
Deposit Microscopic examination:
-Erythrocytes (E)
-Leucocytes (L)
-Crystals (X)
-Debris (D)
-Casts (C)
Semi-quantitative results are expressed on the scale below:
0 = absent
T = trace
1 = small amount
2 = moderate amount
3 = large amount

NEUROBEHAVIOURAL EXAMINATION: Not specified, abnormalities are general denoted

IMMUNOLOGY: No

Sacrifice and pathology:

Terminal observations
All surviving animals were killed at the end of the specified period by carbon dioxide asphyxiation. Post mortems were performed over 2 working days at the first kill and on one day at the second (reversibility) kill.

GROSS PATHOLOGY: Yes
Necropsy
All animals were weighed and then examined externally. A macroscopic examination was then performed by opening the cranial, thoracic and visceral cavities and observing the appearance of the tissues in situ. Abnormalities were recorded with details of location, colour, shape and size.

Organ weights
The following organs from all animals were weighed after trimming of fat and other contiguous tissue.
Adrenals
liver
brain
ovaries
kidneys
testes
Paired organs were weighed separately but combined weights only have been reported.

Tissue preservation
With the exception of the eyes and optic nerves, which were taken into Davidson's fluid, either whole organs or selected samples of the following tissues were preserved in buffered formol saline.

adrenals
brain (4 levels)
colon
eyes (incl. optic nerve)
heart
kidneys
lungs
oesophagus
pituitary
rectum
sciatic nerve
skin
sternum (incl. marrow)
thymus
trachea
vagina
aorta
caecum
duodenum
femur++
ileum
lacrimal gland (exorbital)
mammary gland
ovaries
preputial gland
salivary glands (parotid, sub-lingual, sub-maxillary)
seminal vesicles
spinal cord (3 levels)
stomach
thyroids+++
urinary bladder
bone marrow smear+
cervical lymph node
epididymides
harderian gland
jejunum
liver
mesenteric lymph node
pancreas
prostate
skeletal muscle (thigh)
spleen
testes
tongue
uterine horns
all gross lesions

+ femoral, fixed in anhydrous methanol, not processed further
++ including articular surface
+++ including parathyroids if identified


HISTOPATHOLOGY: Yes
The following tissues were dehydrated, wax embedded, cut at a nominal thickness of 5 microns, stained with haematoxylin and eosin and examined microscopically:
i) All tissues from control and high dose animals killed after 13 weeks treatment.
ii) All tissues from the 2 decedents.
iii) Any tissues showing macroscopic abnormalities, together with the kidneys, liver and lungs from all low and intermediate dose animals killed after 13 weeks treatment.
iv) The stomach, identified as a target organ, was examined from all animals

Statistics:

Statistical analysis
Bodyweight, haematological and organ weight data were analysed by analysis of variance and, if a between groups difference significant at the 5% level occurred, by pairwise t-tests between the control and treatment groups.
Blood chemistry data were analysed by pairwise Kruskal-Wallis testing, and, if a between groups difference significant at the 5% level occurred, significant differences between the control group and the treatment groups were determined using the Wilcoxon rank sum test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The only clinical sign considered to be related to dosing was the rough or oily appearance to the coat noted from the onset of dosing. Animals from all groups, including the controls, were affected but this sign was most pronounced in animals given 100mg/kg/day.
This clinical sign is largely considered to be due to the use of corn oil as the vehicle.
So it can be concluded that the seen effects are not related to the treatment with the test item itself, it could be maximally due to an slightly enhanced regurgitation non-palability of the test item, but not due to a toxic effect.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Animal 41F (control) died on day 87 during the bleed and animal 72F (100mg/kg/day) was killed for humane reasons on the same day, because of ocular damage caused by orbital sinus puncture. The former animal was considered to have died from ether overdosage.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For the first 5 weeks of the study the mean weight gains of treated and control groups were comparable. Thereafter, animals given 100mg/kg/day showed a reduction in weight gain compared to controls. Mean weight gains for this group over weeks 6-14 were 29% (males) and 38% (females) lower than control values and, these reductions were highly statistically significant (p<0.001). As a consequence, the mean weight gains over the whole treatment period for males and females given 100mg/kg/day were 16% and 17% lower respectively than control values (p<0.001 both sexes).
Animals given 1 or 10mg/kg/day gained similar amounts of weight to controls over each of these periods.
Over the reversibility period the weight gains of both sexes previously given 100mg/kg/day were markedly higher (71% males and 40% females), than those of the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no intergroup differences in food intake, during either the treatment or reversibility periods.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ocular changes or abnormalities.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no adverse haematological changes, although the red blood cell values (Hb, RBC and PCV) of both sexes given 100mg/kg/day tended to be higher than those of the controls during both weeks 4 and 13 of treatment.
Tables are attached.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant treatment-related effects on blood chemistry parameters.
Although various blood chemistry parameters showed statistically significant intergroup differences during weeks 4 and 13 of treatment, none are considerd to be of any toxicological significance. This is because the apparent differences either did not represent a trend with increasing dose, or did not show the expected response for diagnostic significance, or reflected unusual control values, or were not consistent on the 2 occasions.
Amongst the statistically significant differences recorded were:
(i) The mean blood urea nitrogen level of females given 1mg/kg/day was statistically significantly lower than the control value at week 4 but higher at week 13. All values were within the expected range.
(ii) The mean plasma total protein levels of males given 10 or 100mg/kg/day were slightly but statistically significantly lower (up to 7%) than the control value at week 4. At week 13 no such differences were apparent.
(iii) At both weeks 4 and 13 fluctuations in plasma electrolytes (calcium, chloride, sodium, potassium and inorganic phosphorus) were noted.
(iv) At week 4 the mean plasma bilirubin levels of all treated female groups were statistically significantly lower than the control value. No statistically significant group differences existed at week 13.
The remaining isolated statistically significant intergroup differences were considered to be coincidental to treatment.
Tables are attached.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At week 12 the urine specimens of males given 100mg/kg/day showed an increased incidence of trace amounts of blood and increased amounts of ketones and protein compared to the controls. This was not apparent on this occasion in other treated groups.
At the end of the reversibility period the urine specimens of males previously dosed at 100mg/kg/day were similar to those of the controls.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related differences in the organ weights of animaIs killed after 13 weeks of dosing.
Animals previously dosed at 100mg/kg/day, killed at the end of the reversibility period, had statistically significant different values to controls for some organ weights. These tended to be the variations that would be expected in animals showing growth retardation, and are not surprising since the bodyweights of the former high dose animals were still significantly lower than those of the controls.
Tables are attached.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The only macroscopic post mortem finding considered related to treatment was a slight increase in the incidence of reddening or thickening of the glandular mucosa of the stomach in animaIs given 100mg/kg/day.
At the week 13 kill 3/10 males and 2/10 females given 100mg/kg/day showed these changes, whereas none of the controls did.
At the end of the reversibility period reddening of the stomach mucosa was noted in 3/10 males and 1/10 females previously dosed at 100mg/kg/day. However, a control male showed stomach thickening and a further control male also showed reddening of the stomach mucosa.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

All animals killed after being dosed for 13 weeks at 100mg/kg/day had a diffuse, acute inflammation of the glandular region of the stomach other than adjacent to the limiting ridge. This lesion varied in severity from minimal to marked. In most of these animals this was associated with aggregations of macrophages containing brown pigment granules and aggregations of lymphocytes in the submucosa of the glandular region of the stomach. Staining with Perl's reagent indicated that the brown pigment granules contained haemosiderin.
One male killed after being dosed at 10mg/kg/day for 13 weeks showed minimal acute inflammation of the glandular region of the stomach.
Among animals dosed at 1mg/kg/day and killed after 13 weeks treatment no such lesions were observed.
One male and 2 females previously dosed at 100mg/kg/day and then killed after 4 weeks off-dose had diffuse acute inflammation of the glandular region of the stomach, but this was graded as minimal only. An increased incidence of aggregations of lymphocytes and pigmented macrophages in the submucosa of the glandular region of the stomach was also noted for this group, compared to controls.

Histopathological findings: neoplastic:

not specified

Effect levels

1

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: non-neoplastic

urinalysis

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Given data allows the conclusion that the test was well performed and that the results are reliable. However, due to the broad dose selection (1, 10, and 100 mg/kg), only a NO(A)EL can be delineated which may be way below the realistic NO(A)EL; no relevant treatment-related effects were noted at 10 mg/kg. The males were considered slightly more susceptible, and based on the effects described above, a NOAEL of 10 mg/kg will be used for further assessment.

Executive summary:

A study to assess the possibly hazard of EC 401-610-3 upon repeated exposure was investigated according to OECD 408 under GLP.

Method: Groups of 10 male and 10 female Sprague Dawley CD rats were treated daily for 13 weeks, by gavage, with either 0, 10, or 100mg of the test item in corn oil/kg. Supplementary control and high dose groups each comprising 10 males and 10 females were similarly treated for 13 weeks then maintained untreated for 28 days to investigate the reversibility of any findings.

All animals were observed daily, bodyweights were recorded twice weekly for the first 4 weeks of treatment and then weekly thereafter. Food consumption was measured weekly. Blood samples were collected during treatment weeks 4 and 13 from all animals to be killed after 13 weeks of treatment. Urine samples were obtained during week 12 from all rats to be killed after 13 weeks of treatment and from males only of the supplementary groups during the final week of the reversibility period. Ophthalmoscopy was performed on all animals before the start of treatment and again during treatment week 12. At the scheduled necropsy time points the animals were killed and a range of tissues was weighed and examined microscopically.

Results: Treatment with the test item did not affect mortality, clinical signs, food consumption, ophthalmology, haematology, blood chemistry or organ weights.

100mg/kg/day reduced body weight gain of both sexes from week 6. At the end of the treatment period increases /in urinary blood, protein and ketones were seen at this dose level together with macroscopic thickening and in males, reddening of the stomach mucosa, which was associated histopathologically, with acute inflammation of the glandular region. Recovery from all these changes was seen at the end of the 4-week reversibility period.

At 10mg/kg/day, one animal alone showed a minimal degree of the same gastric lesion. -

No treatment related changes wore observed at 1mg/kg/day-

Conclusion: The no observable effect level for female rats was 10mg/kg/day and for males 1mg/kg/day, whereas the NOAEL can be set as 10 mg/kg. There was no effect noted triggering a classification as STOT RE.