Diamminediisocyanatozinc

Endpoint summary

Description of key information

Subacute (28d) study, GLP, Sprague-Dawley rats, m/f, 0, 1, 20, 400 mg/kg, oral (gavage): NOEL = 20 mg/kg

Subchronic (13 week) study, OECD 408, GLP, Sprague-Dawley CD rats, m/f, 10/sex/group + recovery, 0, 1, 10, 100 mg/kg in corn oil, oral (gavage): NOAEL = 10 mg/kg (males), NOEL = 10 mg/kg (females)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

caesarian-derived strain of Sprague-Dawley origin, reared by cross-fostering

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited, Manston Road, Margate, Kent, England. These rats, which were from a caesarian-derived strain of Sprague-Dawley origin, were reared by cross-fostering.
- Females (if applicable) nulliparous and non-pregnant: not stated, but assumed
- Age at study initiation: approx. 4 weeks upon arrival
- Weight at study initiation: 130 g resp. 118 g within a weight range of 17 g for males and 11 g for females
- Fasting period before study: not stated
- Housing: On arrival the rats were placed at random in suspended cages with wire mesh floors, according to sex, so that each cage contained 5 rats of the same sex.
- Diet (e.g. ad libitum): Labsure Laboratory Animal Diet No. 1, ad libitum, except prior to Laboratory investigations
- Water (e.g. ad libitum): Tap water, ad libitum, except prior to Laboratory investigations
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to the Study Director to indicate that any non-nutrient substance likely to influence the effect of the test compound could reasonably be expected to be present in the diet, or the tap water, both of which were routinely subjected to chemical analysis.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Animal room temperature controls were set at 21 ± 2°C
- Humidity (%): Relative humidity controls were set at 50 ± 10%
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): lighting was controlled to give 12 hours light (8.00 a.m. to 8.00 p.m.) and 12 hours dark per 24 hours.

Route of administration:

oral: gavage

Details on route of administration:

The animals were dosed at approximately the same time each day where possible, using a suitably graduated syringe and a rubber catheter (Ch 8 or 10) inserted into the stomach. Throughout the dosing procedure suspensions were maintained on a magnetic stirrer. The dosage volume administered to individual rats was adjusted according to the most recent recorded bodyweight.

Vehicle:

corn oil

Remarks:

suspension

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test compound was administered as a suspension in corn oil. A series of suspensions was prepared, the concentrations being chosen to give a constant dosage volume of 5 ml/kg bodyweight. The dosing suspensions were prepared freshly each day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation sampling
Prior to commencement of the study the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable and that homogeneity of the formulation was satisfactory.
Samples of the formulations prepared in weeks 1, 2, 3 and 4 were also analysed to check the accuracy of preparation. The samples were taken in the animal room just prior to dosing of the animals and the dosing suspensions were kept on a magnetic stirrer during sampling. Additional bulk samples of the formulations prepared in week 4 were despatched for analysis. Chemical analysis was carried out by the Sponsor.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

medium dose

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

high dose

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Health check group before commencement of dosing

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels were chosen by the Sponsor with reference to previous studies.
- Rationale for animal assignment (if not random):
After an acclimatisation period of 6 days each animal was weighed and the required number of animals were randomly selected then allocated to the treatment groups by computerised random number generation so that each group contained a similar population of rats, and the initial group mean bodyweights were approximately equal.
- Fasting period before blood sampling for clinical biochemistry: All rats had free access to tap water and Labsure Laboratory Animal Diet No. 1, except prior to Laboratory investigations.

Positive control:

not required

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day and again in the afternoon to look for dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All signs of ill health, together with any behavioural changes or reaction to treatment were recorded for individual animals. These m . detailed examinations of individual animals were carried out daily on every week day, at suitable intervals after dosing. Examinations were carried out immediately post dosing at weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of allocation of animals to groups, on the day of commencement of treatment, twice a week thereafter and on the day of terminal sacrifice.

FOOD CONSUMPTION:
The quantity of food consumed by each cage of rats was recorded on a weekly basis. Food intake per rat (g/rat/week) was calculated using the amount of food given to, and left by, each cage in each group and the number of rats surviving in each cage.

FOOD EFFICIENCY:
Efficiency of food utilisation: Food conversion ratios were calculated, from bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the water bottles was maintained throughout the study. Water consumption was measured accurately, by weight, over daily periods during week 3 for all cages in all remaining groups.

OPHTHALMOSCOPIC EXAMINATION: No

General remarks on laboratory investigations
During the pre-treatment period haematological investigations were performed on rats assigned for health check.
During week 4 samples of blood were withdrawn, under light ether anaesthesia, from the orbital sinus of 10 males and 10 females from each group.
The blood samples collected were divided into tubes as follows:
EDTA anticoagulant for haematological investigations
Citrate anticoagulant for coagulation tests
Fluoride anticoagulant for plasma glucose estimation
Heparin anticoagulant. for the remaining biochemical tests
Food was removed overnight from animals to be sampled for laboratory investigations, except at the pre-treatment investigations.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 4
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, food was removed overnight.
- How many animals: 10 males and 10 females from each group
- Parameters examined:
The following estimations were performed with a Technicon Hemalog 8/90 Micro, using standard Technicon methodology:
Packed cell volume (PCV) %
Haemoglobin (Hb) g/dl
Red cell count (RBC) x10exp6/mmexp3
Absolute indices calculated as follows:
Mean corpuscular haemoglobin concentration (MCHC)
Hb (g/dl) x 100 / PCV (%) %
Mean corpuscular volume (MCV)
PCV (%) x 10 / RBC (x10exp6/mmexp3) fl
Total white cell count (WBC Total) x10exp3/mmexp3
Platelet count (Pits) x10exp3/mmexp3
The following were performed using the appropriate methodology, as described below:
Differential WBC counts - standard microscopy of blood smear, stained with modified Wright's stain, counting 100 cells, expressed x10exp3/mmexp3.
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Cell morphology: If abnormal cells were observed when examining any stained slide, their presence or absence on each such slide examined were recorded and tabulated separately.
Thrombotest (TT) - Owren, P.A. (Lancet, 1959, ii, 754) (Not performed on health check animals)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 4
- Animals fasted: Yes, food was removed overnight.
- How many animals: 10 males and 10 females from each group
- Parameters examined: Biochemistry
The following parameters were analysed with a Technicon SMA 12/60, using standard Technicon SMA methodology:
Total Protein g/dl
Albumin (Alb) g/dllobulin (Glob) - By subtraction Total Protein (g/dl) minus Albumin (g/dl) g/dl
Urea nitrogen (Urea Nitr) mg/dl
Creatinine mg/dl
Sodium (Na) mEq/l
Potassium (K) mEq/l
Calcium (Ca) mEq/l
Inorganic phosphorus (P) mEq/l
Chloride (Cl) mEq/l
The following parameters were analysed using a Roche Cobas Centrifugal analyser, using the appropriate BCL test kit:
Alkaline phosphatase (AP), Reaction temperature 30°C mU/ml
Glutamic-pyruvic transaminase (GPT), also known as 'alanine aminotransferase', Reaction temperature 30°C mU/ml
Glutamic-oxaloacetic transaminase (GOT), also known as 'aspartate aminotransferase', Reaction temperature 30°C mU/ml
Cholesterol (Choi) - (Enzymatic assay) mg/dl
Glucose (Hexokinase mediated assay) mg/dl
Total bilirubin mg/dl

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Post mortem examination
On completion* of 4 weeks of treatment, all remaining rats were killed.
The terminal procedures took 2 days to complete, thus the dosing of individual treated animals was continued until the day prior to being killed. Approximately 5 male and 5 female animals from each group were killed on each day.
All rats were killed by carbon dioxide asphyxiation and subjected to necropsy procedure indicated below:
All superficial tissues were examined visually and by palpation, and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable i1lumination. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

The following organs from all animals killed at the scheduled sacrifice were dissected free of fat and weighed: adrenals, kidneys, spleen, heart, liver, testes, ovaries
The combined weight of paired organs was recorded.
The weights of major organs of individual rats dying or killed during the study were recorded at the discretion of the pathologist.

Preservation of tissues
Samples of all the tissues listed below from all animals were preserved in buffered 10% formalin (except eyes, which were preserved in Davidson's fixative).
adrenals*
alimentary tract (oesophagus, stomach*, duodenum, jejunum, ileum, caecum, colon, and rectum)
aorta
brain (medullary, cerebellar and cortical sections)
eyes
femur (with joint)
Harderian gland
head (to preserve nasal cavity, paranasal sinuses, oral cavity, nasopharynx, middle ear, teeth, lachrymal gland and Zymbal’s gland)
heart*
kidneys*
larynx and pharynx
liver*
lungs* (all lobes and mainstem bronchi)
lymph nodes (cervical and mesenteric)
mammary gland
ovaries
other macroscopically abnormal tissue*
pancreas
pituitary
prostate
salivary gland
sciatic nerve
seminal vesicles
skeletal muscle
skin
spinal column (to preserve samples of spinal cord from cervical, thoracic and lumbar levels)
spleen*
sternum (for bone and marrow)
testes (with epididymides)
thymus (where present)
thyroid (with parathyroid)
tongue
trachea
urinary bladder
uterus (corpus and cervix)
vagina
*Tissues required for histopathological study

In addition, samples of any macroscopically abnormal tissues were routinely preserved, along with samples of adjacent tissue where appropriate.
This extensive list of tissues preserved was intended to satisfy any possible future requirements for further examination of tissues.

HISTOPATHOLOGY: Yes
Tissues required for microscopic examination were embedded in paraffin wax and sections cut at 5 micrometres were stained with haematoxylin and eosin.
Frozen sections of liver, fixed in buffered formalin, were cut on a cryostat at 12 micrometres and stained for fat with Oil Red O (ORO).
Histopathological examination was restricted to macroscopically abnormal tissues in any animal and the specified list of tissues from the control, intermediate and high dosage groups.

Statistics:

Statistical analysis
All statistical analyses were carried out separately for males and females.
Data relating to food and water consumption were analysed on a cage basis. For all other parameters, the analyses were carried out using the individual animal as the basic experimental unit.
Food consumption data were analysed using cumulative cage totals, and water consumption data were analysed as the total recorded intake over selected time periods. Bodyweight data were analysed using weight gains.
The following sequence of statistical tests was used for food consumption, water consumption, bodyweight, organ weight and clinical pathology data:
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 stable variance structure could be obtained.
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.
Analyses of variance were followed by Student's 't' test and Williams* test for a dose-related response, although only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the 't' test and Williams' test (Shirley's test).
For organ weight data, the final bodyweight was used as a covariate in an attempt to allow for differences in bodyweight which might influence the organ weights. Covariance analysis was used where there was a significant (at the 10% level) linear•correlation between organ weight and final bodyweight.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of reaction to treatment were as follows :
At 400 mg/kg/day, salivation (often excessive) with associated brown staining around the muzzle, paddling movements of forepaws, respiratory distress (gasping), lethargy and walking on toes were noted post dosing for most individuals on the majority of occasions.
At 20 mg/kg/day, salivation was noted post dosing for most individuals on a few occasions during the latter half of the study. Isolated episodes of walking on toes were also noted post dosing. These findings were confined to the post dosing period and were considered not to be indicative of any toxicity of the test material.
No signs of reactions to treatment were noted at 1 mg/kg/day. With the exception of signs reported below for mortalities, all other signs were considered not attributable to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

All animals receiving 400 mg/kg/day were sacrificed in moribund condition or found dead during the first 2 weeks of treatment.
Signs of poor condition, in addition to signs reported during the post dosing period, included: pilo-erection, hunched posture, partial closure of the eyes, decreased respiration and unsteady gait.
One female rat receiving 1 mg/kg/day died under ether anaesthesia for laboratory investigations in week 4. Pathological findings were consistent with anaesthetic death and no treatment-related findings were noted.
Factors contributory to death:
For every rat dying or killed in a moribund state during the treatment period the pathologist attempted to determine the major factor or factors contributory to death or moribund condition of the rat.
Gastric lesions were contributory factors in 9/10 male and 9/10 female rats receiving 400 mg/kg/day. These factors were considered to be treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related effect was observed on bodyweight for those animals surviving through to termination. There was a marked reduction in bodyweight gain for rats receiving 400 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no treatment-related effect on food consumption for those animals surviving through to termination. There was a marked reduction in food intake for rats receiving 400 mg/kg/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Efficiencies of food utilisation were essentially similar for all groups surviving through to termination. Inferior efficiency of food utilisation was seen for rats receiving 400 mg/kg/day.

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

no effects observed

Description (incidence and severity):

There was no treatment-related effect on water consumption (only low and mid dose animals were evaluated)

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effect was observed (only low and mid dose animals were evaluated).
Minor statistically significant group differences noted in platelet counts and neutrophil counts were considered not to be treatment-related. Values recorded were well within expected ranges.
The corresponding table is attached.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effect was observed.
Minor statistically significant group differences noted in some parameters were considered not to be treatment-related. Values recorded were well within expected ranges.
The corresponding table is attached.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related effects were noted on organ weights of rats killed at termination of the study.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The examination of the decedent rats treated with 400 mg/kg/day revealed the following changes probably attributable to treatment.
Gastro-intestinal tract: Contents minimal, contents watery, distension, thickening of forestomach limiting ridge, haemorrhagic depressions of stomach mucosa, pallor of stomach mucosa, congestion of stomach mucosa (male rats only).
Spleen: Pallor (female rats only) and small spleen.
Seminal vesicles: Contents minimal.
Adipose tissue: Minimal (female rats only).
No treatment-related findings were seen in rats receiving 1 or 20 mg/kg/day killed at termination of the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

PART A
The following treatment-related changes were observed in rats from the 400 mg/kg/day dosage group:
Stomach:
Mucosal necrosis of glandular region graded focal or areas or generalised in 4/10 male rats and 8/10 female rats;
reduction/degeneration of the parietal cells of the glandular region graded minimal, moderate or marked in 6/10 male rats and 9/10 female rats;
hyperplasia of the epithelial cells of the glandular region of 5/10 male rats and 5/10 female rats;
minimal or moderate inflammation of the glandular region of 9/10 male rats and 10/10 female rats associated with vascular congestion in the majority of the male and female rats and minimal haemorrhage in rat 77female and areas of haemorrhage in rat 72female;
oedema of the glandular stomach graded minimal or moderate in 5/10 male rats and 7/10 female rats;
oedema of the limiting ridge and/or non-glandular stomach of 7/10 male rats and 7/10 female rats.
Spleen:
Minimal reduction of lymphoid tissue of 2/10 male rats and 5/10 female rats.
Similar changes were not observed in rats from the other treatment groups or the control group. The minimal submucosal inflammation > observed in the glandular region of the stomach in a number of treated and contrpl rats was considered to be of no toxicological significance. The incidence of this change in male rats receiving 20 mg/kg/day was considered fortuitous and unrelated to any other microscopic change.

Factors contributory to death
For every rat dying or killed in a moribund state during the treatment period the pathologist attempted to determine the major factor or factors contributory to death or moribund condition of the rat.
Gastric lesions were contributory factors in 9/10 male and 9/10 female rats receiving 400 mg/kg/day. These factors were considered to be treatment-related.

PART B
All other histopathological findings were considered not to be treatment-related.

Histopathological findings: neoplastic:

not specified

Dose descriptor:

LOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

food efficiency

gross pathology

histopathology: non-neoplastic

mortality

Remarks on result:

other: All animals of the high dose group had to killed in moribund state within the first two weeks.

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Remarks on result:

other: mid dose

Critical effects observed:

no

PRE-TREATMENT HEALTH CHECK

Macroscopic examination and haematological investigations of the 10 male and 10 female rats killed prior to the start of treatment for health check purposes revealed no abnormalities.

No signs directly attributable to a specific organ toxicity were noted, rather to a general poor conditions and moribund state.

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, 20, and 400 mg/kg), only a NOEL can be delineated which may be way below the realistic NO(A)EL; no effects were noted at 20 mg/kg, all animals had to be preliminarily killed due to a moribund state. So this 28 day study should not be regarded for DNEL derivation, rather the also available 90 day study with prolonged exposure and narrower dosing intervals.

Executive summary:

In this GLP study related to OECD TG 407, three groups of 10 male and 10 female CD (UK) rats received the test material by daily oral gavage of suspensions in corn oil at a constant dosage volume of 5 ml/kg bodyweight, to give levels of 1, 20 or 400 mg/kg/day. A fourth group (Control), of the same size, received the vehicle alone at the same dosage volume. Treatment was continued for up to 28 consecutive days.

 

The main findings are summarised as follows:

 

400 mg/kg/day:

At 400 mg/kg/day all rats were sacrificed in moribund condition or were found dead during the first 2 weeks of treatment. Post dosing signs of salivation with associated brown staining around the muzzle, paddling movements of forepaws, walking on toes, respiratory distress and lethargy were noted. Signs of poor condition characterised by poor weight gain, hunched posture, pilo-erection, decreased respiration and unsteady gait were noted in many animals.

Post mortem examination revealed; minimal gastro-intestinal tract contents which were watery, thickening of forestomach limiting ridge, pallor and haemorrhagic depressions of stomach mucosa, congestion of stomach mucosa (males only) and small spleen with pallor in a few females.

Histological examination of the glandular region of the stomach revealed; varying degrees of mucosal necrosis, reduction/degeneration of the parietal cells, hyperplasia of the epithelial cells, inflammation with vascular congestion, oedema. In addition, oedema of the Limiting ridge and/or non-glandular stomach was noted. Histological examination of the spleen revealed minimal reduction of lymphoid tissue in a few rats. No other treatment-related histological findings were noted.

 

20 or 1 mg/kg/day:

Post dosing signs of salivation were seen on occasions at 20 mg/kg/day. No persistent or cumulative signs of reaction to treatment indicative of toxicity were seen at 20 or 1 mg/kg/day.

No treatment-related findings were evident for bodyweight, food and water consumption, food conversion, haematology, biochemistry, macroscopic and microscopic pathology, and organ weights at 20 or 1 mg/kg/day.

 

The NOEL was set to 20 mg/kg.

Reference 2

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

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

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

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

medium dose

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

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.

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

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

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

Critical effects observed:

no

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

24

Species:

rat

Quality of whole database:

There is a guideline-conform subchronic study under GLP available, the results are supported by a subacute study. Hence, the tonnage-driven data requirements are fulfilled and the database is of high quality.

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

Mode of Action Analysis / Human Relevance Framework

There is only a subchronic oral key study available, supprted by a subacute one. Hence, a MoA analysis in its classic sense can not be performed.

Justification for classification or non-classification

There were no organ-specific effects noted in the available subchronic key study, as described in Regulation 1272/2008. All effects are considered as general systemic ones, or secondary to local irritation. So no classification as STOT RE is triggered.