Ziram

Endpoint summary

Description of key information

Oral toxicity

Key, report number ZIR 5/901840, subchronic (90 days, rat):

NOAEL: No NOAEL could be set

LOAEL (oral): 100 ppm corresponding to 7.4 mg/kg bw/day (males) and 8.8 mg/kg bw/day (females)

 

Key, report number ZIR 8-G/901813, subchronic (90 days, dog):

NOAEL (oral): 100 ppm corresponding to 4.07 mg/kg bw/day (males) and 4.31 mg/kg bw/day (females)

 

Key, report number ZIR 10/920533, chronic (52 weeks, dog):

NOAEL (oral): 50 ppm corresponding to 1.6 mg/kg bw/day (males) and 1.9 mg/kg bw/day (females)

 

Key, report number ZIR 9/942098, chronic/carcinogenicity (2 years, rat):

NOAEL (general systemic toxicity, oral): No NOAEL could be set

LOAEL (general systemic toxicity, oral): 60 ppm, corresponding to 2.5 mg/kg bw/day (males) and 3.4 mg/kg bw/day (females)

 

Key, report number 83-5121, chronic/carcinogenicity (2 years, rat):

NOAEL (general systemic toxicity, oral): 16 ppm, corresponding to 0.56 and 0.66 mg/kg bw/day for males and females, respectively.

 

Inhalation toxicity

Key, report number UCB 709/003932, subacute (28 days, rat, dust):

NOAEC (respiratory system): 0.1 mg/m³ air (females, males)

NOAEC (general systemic toxicity): 3 mg/m³ air (females, males)

 

Dermal toxicity

Key, report number ZIR 4/89689, subacute (21 days, rat, occlusive):

NOAEL (local, dermal): 1000 mg/kg bw/day (females, males) and corresponding to approx. 11.81 mg/cm²

NOAEL (general systemic toxicity, dermal): 100 mg/kg bw/day (females, males)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May - 24 Aug 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

adopted in 1998

Deviations:

yes

Remarks:

Not all requested organs were weighed. The highest dose level produced one fatality and therefore exceeded the MTD (maximum tolerable dose)

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Consort Limited, Herefordshire, UK
- Age at study initiation: approximately 21 - 24 weeks (males) and 20 - 21 weeks (females)
- Weight at study initiation: 6.2 - 10 kg (males) and 6.8 - 9.4 kg (females)
- Housing: 2 dogs of the same sex were housed in kennels.
- Diet: Standard ground dry diet (Diet A: Special Diets Services) 400 g/animal/day
- Water: Tap water, ad libitum
- Acclimation period: at least 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): not provided
- Air changes (per hr): approximately 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
Fresh treated diet was prepared weekly, usually one day prior to the beginning of the dose week. The required amount of test compound was weighed and ground diet added until the desired concentration of pre-mix was achieved. This was blended in a mixer for a minimum of 2 min. Diets for each group were prepared by direct dilution of the pre-mix. Homogeneity of all diets was achieved by mixing for at least 7 min in a double cone blender. Formulated diets were stored at -20 °C, protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the commencement of the study, the proposed formulations were checked by chemical analysis (spectrophotometry at 435 nm after carbon disulphide extraction) to confirm that the method was acceptable and that the stability of the formulation was satisfactory. Samples of the dietary formulations prepared in Weeks 1 and 13 were also analysed to check the accuracy of preparation. In addition, samples of the 100 ppm formulation prepared in Week 1 were analysed following storage at room temperature for 8 h.
The mean concentrations of the test substance in dose formulations were within 7% of the nominal concentrations. It was also confirmed that the test substance was homogeneously blended in the diet. The stability data indicate that the test substance was stable at 5000 ppm in the diet for up to 14 days storage at ambient temperature. For the 100 ppm diet stability was not affected for an 8 h storage at ambient temperature but after storage periods of 24 h and 14 days, and therefore, a storage at -20 °C was recommended for diet formulations, fed on a daily basis and consumed within 8 h.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously via diet

Dose / conc.:

100 ppm

Remarks:

corresponding to 4.07 mg/kg bw/day for males and 4.31 mg/kg bw/day for females

Dose / conc.:

300 ppm

Remarks:

corresponding to 12.15 mg/kg bw/day for males and 13.04 mg/kg bw/day for females

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 41.81 mg/kg bw/day for males and 41.07 mg/kg bw/day for females

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Regularly throught the working day.
- Cage side observations checked: All signs of ill health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to feeding, once a week throughout the pre-dosing and dosing periods

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was calculated as g food per dog per week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: From the food consumption data obtained, the quantity of the test substance ingested weekly by each group was calculated. The approximate dose equivalents expressed as mg/kg bw/day were calculated at weekly intervals from the mean body weight data.
The quantity of food left by individual animals was recorded daily throughout the experimental period.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before dosing and during Week 13 by means of a Keeler indirect ophthalmoscope
- Dose groups that were examined: all animals
Prior to examination, the pupils of each animal were dilated using a Tropicamide ophthalmic solution.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before dosing and during Weeks 6 and 13
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: erythrocyte count (RBC), haemoglobin, haematocrit, mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), total leukocyte count, differential leukocyte count, platelet count, reticulocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT), cell morphology and packed cell volume (PCV)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before dosing and during Weeks 6 and 13; collection period of 16 h, water was removed from the kennels about 5 h prior to the start of collection
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: total protein, albumin, sodium, potassium, glucose, urea (BUN), alanine aminotransferase (GPT), aspartate aminotransferase (GOT), alkaline phosphatase (AP), creatinine, calcium, phosphorus, chloride, total cholesterol, total bilirubin, gamma-glutamyltransferase (GT), creatine phosphokinase (CPK) and ornithine carbamoyl transferase (OCT)

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: before dosing and during Weeks 6 and 13
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes, overnight
- Parameters checked: volume, specific gravity, glucose, pH, protein, ketones, haem pigments, bile pigments, urobilinogen, total reducing substances, epithelial cells (E), polymorphonuclear leukocytes (P), mononuclear leukocytes (M), erythrocytes (R), organisms (O), renal tubule casts (C), other abnormal constituents (A)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Prior to terminal autopsy, bone marrow was obtained from each animal by sternal puncture and a smear prepared for subsequent staining and examination.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of 13 weeks dosing each animal was killed by exsanguination under pentobarbitone anaesthesia. All animals were fasted overnight prior to post mortem examination. Since the post mortem procedures took 3 days to complete, the dosing of individual treated animals continued until the day prior to their sacrifice.
At autopsy the macroscopic appearance of the tissues was noted and the weights of the following organs recorded (paired organs being weighed separately): adrenals, brain, heart, kidneys, liver, lungs, pancreas, pituitary, spleen, testes or ovaries, thymus, thyroids (and parathyroids) and uterus or prostate.

HISTOPATHOLOGY: Yes
The tissues listed below were examined for all animals by light microscopy: Adrenals, aorta (arch and abdominal), femur and articular surface, sternum, brain (cerebral cortex, thalamic nuclei, mid-brain, medulla and cerebellum), epididymides, oesophagus, gall bladder, heart, kidneys, caecum, colon, rectum, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), ovaries, pancreas, pituitary, prostate, mammary gland, sciatic nerve, duodenum, jejunum, ileum, spleen, skeletal muscle, skin, salivary gland (submandibular), spinal cord (cervical, thoracic and lumbar), stomach, testes, thymus, thyroids/parathyroids, trachea, tongue, eyes, urinary bladder, uterus, vagina and all gross lesions.

The fixative routinely used was 10% buffered formalin. The eyes were preserved in Davidson's fixative whilst additional pieces of liver and kidney were placed in formol calcium. Tissues were embedded in 56 °C M.P. paraffin wax and sections were cut at 4 µm and stained with haematoxylin and eosin. Cryostate sections of liver previously fixed in formol calcium were cut at 12 µm and stained with Oil Red O. Liver sections were stained using PAS for glycogen.

Statistics:

All analyses were carried out both together and separately for male and female.
Data relating to food consumption were analysed as totals over selected time periods, expressed on a weekly basis. Bodyweight data were analysed using weight gains.
The following tests were used for food consumption, bodyweight, organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:
- 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.
- For pre-dose data, analyses of variance were followed by Student’s ‘t’ test. For data from the dosing period, analyses of variance were followed by Williams’ test for a dose-related response. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate, analysis of covariance was used in place of analysis of variance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Convulsive episodes were noted for the dog of the high dose group (1000 ppm) being sacrificed for humane reasons in Week 5 as discussed under "mortality". In addition, a female dog of the high dose group (1000 ppm) was noted to be trembling on one occasion in Week 8.
There were no other clinical signs attributable to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male dosed at 1000 ppm was sacrificed during week 5, because of a seizure without apparent significant recovery. Convulsive periods had previously been observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reductions in body weight were noted for one female dog (1000 ppm) from Week 4 to 6, with subsequent recovery from Week 7 and for one male dog (1000 ppm) from Week 2 until its death in Week 5, and were considered to be related to the treatment. Body weight loss and inappetence were also noted for a male dog of the high dose group (1000 ppm) during Week 3 and 4, however, as this was associated with a retropharyngeal abscess it was considered to be unrelated to treatment. With resolution of the abscess from Week 5, appetite restoration and body weight gain were noted for this animal. It was concluded that treatment with the test substance produced a slight reduction in body weight gain for the 1 male and 1 female dog at 1000 ppm.
For details, please refer to attachment 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

From weeks 3 to 5, a gradually reduced food intake was recorded for one female dog of the high dose group (1000 ppm) with some subsequent recovery from Week 6. Inappetence was also noted for one male dog (1000 ppm) from Week 2 until its death in Week 5. These changes were considered to be associated with treatment. In addition, inappetence and associated body weight loss were noted for a male dog of the high dose group (1000 ppm) during weeks 3 and 4 and was associated with a retropharyngeal abscess, as previously discussed, and was considered to be unrelated to treatment. It is concluded that treatment with the test substance was associated with a slightly reduced food intake for 1 male and 1 female animal receiving 1000 ppm.
For details, please refer to attachment 2 under "Overall remarks, attachments".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no changes observed during Week 13 that were considered to be related to the treatment with the test substance.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At Weeks 6 and 13, group mean red cell indices (PCV, Hb, RBC) were slightly reduced in comparison to concurrent control values, for males and females receiving 1000 ppm. However, these differences were not progressive and did not always attain statistical significance. In addition, during Week 6, slightly elevated circulating reticulocyte counts, with associated slight polychromasia, hypochromasia and/or moderate anisocytosis, were noted for one male and one female animal at 1000 ppm. Slightly elevated circulating reticulocyte count was also noted for one female dog (1000 ppm) during Week 13. Group mean APTT values for males receiving 300 ppm during Week 13 and 1000 ppm during Weeks 6 and 13 were slightly but statistically significantly higher than corresponding control values. A similar change was apparent for females at 1000 ppm during Weeks 6 and 13, although these did not attain statistical significance.
There were no other changes considered to be attributable to treatment. As the EDTA sample for haematological investigations was clotted, no haematology results were obtained for the animal sacrificed at Week 5. However, values for PT and APTT were slightly lower than pre-dose values.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

During Week 6, high bilirubin, AP, GPT, GOT, OCT and cholesterol values were recorded for one female dog (1000 ppm), with the enzyme levels remaining clearly elevated, though to a lesser extent, on repeat investigation in Week 7. These values, however, were essentially unremarkable at Week 13, with the exception of AP which was still slightly elevated. Mean AP values were increased among females receiving 1000 ppm at Weeks 6 and 13 in comparison with values for concurrent controls, attaining statistical significance in Week 13. In addition, there was an apparent dose-related increase in cholesterol for males and females at Weeks 6 and/or 13, attaining statistical significance at 300 and 1000 ppm. Group mean albumin concentrations were slightly, but statistically significantly lower than corresponding control values for animals at 1000 ppm during Week 6 and for animals from all treated groups during Week 13. This was associated with a corresponding increase in globulin concentrations, such that total protein levels were unaltered. There were no other changes considered to be attributable to treatment.
The male dog sacrificed at Week 5 showed slightly higher serum GPT, sodium and chloride levels and markedly higher serum AP, GOT, CPK and cholesterol
values than the corresponding pre-dose values.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

A high urinary SG, protein and traces of total reducing substances were noted in the urine of the male dog killed for humane reasons in Week 5. There were no other effects on urinalysis parameters that were considered to be related to the treatment with the test substance.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean liver weights for males and females at 1000 ppm were statistically significantly higher than control values, with individual liver weights for 3 males and 1 female at this dose exceeding the normal upper limit of 4% of body weight, and this was considered to be related to treatment. A slightly high liver weight for one male dog at 300 ppm, equaling the normal upper limit of 4% body weight, was also considered to be related to treatment. The individual liver weight for one female dog at 100 ppm also exceeded the normal upper limit of 4% of body weight. In isolation, this finding is considered to be of uncertain relationship to treatment.
In addition, a statistically significantly lower group mean heart weight for males at 1000 ppm and lower group mean lung weight for females at this dose was also noted in comparison to controls. However, as differences were slight, not dose-related and confined to 1 sex only and as all individual values were within normal limits for dogs of this age, these findings are considered to be incidental and unrelated to the treatment with the test substance.
In the male animal of the high dose group (1000 ppm) killed for humane reasons in Week 5, liver weight was increased, which at 4.33% of body weight exceeded the normal upper limit of 4.0%. Spleen and prostate weights were also considered to be lower than expected in this animal.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopically, multiple depressed pale areas were noted on the liver of one female dog receiving 1000 ppm, which correlated with histopathological findings.
In the male dog sacrificed at Week 5 (1000 ppm group) pallor of the endocardium, extending into the myocardium of the left ventricle and an area of red endocardial discolouration on the left atrioventricular valve of the heart were noted. Further pallor of the liver, with accentuation of the lobular markings, and a friable cut surface, was seen grossly. All these macroscopic findings were accompanied by histopathological changes.

There were no other findings considered to be of toxicological importance, in particular no treatment-related changes were seen in the hearts of any dogs killed at termination.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a slight relative increase in myeloid cells in the smear from a male dog of the control group. For all other animals sacrificed after 13 weeks of treatment, all smears were normal in cellularity, distribution or cell morphology.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings were noted in liver and lungs as follows:
Liver:
Corresponding to the macroscopic observations in the liver, one female dog receiving 1000 ppm showed focal necrosis, together with loss of cells and dilated sinusoids. This finding correlates with the high individual AP, GPT, GOT and OCT values previously noted for this dog. Focal necrosis was also seen in one male dog treated with 300 ppm. Minimal amounts of pigment were seen in the Kupffer cells of one male and one female animal receiving 300 ppm and for two males and one female at 1000 ppm. These changes were not seen in control dogs or dogs receiving 100 ppm.

Lungs:
Minimal foci of eosinophilic homogeneous material were seen in the alveoli of the lungs of one male and one female animals receiving 100 ppm, one male animal receiving 300 ppm and one male animal receiving 1000 ppm. In the male dog receiving 1000 ppm, this change was associated with acute inflammatory cell infiltration. The toxicological significance of these findings, which were not related to dose, is not clear.

There were no other findings considered to be of toxicological importance.

In the male dog sacrificed in Week 5, focal myocardial necrosis with acute inflammatory cell infiltration and haemorrhage was seen histologically. Further, centrilobular hepatocyte degeneration was reported histologically. This was possibly secondary to the heart changes seen. These changes were considered likely to be related to convulsions seen clinically rather than a direct effect of the test compound. Areas of polymorphonuclear leucocyte infiltration into the alveoli were also noted for this dog.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 4.07 mg/kg bw/day for males and 4.31 mg/kg bw/day for females

Key result

Dose descriptor:

LOAEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: additionally, clincial signs, mortality, changes in body weight, food consumption, clinical biochemistry, and gross pathology were observed at 1000 ppm.

Remarks on result:

other: corresponding to 12.15 mg/kg bw/day for males and 13.04 mg/kg bw/day for females

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The present study was conducted to assess the sub-chronic toxicity of the test substance on dogs when given for a time frame of approximately 13 weeks. The study was similar to the OECD guideline 409 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 4 male and 4 female dogs at dose levels of 100, 300 and 1000 ppm corresponding to approximately 4.07, 12.15 and 41.81 mg/kg bw/day for males and 4.31, 13.04 and 41.07 mg/kg bw/day for females. Under the conditions of the test, the test substance caused clinical signs, reduced body weights and food consumption and changes in haematology and clinical biochemistry parameters, organs weights, gross pathology and histopathology at 300 and/or 1000 ppm. Therefore, the NOAEL was set at 100 ppm for males and females (corresponding to 4.07 and 4.31 mg/kg bw/day respectively).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jul - 26 Oct 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

Animals not observed daily after Week 4 and histopathology was carried out on a limited number of organs. Further deviations were noted under “Principles of methods if other than guideline”.

Principles of method if other than guideline:

Deviations to the current guideline:
Urinalysis and functional observations were not performed and thyroid hormones were not investigated as the protocol was similar to the OECD 408 guideline of 1981. No rational was provided for dose selection.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Portage, Michigan, USA
- Age at study initiation: Approximately 28 days
- Weight at study initiation: 157 - 188 g (males) and 117-144 g (females)
- Housing: Groups of 5 animals/sex were housed in suspended cages with wire meshfloors.
- Diet: Powdered SDS Rat and Mouse No. 1 modified maintenance diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
On a weekly basis, a pre-mix was prepared by grinding the test substance directly into diet and mixing for a minimum period of 2 min. The required concentrations were then prepared by direct dilution of the pre-mix with further quantities of untreated diet; homogeneity being achieved by mixing in a double cone blender for a minimum period of 7 min. The concentration of test substance in the low-dose level was increased by 15% above nominal to 115 ppm in order to compensate for losses during storage already observed in pre-dosing analysis. On completion of the mixing procedure, the diets to be fed at each (dose) level were divided into 3 aliquots for use during the following week. Animals were offered fresh diet daily and all aliquots were stored at +4°C until immediately before and again after feeding.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary samples were analysed before start of treatment as well as during the present study sampled in Weeks 1, 6 and 13. Analysis of diets prepared for use in this study showed good agreement with the nominal inclusion levels required, under the conditions used. It was also demonstrated that at the lowest inclusion level the fortification adequately compensate for any degradation.

1) Prior to the start of treatment the proposed diet mixing procedures were checked by chemical analysis of trial diets to confirm that the proposed procedures produced homogeneous diet, that the accuracy of mixing was acceptable and that the concentration of test substance in the diet remained unchanged between preparation and administration. Based on this analysis, it was apparent that the test substance was not sufficiently stable in rodent diet to permit storage at room temperature for weekly or twice weekly feeding. Following further analytical work satisfactory levels were achieved by preparation of diets weekly and storage at +4 °C prior to use and 15% fortification of the low inclusion levels of test substance.

2) Samples of diets of the present study prepared in Weeks 1, 6 and 13 were also analysed to check the accuracy of preparation. On each occasion the diets were analysed immediately on preparation and again after storage for 7 d at +4 °C followed by exposure to the animal room environment for 24 h. Therefore, representative samples of dose formulations were taken at discharge from the blender and riffled to produce duplicate sub-samples for analysis and post-dose samples of dose formulations were received from the animal rooms after appropriate storage and riffled to produce duplicate sub-samples.

Mean results for pre-dose samples were all within 5% of nominal concentrations. Mean results for post-dose samples were all within 10% of nominal concentrations. The results confirm that, at nominal concentrations of 70 ppm and 5000 ppm, the test substance can be homogeneously blended in rodent diet. Results for Trial 1 show that at 5000 ppm no significant change in concentration occurred over the 15 day storage period, however, at 70 ppm a reduction of 15.5% occurred after 4-day storage and the concentration continued to drop after 8-day (-26.3%) and 15-d (-34.5%) storage. The results for Trial 2 show a much less significant loss in concentration (-10.9%) after 1-day storage but with further reduction (-15.7 %) after 2-day storage. The Day 0 data from Trials 1 and 2 confirm the stability of the 70 ppm formulations during storage at -20 °C for 59 days. The results obtained for Trial 3 confirmed that no significant change (100 ppm: -9.9%, 300 ppm: -9.2%) in concentration occurred during this period. Diets formulated for dosing during the study were prepared on a weekly basis, stored at +4°C and fed daily. The procedural recovery data obtained during method validation and the determination of stability confirm that the method of analysis described provides good extraction efficiency for the test substance from the rodent diet matrix. A mean procedural recovery value of 92.9% + 2.72 SD (n = 19) was obtained at 70 ppm and 92.2% + 5.25 SD (n = 16) at 5000 ppm. All results were corrected for the appropriate procedural recovery value at analysis.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

100 ppm

Remarks:

The concentration of test material in the low dose level was increased by 15% above nominal (115 ppm) to compensate losses during storage. In total this corresponded to 7.4 and 8.8 mg/kg bw/day in male and female rats, respectively.

Dose / conc.:

300 ppm

Remarks:

corresponding to 21.4 and 24.2 mg/kg bw/day in male and female rats, respectively

Dose / conc.:

1 000 ppm

Remarks:

corresponding to 67.8 and 76.9 mg/kg bw/day in male and female rats, respectively

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule and cage side observations checked: Two checks were made on a daily basis for dead or moribund animals. Individual animals were observed for any signs of behavioural changes, reaction to treatment or ill health once daily on working days for the first four weeks and then once weekly.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of allocation of animals to groups, on the day of commencement of treatment, and once a week thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was calculated as g/rat/week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Compound intake was calculated on a weekly basis based on the ppm inclusion level, the mean food consumption and the mean body weight. The weekly achieved compound intake was then used for the calculation of the mean compound intake over the whole treatment period.
- Time schedule: The quantity of food consumed by each cage of rats was recorded on a daily basis.

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: Food conversion ratios were calculated from body weight and food consumption data as weight of food consumed per unit gain in body weight.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: Before treatment commenced the eyes of all allocated rats were examined. The eyes of all animals in the control and high dosage level groups were examined by indirect ophthalmoscope during the final study week (Week 13).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: haematocrit, erythrocyte count, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration, platelet count, total leukocyte count, differential leukocyte count, cell morphology, thrombotest

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13
- Animals fasted: Yes, overnight
- How many animals: all rats
- Parameters checked: glucose, blood urea nitrogen, creatinine, total bilirubin, total cholesterol, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, calcium, phosphorus, sodium, potassium, chloride, albumin, total protein, albumin/globulin ratio

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At scheduled sacrifice, surviving animals were sacrificed by CO2 asphyxiation.
At scheduled necropsy, designated organs of all surviving animals were dissected free of fat and weighed: Adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroid, uterus.

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 illumination. The liver was sectioned at intervals of a few millimeters; 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. Gross lesions were examined from all animals. The following tissues were removed from all animals and preserved in buffered 10% formalin (except eyes: Davidson’s fixative): adrenals, aorta, brain (medullary, cerebellar, cortical sections), caecum, colon, duodenum, eyes, femur (with joint), Harderian gland, head (nasal cavity, paranasal sinuses, oral cavity, nasopharynx, middle ear, teeth, lachrymal gland, Zymbal’s gland), heart, ileum, jejunum, kidneys, larynx, liver, lungs (all lobes and mainstem bronchi), lymph nodes (cervical, mesenteric), mammary glands, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach, testes with epididymides, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus (corpus, cervix), vagina and other macroscopically abnormal tissues.

HISTOPATHOLOGY: Yes
The following tissues were embedded in paraffin wax and sections were cut at 4 µm stained with haematoxylin and eosin except for the liver. Frozen sections of the liver were fixed in formalin and were cut on a cryostat at 12 µm and stained with Oil Red O. Sections of kidney were stained with Oil Red O or Periodic Acid-Schiff reagent.
In the first instance, histopathological examination was restricted to:
1. The following list of tissues including all macroscopically abnormal tissues from all animals from the control and high-dose group:
adrenals, aorta, brain (medullary, cerebellar, cortical sections), caecum, colon, duodenum, heart, ileum, jejunum, kidneys, liver, lungs (all lobes and mainstem bronchi), lymph nodes (cervical, mesenteric), oesophagus, ovaries, pancreas, pituitary, rectum, salivary glands, sciatic nerve, spleen, sternum, stomach, testes with epididymides, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus (corpus, cervix).
2. Any macroscopic abnormal tissue in any animal.
3. Lungs, liver, kidneys and stomach from all rats in the low and intermediate dose groups.

Statistics:

All analyses were carried out separately for male and female.
Data relating to food and water consumption were analysed on a cage basis. All other parameters were carried out using the individual animal as basis.
Histopathological findings were analysed using Fisher’s exact analysis.
The following tests were used for food and water consumption, bodyweight, relative organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:
- 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.
- Except for pre-dose data, 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 has been reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate, during absolute organ weight analysis, analysis of covariance was used in place of analysis of variance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slightly higher incidence of hair loss in males/females given 300 and 1000 ppm. Not apparent among controls or low-dose group and not confirmed at post mortem examination.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control female died during week 13. Examinations revealed congestion in the lungs, the smell of ether in the thoracic cavity and a pale subcapsular area on the median liver-lobe.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Marked and dosage-related reduction of body weight gains was noted during Week 1 for male and female rats given 1000 ppm and males given 300 ppm. The trend continued for rats given 1000 ppm less markedly.
For details, please refer to attachment 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Marked and dosage-related reduction in food intake was noted during Week 1, for male and female rats given 300 or 1000 ppm. The trend continued over the remainder of the study (lesser degree).
For details, please refer to attachment 2 under "Overall remarks, attachments".

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

A dosage-related impairment in efficiency of food utilisation was noted for male and female rats given 300 or 1000 ppm over Week 1. Thereafter food conversion ratios were similar for control and treated rats.
For details, please refer to attachment 2 under "Overall remarks, attachments".

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

no effects observed

Description (incidence and severity):

There were no treatment-related differences in water consumption observed in any of the treatment groups in comparison to the control animals.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There was no evidence of an effect of treatment on the eye.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in mean RBC (red blood cell) values were observed for male and female animals given 1000 ppm. Corresponding increases in mean corpuscular haemoglobin concentration (MCHC) values were also noted reaching statistical signifiance at 300 ppm and above for males and at 100 ppm and above for females. There were no corresponding changes noted in other haematological parameters or in histopathological examination to account for these changes. The majority of values were within the expected background range and this finding is of doubtful toxicological significance. Other intergroup variations were considered to have arisen fortuitously and to be of no toxicological significance.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Group mean values for urea nitrogen were higher in male and female rats given 1000 ppm and in females given 100 and 300 ppm, when compared to the control values. There was a dose-related and statistically significant decrease in group mean total protein values, mainly as a result of reduced albumin, for treated females and a decrease in mean serum calcium values for all treatment groups when compared to the controls. Group mean serum alkaline phosphatase (AP) in females given 1000 ppm was also statistically significantly reduced. There were no corresponding changes in other biochemical or organ weight parameters and no histopathological changes to account for these findings. The majority of individual values were within the expected background ranges and thus, these findings are considered of doubtful toxicological significance.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Concerning organ weights, higher relative brain weights were noted for male and female rats given 1000 or 300 ppm. Higher relative spleen weights were noted for male and female rats given 1000 ppm and females given 300 ppm. Higher relative testes and ovary weights were also noted at 1000 ppm. There were no macroscopic or microscopic findings to account for these changes and they may possibly be attributed to body weight effects of the test substance.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross necropsy revealed no lesions apparent at post mortem that were considered to be related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings included minimal epithelial hyperplasia of the non-glandular stomach, proximal to the limiting ridge, which was noted among a small number of males (1/10) and females (3/10) given 1000 ppm and a female (1/10) given 300 ppm. The changes noted did not achieve statistical significance, were minor in degree and probably resulted from minor irritation of the mucosa.
All other histological changes were within the normally expected background ranges for rats of this strain and age-range and were not considered to be related to the treatment with the test substance.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

< 100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No NOAEL could be derived since treatment-related adverse effects were observed on all dose levels including the lowest dose level tested.

Remarks on result:

other: 100 ppm corresponded to 7.4 and 8.8 mg/kg bw/day in male and female rats, respectively

Key result

Dose descriptor:

LOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

other: additionally, changes in body weight gain, food consumption and organ weights were observed at 300 and/or 1000 ppm.

Remarks on result:

other: corresponding to 7.4 and 8.8 mg/kg bw/day in male and female rats, respectively

Key result

Critical effects observed:

not specified

Conclusions:

The present study was conducted to assess the sub-chronic toxicity of the test substance on Sprague-Dawley rats when given for a time frame of approximately 13 weeks. The study was similar to OECD guideline 408 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 10 male and female rats at dose levels of 100, 300 and 1000 ppm corresponding to approximately 7.4, 21.4 and 67.8 mg/kg bw/day for males and 8.8, 24.2 and 76.9 mg/kg bw/day for females. Under the conditions of the test, the test substance caused changes in haematology and clinical biochemistry parameters at the lowest dose tested, while at higher dose level also clinical signs, reduced body weight gain and food consumption, decreased organs weights and histopathology changes were observed. Therefore, a NOAEL could not be established and the LOAEL was set at 100 ppm for males and females (corresponding to 7.4 and 8.8 mg/kg bw/day respectively).

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Sep 1991 - 01 Oct 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

Version / remarks:

adopted in 1984

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

The lowest dose level produced toxicity and was therefore too high.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source : Charles River Breeding Laboratories, Michigan, USA
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 157 - 192 g (males), 114 - 175 (females)
- Housing: 5 animals of the same sex were housed in stainless steel, wire-mesh cages. Each cage measured 36.5 cm wide, 55 cm deep and 25 cm high.
- Diet: SDS Rat and Mouse No. 1 modified maintenance diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 6 day acclimation period after delivery, further 7 days after allocation of the animals in groups and before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
A pre-mix was prepared each week by grinding the test substance directly into untreated sieved basal diet and mixing in a Turbula Mixer for a minimum period of 2 minutes. The required concentrations were then prepared by direct dilution of the pre-mix with further quantities of untreated diet; homogeneity being achieved by further mixing in a double-cone blender for a minimum period of 7 minutes. The total volumes of diets required were such (i.e. so large) that two mixes (a and b) for each inclusion level were made from one pre-mix. The concentration of test material in the low dose level was increased by 10% above nominal in order to compensate for losses during storage observed in predosing analytical chemistry. On completion of the mixing procedure for each group, the diets to be fed at each level were divided into 8 aliquots labelled, either "a" or "b", for daily use during the following week. All aliquots were transferred as quickly as possible to storage at +4 °C until immediately before feeding. An aliquot was removed daily from +4 °C storage, allowed time to reach ambient temperature and fed to the animals. Whether diet fed was from mix "a" or "b" was documented.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary samples were analysed (spectrophotometry at 435 nm after carbon disulphide extraction) before start of treatment as well as with dietary samples from the present study.

1) Prior to the start of treatment the proposed diet mixing procedures were checked by chemical analysis to confirm that the method was acceptable and the homogeneity and stability of the formulation was satisfactory under the conditions of the study. This analysis revealed that the test material was not sufficiently stable in rodent diet to permit storage at room temperature for twice weekly feeding. Therefore, diets were prepared weekly and stored at +4 °C prior to use.

2) Samples of diets of the present study prepared in Weeks 1, 3, 13, 26, 39, 41, 52, 65, 78, 91 and 104 were also analysed to check the accuracy of preparation. Samples were taken at preparation and further samples stored for 7 days at +4 °C followed by exposure at room temperature for 24 h. Samples were taken from both mixes a and b at each dose level. Either mix a or b was analysed and the other was retained frozen for possible further analysis. Analysis of the samples taken showed good agreement with nominal values at the start of each sampling week. Tolerable agreement was achieved for samples obtained at the end of the sampling weeks indicating that the fortification procedure applied to the lowest dietary inclusion level was successful in compensating for degradation. Mean results in the post-dose samples were all within +6% and -17% of nominal concentrations. Further, the test substance was confirmed to be homogeneously blended in rodent diet. The stability in rodent diet showed a maximum concentration loss of 23% at 25 ppm and 13% at 50 ppm during the 8-day storage period. In total, the results indicate that the formulations were homogeneous and stable from the time of preparation to completion of dosing under the study conditions.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

Continuously via diet

Dose / conc.:

60 ppm

Remarks:

The concentration of test material in the low dose level was increased by 10% above nominal (66 ppm) to compensate losses during storage. In total this corresponded to 2.5 mg/kg bw/day for males and 3.4 mg/kg bw/day for females.

Dose / conc.:

180 ppm

Remarks:

corresponding to 7.7 mg/kg bw/day for males and 10.2 mg/kg bw/day for females

Dose / conc.:

540 ppm

Remarks:

corresponding to 23.7 mg/kg bw/day for males and 34.6 mg/kg bw/day for females

No. of animals per sex per dose:

50 per sex (104 week sacrifice)
20 per sex (52 week sacrifice)

Control animals:

yes, plain diet

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for the first 4 weeks thereafter once weekly for any signs of behavioural changes, reaction to treatment or ill health; twice daily for moribund or dead animals
- Cage side observations checked: signs of behavioural changes, reaction to treatment or ill health an d or moribund or dead animals

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of allocation of animals to groups, on the day of commencement of treatment and once a week thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: The quantity of food consumed by each cage of rats was recorded daily and reported on a weekly basis. Food consumption was calculated from the raw data using the total amount of food given to and left by each cage in each group and the number of rats surviving in each cage and presented as weekly food consumption in g/rat/week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Substance intake (mg/kg bw/day) was calculated from the group mean body weight and food consumption and the nominal dietary inclusion levels of the test material.

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: No, but food conversion ratios were calculated, where possible, over the period Weeks 1 to 26 from the body weight and food consumption data as weight of food consumed per unit gain in body weight.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily by visual appraisal of the water bottles and measured by weight for 7 consecutive days during Weeks 12, 25 and 51 for all satellite group cages.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: Before treatment (all animals), during Week 52 (satellite and main group) and at termination (main group only during Week 104), the eyes of all surviving control and high dose level group animals were examined.
Prior to examination, the pupils of each animal were dilated using a Tropicamide ophthalmic solution.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Weeks 13, 26, 52 and 78 and at termination
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- How many animals: 10 male and 10 female animals from each group
- Parameters checked: Packed cell volume (PCV), Haemoglobin (Hb), Red blood cell count (RBC), Total white cell count (WBC Total), Platelet count (Plts), mean corpuscular haemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Thrombotest (TT), Differential WBC counts and Cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Weeks 13, 26, 52 and 78 and at termination
- Animals fasted: Yes, overnight
- How many animals: 10 male and 10 female animals from each group
- Parameters checked: Total protein, albumin (Alb), globulin (Glob), urea nitrogen, creatinine, sodium (Na), Potassium (K), Calcium (Ca), inorganic phosphorus, chloride (Cl), total cholesterol (Chol), alkaline phosphatase (AP), total bilirubin (bilirubin), glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), tri-iodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH).

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 13, 26, 52, 78, 104
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight
- Parameters checked: Volume, pH, specific gravity and protein content were determined quantitatively. Furthermore, total reducing substances (TRS), glucose, ketones bile pigments, urobilinogen and haem pigments were determined qualitatively. The deposit of a urine sample after centrifugation was examined microscopically for the presence of different cell types and organisms.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the treatment period all surviving main group animals were sacrificed. Since the terminal procedures took 10 days to complete, the treated animals continued to receive test substance in their diet until the day on which they were sacrificed. All animals were sacrificed by carbon dioxide asphyxiation. The weights of major organs of individual animals dying or sacrificed during the study were recorded at the discretion of the pathologist. The following organs from all animals killed at the scheduled sacrifices were dissected free of fat and weighed: adrenals, kidneys, pituitary, brain, liver, testes (with epididymides), heart, ovaries and thyroid. All superficial as well as subcutaneous tissues and all organs were visually examined.

HISTOPATHOLOGY: Yes
The samples of the following tissues from all animals were preserved and histopathologically examined: Adrenals, aorta (arch and abdominal), bones (sternum and femur), bone marrow (sternum), brain (cerebral cortex, thalamic nuclei, mid-brain, medulla and cerebrum), caecum, colon, duodenum, eyes, femur, heart, jejunum, ileum, kidneys, liver, lungs (all lobes and mainstem bronchi), lymph nodes, mammary gland, other macroscopic abnormalities, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal column, spleen, sternum, stomach, testes (with epididymides), thymus, thyroids/parathyroids, trachea, urinary bladder and uterus (corpus and cervix).

Tissues were preserved in buffered 10% formalin (except eyes, which were preserved in Davidson's fixative).Further tissues were embedded in paraffin wax and sections were cut at 4 µm and stained with haematoxylin and eosin. Frozen sections of the liver were fixed in buffered formalin and cut on a cyrostat at 12 µm and stained for fat with Oil Red O. Liver sections were also stained using PAS for glycogen.

Statistics:

All analyses were carried out both together and separately for male and female.
Data relating to food and water consumption were analysed on a cage basis. For all other parameters, analyses were carried out using the individual animal as the basic experimental unit.
Food consumption data were analysed using cumulative totals and water consumption data were analysed as the total recorded intake over selected time periods, expressed on a weekly basis. Bodyweight data were analysed using weight gains.
The following tests were used for food consumption, bodyweight, organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by Fisher and Mantel. Otherwise:
- 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. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate, analysis of covariance was used in place of analysis of variance. Mortality was analysed using log rank methods (Mantel, 1966).

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs clearly indicative of a reaction to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no indication of an adverse treatment-related effect when considering the incidence or distribution of decedents throughout treatment. The mortalities observed in the present study were as follows:

Main groups:
Control: 19/50 (males) and 34/50 (females)
60 ppm: 22/50 (males) and 29/50 (females)
180 ppm: 27/50 (males) and 25/50 (females)
540 ppm: 19/50 (males) and 26/50 (females)

Satellite groups:
Control: 2/20 (males) and 0/20 (females)
60 ppm: 1/20 (males) and 1/20 (females)
180 ppm: 1/20 (males) and 1/20 (females)
540 ppm: 3/20 (males) and 0/20 (females)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first week of treatment, a marked dosage-related reduction in body weight gain was noted for males and females receiving 180 or 540 ppm and this was associated with a reduction in food consumption. Subsequently, body weight gain of males and females receiving 540 ppm, improved noticeably but continued to be lower than that of controls so that absolute mean body weight remained substantially depressed throughout the study. After Week 1, males receiving 180 ppm had a mean body weight gain comparable to that of controls for the majority of the dosing period. Intergroup differences in absolute mean body weight arising from the sacrifice of satellite animals were noted at Week 52 but on statistical evaluation were not of toxicological significance. In the final weeks of the study, however, the mean body weight of this group dropped to a greater extent than that of the concurrent controls resulting mainly from differences in group mean weights caused by decedents and large weight losses prior to their deaths. Females receiving 180 ppm showed a lower mean body weight gain than controls throughout the majority of the study period. In the final weeks of the study, the mean body weight gain of this group was comparable to that of controls. The body weight gain of males and females receiving 60 ppm showed no adverse effect of treatment.
For details, please refer to attachment 1 and 2 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Consistent with the aforementioned reduction in body weight gain, food intake by animals receiving 540 ppm, and to a lesser degree, those receiving 180 ppm was lower than that of controls during the first week of treatment. Subsequently, the food intake of males and females receiving 540 ppm and females receiving 180 ppm, improved although it continued to be generally lower than that of controls. After Week 1, the food intake of males receiving 180 ppm improved, such that overall mean food intake was considered to be essentially similar to that of controls. The food intake of males and females receiving 60 ppm showed no adverse effect of treatment.
For details, please refer to attachment 3 and 4 under "Overall remarks, attachments".

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food utilisation as assessed by the food conversion ratios was assessed over the first 26 weeks of treatment, the period of fastest growth. Consistent with the effect on body weight gain, which was more marked than the reduction in food intake during Week 1, food utilisation was impaired for both males and females receiving 540 or 180 ppm. Although body weight and food intakes for rats given 540 ppm remained lower than controls, the percentage differences were such that an overall effect on food utilisation was not apparent.
For details, please refer to attachment 3 under "Overall remarks, attachments".

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

no effects observed

Description (incidence and severity):

The measured water consumption during Weeks 12, 25 and 51 of treatment did not reveal any clear treatment-related effects, variations were considered to largely reflect effects on food consumption.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No lesions likely to prejudice the later evaluation of any toxic effect were noted in the eyes of the rats when an ophthalmoscopic examination was performed prior to the commencement of treatment. No lesions indicative of a reaction to treatment were noted in the eyes of rats receiving 540 ppm in comparison with the controls at the Week 52 or 104 examinations, the lesions seen were those common to the age and strain of rat employed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological investigations revealed a reduction in erythrocyte numbers in all treated groups of a nimals at the first investigation (Week 13) with the effect persisting in females given 60 ppm to Week 26 and in those given 180 or 540 ppm to termination at Week 104. Consequent to these changes in erythrocytes, a reduced packed cell volume was apparent for females given 180 ppm in Week 26 and 52 and in those receiving 540 ppm at Weeks 26, 52, 78 and 104. Additionally, reduced haemoglobin concentrations were apparent for males given 540 ppm at Week 13 only and females of this group from Week 26 to termination. Females given 180 ppm were also affected at Weeks 26 and 52. Changes in the calculated indices, consistent with the effects noted above, were also apparent. Reductions in thrombotest times were noted for treated groups of males at Weeks 13 and 26 only. Other statistically significant differences noted from control were assessed as random occurrences and considered to be of doubtful toxicological importance at this stage.
In summary, consistent changes in haematological parameters were observed in both males and females receiving 180 or 540 ppm. At 60 ppm, an isolated statistically significant change was observed in males only. Since all values were within background ranges, the small intergroup differences seen for this dose level were considered not to be of toxicological significance.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Measurement of T3, T4 and TSH in Weeks 4, 13, 26, 52, 78 and 104 revealed a dose-related reduction in T3 and T4 values at the Week 4 investigations. The reduction in T4 values was still apparent for treated males at Weeks 13 and 26 but no consistent differences in any of these parameters were apparent at later investigations. Full biochemical investigations in Weeks 13, 26, 52, 78 and 104 showed reductions in albumin values at Week 13 amongst males and females receiving 180 or 540 ppm with a corresponding reduction in total protein (although not statistically significant for females receiving 180 ppm). A similar effect was apparent at Week 26 but limited to males and females receiving 540 ppm. At the Week 52 and subsequent investigations there was a marginal decrease in total protein (not statistically significant in Week 104) in males and a lower protein albumin fraction in females receiving 540 ppm (except in Week 104). Blood urea nitrogen was increased at Week 13 amongst males and females receiving 540 ppm (not statistically significant for females) and at Week 26 for males receiving 180 or 540 ppm. At the Week 52 and subsequent investigations an increase in urea nitrogen was apparent for females receiving 540 ppm (not statistically significant in Week
104). Decreased GPT was noted at the Week 13 and subsequent investigations to termination for males and females receiving 540 ppm (not always statistically significant) and at Weeks 13 and 26 for males given 180 ppm. Increased alkaline phosphatase values were noted at the Week 13 and subsequent investigations to termination for all treated groups of males and females receiving 180 or 540 ppm (although rarely attaining a formal level of statistical significance). Reduced calcium values were apparent at Weeks 13 and 26 for males receiving 540 ppm and females receiving 180 or 540 ppm. Although calcium levels in Week 52 were again apparently lower for females receiving 540 ppm, the importance of this finding at this investigation is debatable, since the value recorded for females receiving 180 ppm exceeded that of controls for this same parameter. A slight, but statistically significant reduction was also noted in calcium levels of treated males in Week 78 (not statistically significant for males receiving 60 ppm). At the Week 13 and subsequent investigations of the study increased chloride values were apparent among females, lower doses being affected at Week 13 and 26 but only those given 540 ppm being affected at Weeks 52 and 78. However, this finding was considered not to be of toxicological significance as intergroup differences were slight and with in background ranges. Values at Week 104 were similar to control. The magnitude of these effects appeared to generally decrease as the actual achieved intake decreased. Other statistically significant differences noted from control were minor in degree and considered not to be of toxicological importance at this stage.
In summary, minor but consistent changes in biochemical parameters occurred in both males and females receiving 180 or 540 ppm. At 60 ppm, values were within the expected ranges and changes were considered not to represent toxicological significance.
For details, please refer to attachment 6 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis revealed a slight increase in urinary pH amongst males receiving 540 ppm with a similar finding noted at 180 ppm during Weeks 26 and 52. Among females a statistically significant increase in urinary pH was only attained in Week 78 for those receiving 540 ppm. A slightly lower volume of urine was noted for females receiving 540 ppm during Week 52. Protein concentrations appeared to be decreased in all treated groups in Week 52, however, upon closer examination this was seen to be caused by a few control animals having very high values. In Week 104, however, there was a statistically significant decrease in protein concentrations of females receiving 540 ppm.
In summary effects on the urinalysis parameters measured were seen at 180 or 540 ppm, were noted at all investigations, but were not considered to be associated with any histopathological changes in the kidney. At 60 ppm no changes were observed.
For details, please refer to attachment 7 under "Overall remarks, attachments".

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A decrease in group mean adjusted adrenal weights of males receiving 180 or 540 ppm was noted at the interim kill, with reductions in mean absolute adrenal weight seen in all treated males at termination. These decreases at Week 105 were not strictly dose-related in degree and achieved statistical significance in males receiving 540 ppm only. The group mean adjusted liver and thyroid weights of all treated groups of females showed a generally dose-related increase, compared to controls, at the interim and terminal kills (although not always statistically significant). Although not attaining statistical significance, a greater mean thyroid weight was noted for males receiving 540 ppm. Additionally, at the interim kill, a dose-related increase in heart weight was apparent for females receiving 180 or 540 ppm, and at the terminal kill decreases were noted for pituitary and kidney weights of males receiving 540 ppm. Changes in relative organ weights reflected those seen in absolute values. Other statistical differences were a reflection of the lower body weights of the animals for organs not sensitive to body weight changes e.g. brain and heart.
For details, please refer to attachment 8 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of main group rats found dead, sacrificed in extremis or killed at termination revealed the following changes:

Forestomach:
Depressions were observed in a greater number of male and female rats treated with 540 ppm, and male rats treated with 60 or 180 ppm compared with the control rats. Raised areas were observed in 4/50 male rats treated with 540 ppm and 3/50 treated with 180 ppm compared with 0/50 control rats. Thickening was observed in a greater number of terminal male rats treated with 540 ppm, and in a greater number of female rats treated with 540 ppm, compared with the control rats. White discolouration was observed in a greater number of female rats treated with 540 ppm, compared with the control rats.

Stomach antrum mucosa:
Depressions were observed in 8/50 female rats treated with 540 ppm, compared with 2/50 control rats.

Adrenals:
Cysts/cystic adrenals were observed in 4/24 female rats, killed at termination, treated with 540 ppm, compared with 0/16 control rats killed at termination. Cystic enlargement was observed in 2/24 female rats, killed at termination, treated with 540 ppm, compared with 0/16 control rats.

Skeletal muscle:
At termination the incidence of atrophied hind limbs was greater among males given 60 or 540 ppm and females given 540 ppm than controls.

Ovaries:
A greater number of terminal female rats treated with 540 ppm had no corpora lutea visible compared with control rats. There were no statistically significant histopathological findings as this finding is considered not to be of toxicological importance. The incidence and distribution of the remaining macroscopically observed lesions, chiefly attributed to senility, early neoplastic change, habitat and changes secondary to chronic disease and neoplasia were within the expected background range, and therefore were considered to be unrelated to treatment with the test substance.
For details, please refer to attachment 9 and 10 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Skeletal muscle:
An increase in the incidence of adipose replacement and narrowing of myofibres in peripheral muscle bundles was recorded in both male and female animals receiving 540 and 180 ppm, which was considered to be related to the treatment with the test substance. Some increase in the incidence of narrowed myofibres was also noted in male animals receiving 60 ppm. At the low dose level of 60 ppm similar changes were observed in a small number of females. Although they were absent in female controls, the incidence at 60 ppm was comparable to the control incidence for males and it is unlikely that a treatment-related effect was present for females at this low dose level. In occasional control and treated animals similar, but minimal, changes were seen as part of the normal background pathology of ageing, in which instances the changes were considered to be generally more diffuse in nature and not confined to the peripheral bundles.

Sciatic nerve/spinal cord:
An increase in the incidence of foci or areas of axonal degeneration was seen in the sciatic nerves of female animals receiving 540 ppm. An increase was also noted in male animals of this group but statistical significance was not achieved. However, the axonal degeneration in sciatic nerves observed at 540 ppm was considered to be related to the treatment with the test substance. It is probable that the increase in sciatic nerve lesions was associated with the increase in skeletal muscle changes reported above. Minor degrees of axonal degeneration comparable to that seen as part of the background pathology of ageing in control animals was noted in the sciatic nerves of rats from the low and intermediate dosage groups. In the spinal cords of animals at all dosage levels some degree of minor axonal degeneration was noted, but there was no clear dose relationship in degree or incidence and it was not considered that any treatment effect was involved.

Spleen:
An increase in the incidence and amount of Perls' positive brown pigment aggregation (haemosiderosis) was seen in the spleen of both sexes at 540 ppm and in males receiving 180 and 60 ppm. This pigment is a common background finding in a proportion of ageing animals, however this represents an exacerbation of the background occurrence and was therefore considered to be related to the treatment with the test substance. The effect was generally more pronounced in males than in females.

Liver:
A treatment-related and significantly increased incidence of rats with accumulation of brown pigment in a small number of sinusoidal cells was recorded in both sexes at the 540 and the 180 ppm dose levels when compared to the control levels. Occasional animals from the 60 ppm group also possessed similar pigment at comparable levels of incidence to the control group animals. Sample sections were demonstrated to stain positive by Perls' method for iron indicating that this pigment was probably haemosiderin and thus similar to the pigment seen in the spleens. It is noted that although there was an increase in the number of animals in which this was seen the actual amount of pigment and the number of involved sites/cells in individual animals was in no instance particularly extensive. Bile ducthyperplasia was seen in both control and treated animals at all dose levels but there was a statistically significantly increased incidence in both sexes only at the 540 ppm dose level. An increase was also noted in male animals of the 180 ppm dose group, but this increase did not achieve statistical
significance. In almost all cases the degree of hyperplasia was minimal to moderate in both control and treated rats. The increased liver weights reported at the terminal kill may be associated with the above observations.

Kidneys:
An increase in the incidence of brown pigment was seen in the cortical tubular epithelial cells of kidneys in female rats receiving 540 ppm. Although occasionally seen in male animals at this dose level and in both male and female animals at lower dose levels as well as in control animals these other groups showed broadly comparable occurrences. In the affected animals from the highest dose level the amount of pigment was not pronounced. This pigment was (spleen and liver) negative with Perls'stain but was positive with Schmorl's stain for lipofuscin. The incidence of lipofuscin was considered to be related to treatment with the test substance. These minor changes are unlikely to be associated with the urinalysis effects reported above.

Pancreas:
An increase in the incidence and severity of adipose tissue replacement of exocrine pancreas was noted in a proportion of male animals at the 540 and 180 ppm dose levels. A similar increase in male animals of the 60 ppm dose level did not quite achieve statistical significance. No such effect was seen in female animals. There was no concomitant treatment-related effect seen in respect of exocrine atrophy.

Adrenals:
An exacerbation and increase in the incidence and severity of adrenal cortical hypertrophy with varying degrees of cellular vacuolation or, more often, degeneration was seen at all dose levels. Hypertrophic and degenerative lesions of the adrenal cortex are a normal occurrence in ageing rats of this strain and are particularly evident in female animals. This effect was particularly evident in female animals at the 540 ppm dose level where an increase in the incidence and the severity of the lesion from hypertrophy with degeneration to full cystic degeneration was characteristic. An increase, albeit not statistically significant, was also seen in females at the 180 ppm level. In male animals at all dose levels the effect was confined to an increase in the number of animals with cortical hypertrophy and vacuolation. These changes probably represent an exacerbation of normal age-related lesions commonly encountered in rats of this strain and age range.

Thyroids:
An increase in the incidence and prominence of ultimobranchial cysts of the thyroid glands was noted in both sexes at the 540 ppm and 180 ppm dose levels and also in female animals receiving 60 ppm. Small numbers of control animals and males receiving 60 ppm also showed this change. A small number of high dose animals only showed this change at the 52 week interim stage. The ultimobranchial cyst is a developmental anomaly occasionally seen in a proportion of normal rat thyroid glands and represents the remnants of the embryonic ultimobranchial body, responsible for the distribution of calcitonin secreting C-cells in the thyroid. A small increase in C-cell hyperplasia was seen in occasional male animals (primarily at termination) at the 540 ppm dose level but was not detected in females and thus the aetiology of this change remains equivocal. No treatment-related effects were detected in thyroid follicular tissue.

Stomach:
Increased incidence and severity of hyperplastic and/or ulcerative lesions of the nonglandular stomach epithelium with associated changes, such as subepithelial oedema, were noted in both sexes at the 540 and 180 ppm dose levels and in male animals receiving 60 ppm. In general these changes were more pronounced in male than in female animals. These changes accord with the macroscopic observations for this tissue. These gastric epithelial changes are thought to be the consequence of the irritant action of the compound.

All other changes were within the normal background range of incidence for rats of this strain and age range in this laboratory and were not considered to be of any toxicological significance.
For details, please refer to attachment 11 and 12 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mesenteric lymph nodes:
Benign haemangiomata were seen in the mesenteric lymph nodes (draining the intestinal tract) of five male terminal kill rats receiving 540 ppm. This incidence was statistically significant in comparison to the control group.

Spleen:
A haemangioma was seen in the spleen of a single male decedent animal from the same 540 ppm dose group. Haemangiomata were not observed in other tissues in these animals. They were not seen in any male rats receiving lower doses of the test substance, in any of the female rats, nor in control rats. A haemangiosarcoma was seen in the spleen of a single female intercurrent death animal of the control group. Angiectasis was not an associated finding. Statistical analysis of the relative group incidence for this tumour type by the time-to-tumour methods recommended by the International Agency for Research on Cancer (IARC) demonstrated a statistically significant dose-related trend in the number of animals with tumours and a significant difference between the control and the 540 ppm dosage group. Haemangiomata are benign multilocular tumours occasionally seen in low numbers in rats of this strain and age range, mesenteric lymph nodes and the spleen being the commonest sites for their detection. The incidence which was recorded in the 540 ppm dose group of this study of some 5/50 animals in single site (6/50 in total) is outside the normal background incidence for this laboratory where a range from 0/50 in the majority of instances to a maximum of 2/50 in control animals is recorded for a series of recent and concurrent studies utilising this strain of rat. It is, therefore, considered that the presence of these haemangiomata was a consequence of the administration of the test substance at a dose level of 540 ppm to male animals.

All other changes were within the normal background range of incidence for rats of this strain and age range in this laboratory and were not considered to be of any toxicological significance.
For details, please refer to attachment 11 and 12 under "Overall remarks, attachments".

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

60 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: additionally, changes in body weight, food consumption and efficiency, and urinalysis were observed at the next higher dose level.

Remarks on result:

other: other: corresponding to 2.5 mg/kg bw/day for males and 3.4 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

540 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no tumorigenic effects observed at this dose level

Remarks on result:

other: corresponding to 34.6 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

180 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no tumorigenic effects observed at this dose level

Remarks on result:

other: corresponding to 7.7 mg/kg bw/day for males

Key result

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

540 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: corresponding to 23.7 mg/kg bw/day for males

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

60 ppm

System:

other: hepatobiliary, haematopoetic, musculoskeletal, nervous system, endocrine system

Organ:

adrenal glands

blood

liver

spinal cord

spleen

thyroid gland

other: skeletal muscle

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The present study was conducted to assess the carcinogenicity of the test substance in rats when given for a time frame of approximately 104 weeks. The study was similar to the OECD guideline 453 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 50 male and 50 female rats at dose levels of 0, 60, 180, and 540 ppm corresponding to approximately 0, 2.5, 7.7 and 23.7 mg/kg bw/day for males and 0, 3.4, 10.2 and 34.6 mg/kg bw/day for females. Under the conditions of the study, the test substance caused general toxicity as evident by lower body weight gains and food intake, disturbances in food utilisation, haematological, biochemical, urine and organ weight parameters as well as gross pathological and histopathological findings at 60 ppm and/or above. Therefore, no NOAEL with regards to general toxicity was set. Tumorigenic effects were only observed in male animals receiving 540 ppm of the test substance and were limited to haemangiomata with no evidence of malignancy. Thus, the NOAEL for carcinogenicity in male rats was set to 180 ppm corresponding to 7.7 mg/kg bw/day and to 540 ppm corresponding to 34.6 mg/kg bw/day for female rats.

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Dec 1990 - 06 Feb 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

The highest dose level produced one fatality and had therefore to be adjusted.

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna UK Limited, Huntingdon, UK.
- Age at study initiation: 3 - 5 months
- Weight at study initiation: 8.8 - 11.5 kg (males), 8.2 – 10.7 kg (females)
- Housing: 2 dogs of the same sex were housed in kennels
- Diet: Standard ground dry diet (Diet A: Special Diets Services) 400 g/animal/day
- Water: Tap water, ad libitum
- Acclimation period: at least 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24
- Humidity (%): not reported
- Air changes (per hr): approximately 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The test compound was administered by admixture with the diet. A premix of suitable strength was prepared by grinding the required quantity of test substance with untreated basal diet. Blending of the premix was achieved by mixing in a turbula mixer for a minimum period of two minutes. The premix was used to prepare diets for all three inclusion levels. The required concentrations were prepared by direct dilution of the prepared premix. Blending of the inclusion levels for feeding was achieved by mixing in a Gardner double-cone blender for a minimum period of seven minutes. Diets were usually prepared on a weekly basis. Formulated diets were stored at -20 °C protected from light.
While the diets for intermediate and high dose groups were prepared without correction for chemical purity, the concentration of test substance in the low-dose level was increased by 15% above nominal to 57.5 ppm in order to compensate for losses during storage already observed in pre-dosing analysis.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dietary samples were analysed (spectrophotometry at 435 nm after carbon disulphide extraction) before start of treatment as well as with dietary samples from the present study.
1) Prior to the start of treatment the proposed diet mixing procedures were checked by chemical analysis of trial diets to confirm that the proposed procedures produce homogeneous diet, that the accuracy of mixing was acceptable and that the concentration of test substance in the diet remained unchanged between preparation and administration. Initial validation work showed satisfactory homogeneity at 100 and 5000 ppm in dog diet. Stability trials showed significant losses at room temperature over 8 days at 100 ppm, further trials showing that losses at this dose level over 8 h were minimal. Subsequently, homogeneity was shown to be good at 50 ppm, however, there was an approximately 15.8% loss of the test substance during the preparation of this dietary level. No significant changes in concentration occurred thereafter over an 8-h storage period at room temperature. Based on these results, it was decided that the dietary formulations would be stored frozen (-20 °C), fed on a daily basis and consumed within 8 h. Additionally, the low dose level would be fortified to 57.5 ppm to compensate for losses during preparation.
2) Samples of diets of the present study prepared in Weeks 13, 26, 39 and 52 were also analysed to check the accuracy of preparation. The mean concentration of the test substance in the dose formulations analysed during the study were within +10% to -14% of nominal values. Results also confirm that at nominal concentrations used, the test substance was homogeneously blended in dog diet. The test substance in the dog diet formulations was stable at 5000 ppm for up to 14 days during storage at ambient temperature. At 100 ppm, significant losses occurred (14% after 24 h, 30% after 8 and 14 days). No changes in concentration occurred at 50 or 100 ppm diet formulations during an 8-h storage period at ambient temperature, which reflects the approach used in the present study.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

continuously via diet

Dose / conc.:

50 ppm

Remarks:

The concentration of test material in the low dose level was increased by 15% above nominal (57.5 ppm) to compensate losses during storage. In total this corresponded to 1.6 mg/kg bw/day for males and 1.9 mg/kg bw/day for females.

Dose / conc.:

185 ppm

Remarks:

corresponding to 6.6 mg/kg bw/day for males and 6.7 mg/kg bw/day for females

Dose / conc.:

700 ppm

Remarks:

The dose was reduced to 500 ppm from Day 3 of Week 12. In total this corresponded to 17.4 mg/kg bw/day for males and 20.6 mg/kg bw/day for females.

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Regularly throughout the working day.
- Cage side observations checked: All signs of ill health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to feeding, once a week throughout the study period

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was calculated as g food per dog per week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: The quantity of the test substance ingested weekly by each group was calculated. The approximate dosage equivalents expressed as mg/kg bw/day were calculated at weekly intervals from the mean bodyweight data.
The quantity of food left by individual animals was recorded on a daily basis throughout the experimental period.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment commenced and during Weeks 13, 26 and 52
- Dose groups that were examined: all animals
The eyes of all animals were examined by means of a Keeler indirect ophthalmoscope. Prior to examination, the pupils of each animal were dilated using a Tropicamide ophthalmic solution.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-exposure and in weeks 13, 26 and 52
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: packed cell volume (PCV), erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), platelet count, reticulocyte count, total leukocyte count, differential leukocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT) and cell morphology. Erythrocyte sedimentation rate (ESR) was performed only in two cases as part of diagnostic screen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre-exposure and in weeks 13, 26 and 52
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: total protein, albumin, sodium, potassium, glucose, urea (BUN), alanine aminotransferase (ALT, GPT), aspartate aminotransferase (GOT), alkaline phosphatase (AP), creatinine, calcium, phosphorus, chloride, total cholesterol, total bilirubin, gamma-glutamyltransferase (GT), creatine phosphokinase (CPK), ornithine carbamoyltransferase (OCT), alpha-hydroxybutyrate dehydrogenase (HBDH), tri-iodothyronine (T3) and thyroxine (T4).

PLASMA/SERUM HORMONES/LIPIDS: Yes, please refer to "clinical chemistry".

URINALYSIS: Yes
- Time schedule for collection of urine: pre-exposure and in weeks 13, 26 and 52. Urine samples were collected over a period of approx. 16 hrs, water having been withheld from the animals about 5 hrs prior to the start of collection.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes, overnight
- Parameters checked: volume, pH, specific gravity, protein, glucose, ketones, haem pigments, bile pigments, urobilinogen, total reducing substances (TRS), epithelial cells (E), polymorphonuclear leukocytes (P), mononuclear leukocytes (M), erythrocytes (R), organisms (O), renal tubule casts (C) and other abnormal constituents (A).

NEUROBEHAVIOURAL EXAMINATION: Yes, please refer to "other, neurological examination" below.

IMMUNOLOGY: No

OTHER:
NEUROLOGICAL EXAMINATION: Yes
Full neurological examinations were performed on all animals once during the pre-dosing periods and again during Weeks 34 and 50 of treatment. During the treatment period neurological examinations were performed prior to food being offered on that day. Examination included general physical examination, behaviour and gait, general examination of the animal while standing and moving with particular reference to strength and co-ordination and cranial nerve function.
Examination on cranial nerve function involved head tilt, facial muscle, muscles of mastication, blink reflex, pupillary light reflex, eye position, strabismus, abnormal nystagmus, corneal reflex, palpebral reflex, ear movement, position of philtrum, commissure of lips, jaw closure, open jaw resistance, tongue, gag reflex.
The examination of spinal reflexes involved muscle tone, patellar reflex, triceps reflex, flexor reflex (all 4 limbs individually), crossed extensor reflex and perineal reflex.
Postural and attitudinal reactions included wheelbarrowing, thoracic hopping, pelvic hopping, extensor postural thrust, tactile placing, visual placing, tonic neck reaction, righting reaction.

BONE MARROW EXAMINATION: Yes
Prior to necropsy, bone marrow was obtained from each animal by sternal puncture and a smear prepared, stained (using modified Wright's stain) and examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of 52 weeks of treatment, all surviving dogs were sacrificed. Since the terminal procedures took several days to complete, the dosing of individual treated animals continued until the day prior to being sacrificed. The duration of the dosing period, however, is reported as 52 weeks. All animals were sacrificed by exsanguination under pentobarbitone anaesthesia (200 mg/mL pentobarbitone sodium). The animals were starved overnight prior to post mortem examination.
The following tissues and organs were dissected out and weighed from all animals, paired organs being weighed separately: adrenals, brain, heart, kidneys, liver, lungs, pancreas, pituitary, spleen, thymus, thyroids (with parathyroids), uterus or prostate and testes (with epididymides) or ovaries.

HISTOPATHOLOGY: Yes
The samples of the following tissues from all animals were preserved in neutral buffered 10% formalin (except eyes which were preserved in Davidson's fixative): adrenals, aorta (arch and abdominal), bones (sternum and femur plus joint), bone marrow (sternum), brain (cerebral cortex, thalamic nuclei, mid-brain, medulla and cerebellum), caecum, colon, duodenum, eyes (with optic nerve), gall bladder, heart, jejunum, ileum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), mammary gland, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submandibular), sciatic nerves, semilunar ganglia, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar, dorsal and ventral roots and dorsal root ganglia), spleen, stomach, testes (with epididymides), thymus, thyroids/parathyroids, tongue, trachea, urinary bladder, uterus and vagina.

Tissues were embedded in paraffin wax and sections were cut at 4 µm and stained with haematoxylin and eosin. Frozen sections of the liver were fixed in formol calcium and cut on a cyrostat at 12 µm and stained for fat with Oil Red O. Liver sections were also stained using PAS for glycogen.

Statistics:

All analyses were carried out both together and separately for male and female animals. In view of the small number of animals and to confirm possible effects, analyses were also performed on combined data.
Data relating to food consumption were analysed as totals over selected time periods, expressed on a weekly basis. Bodyweight data were analysed using weight gains.
The following tests were used for food consumption, bodyweight, organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:
- 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.
- For pre-dose data, analyses of variance were followed by Student’s ‘t’ test. For data from the dosing period, analyses of variance were followed by Williams’ test for a dose-related response. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate, analysis of covariance was used in place of analysis of variance. Statistical analysis in histopathological examination was performed using Fisher’s exact and Mantel’s tests.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

With the exception of the clinical signs (collapsed state with excessive salivation, trembling, champing of the jaws and jerking of the limps) noted prior to sacrifice of the female Dog (700 ppm), there were no signs considered to be indicative of a reaction to treatment with the test material. The major sign, noted in all study groups, including controls, was occasional liquid faeces. However, the overall incidence for this sign was not dose-related and it was therefore considered unrelated to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two female dogs treated with 700 ppm were sacrificed during the course of the study, prior to the scheduled sacrifice after 52 weeks of treatment.
The first female dog was sacrificed for human reasons on Day 7 of Week 11. The animal was found in a collapsed state, laterally recumbent, on the morning of Day 7 in Week 11, prior to diet administration on that day. The dog showed excessive salivation, trembling and occasional champing of the jaws and jerking of the limbs. The condition of this animal worsened in the 4-h period after its discovery in the collapsed state. This mortality was considered to be related to treatment, the high dosage level in the study was reduced from 700 to 500 ppm from Day 3 of Week 12.
The second female dog was sacrificed on Day 1 of Week 6 due to suspected polyarteritis. Prior to sacrifice, periods of pyrexia, reduced food intake, weight loss, stiff gait, subdued behaviour and haematological/biochemical changes consistent with a polyarteritic condition were noted. Polyarteritis has been described in the beagle dog by several authors and the clinical picture for this animal was consistent with polyarteritis. This condition was considered coincidental, and unrelated to treatment, but in view of the potential recurring nature of the disease, a decision was taken to remove the dog from the study. The presence of such a condition in a high dose level animal was considered likely to affect the integrity of the study. Therefore, it was decided to remove the dog from the study and to replace it with another animal. Subsequent pathological examination did not confirm the presence of polyarteritis, but synovitis was diagnosed as a factor contributory to the animal's clinical condition. A replacement dog was obtained, and commenced treatment 8 weeks behind the other remaining animals on the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Intergroup differences in group mean weight gain amongst males showed no dose relationship, and there was no effect of treatment. In females, no effect of treatment on weight gain was apparent during the earlier part of the study, although after 26 weeks, body weight gain of females at 500 ppm was lower than that of the control animals. Thereafter, however, all female animals at both 185 ppm and 500 ppm showed some weight loss, such that overall gain after 52 weeks was only 31% and 19% of control values at 185 and 500 ppm, respectively. These intergroup differences in weight gain amongst females resulted in slightly lower mean body weight at 500 ppm at termination of the study (Week 52), although none of the intergroup differences in absolute body weight were statistically significant after either 26 or 52 weeks of treatment.
For details, please refer to attachment 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

While food intake of females receiving 185 or 500 ppm was marginally lower than for controls, the differences, when considered in terms of a g/dog/day (392 and 384 g at 185 and 500 ppm respectively, compared to 399 g for female controls) were minimal, and, in addition, females in the 500 ppm dose group also showed less than maximal intake in the pre-dose period. It was concluded that there was no effect of treatment on food intake. Therefore, no clear explanation for the reduced overall weight gain of females at 185 or 500 ppm was evident, an effect of treatment on efficiency of food utilisation being the apparent cause of the bodyweight effect at these doses.
For details, please refer to attachment 2 under "Overall remarks, attachments".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no changes at Weeks 13, 26 or 52 considered to be related to treatment. All findings were considered typical of the age and strain of animals employed.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some statistically significant intergroup differences were recorded in haematology parameters during the study. In particular, mean MCV values were elevated at each set of investigations for animals receiving 500 ppm, a trend towards increased lymphocyte counts was seen amongst treated groups, and APTT values were increased for animals receiving 500 ppm in Week 13. However, these changes were generally small, and did not always show clear relationship to dose, and since most values were within the expected historical control range for dogs of the age employed, were considered unrelated to the treatment with the test substance. In Week 52, a number of dogs from treated groups showed reticulocyte counts greater than 2.0%, but in the absence of any clear effects of treatment on haemoglobin and red cell count values, these changes, in addition to the change in MCV values mentioned above, were considered of no toxicological importance.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were a number of intergroup differences amongst biochemical parameters. The principal parameters affected were GPT, AP, albumin and cholesterol. The intergroup differences in GPT values amongst males did not achieve statistical significance, however, examination of individual values revealed that one male dog given 185 ppm as well as two male dogs given 500 ppm showed elevated GPT values compared to pre-dose in Weeks 26 and 52. Since these animals also showed the most marked changes in the liver at microscopic examination, it was considered that the changes for these animals were treatment-related. GOT values for these animals were generally similar to those recorded in the pre-dose period, with the possible exception of the slightly elevated value recorded for one male dog in Week 26. There were no effects of treatment on GPT or GOT values amongst females. AP values showed an apparent dose-related increase amongst males receiving 185 or 500 ppm in Weeks 26 and 52, although differences were not statistically significant. Examination of individual values once again indicated that these differences were mainly due to higher than expected values (compared to historical control data for dogs of the age employed), for male dog at the 500 ppm dosage level. Although the individual data for these animals were similar to pre-dose values in Weeks 26 and 52, historical control data indicate that AP values generally fall as the animals grow older, as was demonstrated by data for other dogs on the study, therefore it was considered that the AP values recorded for these dogs in Weeks 26 and 52 were elevated as a result of treatment, and are probably associated with the pathological findings seen in the liver of these dogs at termination. Intergroup differences in AP values recorded for females were considered unrelated to treatment. A consistent decrease in albumin values, compared to controls, was observed for male dogs at 500 ppm in Weeks 26 and 52, all male dogs in this treatment group showing a general decrease compared to both concurrent controls and pre-dose values. Some statistically significant intergroup differences in total protein and albumin values were recorded for females during the study. However, the differences were not always clearly dose-related, individual values for treated dogs generally fell within the expected historical control range, and since the intergroup differences at 50 and 185 ppm were not supported by any significant corroborative pathological findings at termination, these protein changes in females were considered of equivocal toxicological significance. A consistent increase in cholesterol values was seen for male dogs at 500 ppm, compared to controls, at Weeks 13, 26 and 52. This change was evident in the individual data for all dogs in this dose group, most values recorded at each set of investigations during the treatment period being above the upper limit of the historical control range for dogs of the age under consideration. There were no effects of treatment on cholesterol values in females.
Therefore, in summary, changes in plasma biochemistry values (principally GPT, AP, albumin and cholesterol) were observed amongst male dogs at 185 and 500 ppm, that were considered of toxicological importance, and were probably associated with the changes seen in the liver at termination.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no changes at Weeks 13, 26 or 52 considered indicative of a reaction to treatment.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

For detail, please refer to "Other, neurological examination" below.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The group mean liver weight for males receiving 500 ppm was increased compared with the control value, the difference attaining statistical significance. This was largely due to the liver weights for 2 female dogs, which were at, or approaching, the normally expected upper limit of 4 % of terminal body weight. There were no histological changes in the livers of these dogs to account for the marginally higher liver weights. Liver weights for other treated animals, which showed histopathological lesions appeared normal. There were no other intergroup differences in organ weights, which were considered to be related to treatment. Group mean heart weights were decreased at 500 ppm compared with the controls, the difference attaining statistical significance for combined male and female data. In addition, group mean prostate weights showed a dose-related decrease compared with the control value, although the differences did not attain statistical significance. As there were no toxicologically significant macroscopic or microscopic pathological findings for these tissues, these apparent organ weight changes were considered to be of no toxicological importance. Uterus and ovary weights were also generally decreased in treated animals compared with controls. Histopathological examination revealed that these differences were related to the relative stages in the oestrous cycle of the individual animals and were, therefore, unrelated to treatment.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Multiple red discoloured linear depressions up to 25 x 3 mm were noted on the mucosal surface throughout the stomach body and antrum for one female dog given 185 ppm (confirmed as focal erosion of the superficial gastric mucosa histologically). Dark ingesta was also present throughout the gastro-intestinal tract for this dog, yet no dark stained faeces were noted in the week prior to sacrifice. Single oval, flat choleliths, which were firm but friable were present in the gall bladders of two male dogs receiving 185 ppm. Although one of those dogs did show markedly elevated AP and GPT values in Week 52, there were no similar findings in the dogs receiving 500 ppm and the toxicological significance of these changes remains equivocal. There were no other noteworthy findings.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were confined to the liver and spleen. Foci of degenerate hepatocytes were seen to a moderate degree in 1/4 male and 2/3 female dogs treated with 500 ppm and in 1/4 females receiving 185 ppm, but only to a trace or minimal degree in both control and 50 ppm dogs. Single cell necrosis was seen in one male dog treated with 500 ppm, but not in any control dog. Inflammatory cell infiltration around central veins and sometimes the branches of the vein were seen in a proportion of these dogs. An apparent increase in centrilobular fibrocytes was seen in 3/4 male dogs treated with 500 ppm and 1/4 male dogs treated with 185 ppm.
There was an increased incidence and degree of aggregates of pigmented Kupffer cells and macrophages in the livers of male and female dogs and an increased incidence of pigmented macrophages in the spleen of male dogs treated with 500 ppm or 185 ppm when compared to control male dogs. The incidence of aggregations of pigmented Kupffer cells and macrophages in the livers of male dogs treated with 500 ppm and female dogs treated with 185 ppm achieved statistical significance.

Other findings
The toxicological significance of the single incidence of focal erosion of the superficial gastric mucosa in one male dog treated with 185 ppm, and choleliths seen in 2/4 male dogs treated with 185 ppm remains equivocal. No changes were seen in the thyroids of dogs treated with 500 ppm to account for the increased T3 values in males. No changes were seen in the prostate to account for the decreased weights recorded. No toxicologically significant findings were recorded for the uterus and ovaries and their decreased weights may be explained by the distribution of stages of oestrus cycle. No changes were seen in the hearts to account for the decreased heart weights for dogs treated with 500 ppm.
In the female dog given 700 ppm of the test substance sacrificed during the study, the areas of recent haemorrhage seen in the heart were considered to be related to the convulsions seen clinically. The moderate fat droplets seen in the liver were considered to be related to the collapsed state of the dog.

Discussion of pathology findings
The histopathological examination clearly identified the liver and the spleen as target organs. The initial process involved in the changes in these tissues appeared to be increased pigmentation of Kupffer cells and/or macrophages with Perls' positive material, possibly reflecting increased iron metabolism and turnover. These changes were evident at all doses in the liver, and for males at 185 or 500 ppm in the spleen. At doses of 185 and 500 ppm, more marked associated changes were seen in the liver, chiefly hepatocyte degeneration and inflammatory cell infiltration. The only change identified at the low dose level (50 ppm) that could be possibly related to treatment, was aggregates of pigmented Kupffer cells and macrophages in one male and one female. No other changes considered to be treatment-related were identified at the 50 ppm treatment level, in particular, there were no biochemical or other pathological changes indicating an effect on the liver at this dosage. In addition, in view of the minor degree of the change observed, the association of the change with a normal physiological process (iron turnover), and the lack of statistical significance, it was considered that the Kupffer cell changes at 50 ppm were of minor toxicological importance.
For details, please refer to attachment 5 and 6 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Neurological examination:
At the pre-dose investigations and those performed during Weeks 34 and 50, examination for general physical condition and observation for behaviour and gait revealed no abnormalities. Tests for cranial nerve function, spinal reflexes and postural reactions elicited responses considered to be within the normal range. There was no evidence of neurological abnormality or deficit in any animal.

Bone marrow examination:
All bone marrow smears were considered normal for cellularity, cell morphology and distribution of cell types.

Key result

Dose descriptor:

NOAEL

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 1.6 mg/kg bw/day for males and 1.9 mg/kg bw/day for females

Key result

Dose descriptor:

LOAEL

Effect level:

185 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

histopathology: non-neoplastic

other: additionally, clincial signs, mortality and changes in organ weight were observed at 500/700 ppm.

Remarks on result:

other: corresponding to 6.6 mg/kg bw/day for males and 6.7 mg/kg bw/day for females

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

185 ppm

System:

other: hematopoietic and hepatobiliary

Organ:

liver

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The present study was conducted to assess the toxicity of test substance on dogs when given for a time frame of approximately 52 weeks. The study was similar to the OECD guideline 452 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 4 male and 4 female dogs at dose levels of 50, 185 and 500/700 ppm corresponding to approximately 1.6, 6.6 and 17.4 mg/kg bw/day for males and 1.9, 6.7 and 20.6 mg/kg bw/day for females. Under the conditions of the test, the test substance caused clinical signs, mortality, reduced body weights and changes in clinical biochemistry parameters, organs weights, gross pathology and histopathology at 185 and/or 500/700 ppm. Therefore, the NOAEL was set at 50 ppm for males and females (corresponding to 1.6 and 1.9 mg/kg bw/day respectively).

Reference 5

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Apr 1980 - 01 May 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

no analytical data on achieved doses, only 8 animals/sex/dose for chronic phase, 10/14 h dark/light cycle, no detailed clinical observations, blood and urine not sampled at 3 month time point, continued under "Principles of method if other than guideline"

Principles of method if other than guideline:

Deviations to the OECD guideline 453:
some blood parameters and histopathology missing (prothrombin time, activated partial thromboplastin time, creatine value, histopathology of vagina and cervix)

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Remarks:

(F344/DuCrj) (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: approximately 4 weeks
- Weight at study initiation: 55 - 65 g (males) and 55 - 70 g (females)
- Housing: 5 animals of the same sex in iron mesh-type cage, W310 x D440 x H230 mm
- Diet: Powdered food M, Oriental Yeast Co., Ltd., ad libitum
- Water: Tap water provided, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 25
- Humidity (%): 50 - 60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES:
Males: From: 16 Apr 1980 To: 22 Apr 1982
Females: From: 23 Apr 1980 To: 01 May 1982

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The dietary admixture was prepared every week. The food used in this study was analyzed regularly for contaminants which might affect the results of the study during the study period.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

104 weeks (interims-sacrifice after 26, 52 and 78 weeks)

Frequency of treatment:

continuously via diet

Dose / conc.:

20 ppm

Remarks:

corresponding to 0.70 mg/kg bw/day for males and 0.83 mg/kg bw/day for females

Dose / conc.:

200 ppm

Remarks:

corresponding to 6.9 mg/kg bw/day for males and 8.5 mg/kg bw/day for females

Dose / conc.:

2 000 ppm

Remarks:

corresponding to 74 mg/kg bw/day for males and 91 mg/kg bw/day for females

No. of animals per sex per dose:

80/sex/dose

Control animals:

yes, plain diet

Details on study design:

The males and females of each group were sacrificed by eights in Week 26, 52 or 78 of administration to conduct haematological, blood biochemical and pathological examinations. At the end of the study (Week 104) the haematological examination and blood biochemical examination were performed in 10 males and 10 females of each group.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General signs were observed daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once a week from Day 1 to Week 26 of administration and thereafter once every other week until Week 104 of administration during the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: calculated as g food/rat/day
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was measured in males and females twice a week for 8 cages of each dose group specified at the start of the study.

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: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was measured in males and females twice a week for 8 cages of each dose group specified at the start of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 26, 52, 78 and 104
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 8 males and 8 females of each group in Week 26, 52 and 78 of
administration and in 10 males and 10 females in week 104 of administration
- Parameters checked: haematocrit (Ht), haemoglobin (Hb), red blood cell count (RBC), mean corpuscular volume (MCY), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (Platelet) and white blood cell count (WBC). Differential count was determined by multiplying differential white blood cell count (obtained by blood smear specimen stained by May-Grunwald Giemsa method) by WBC count. Reticulocyte count (Retics) was calculated by the blood smear specimens stained with ultravital stain for males in Week 78 of administration and for females in Week 52 and 78 of administration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 26, 52, 78 and 104
- Animals fasted: Not specified
- How many animals: 8 males and 8 females of each group in Week 26, 52 and 78 of
administration and in 10 males and 10 females in week 104 of administration
- Parameters checked: alkaline phosphatase (AL-P), lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), gamma-Glutamyl Transpeptidase (y-GTP, GGTP), total protein (TP), albumin (Alb), globulin (Glob), albumin/globulin ratio
(A/G ratio), total bilirubin (T. Bil), direct bilirubin (D. Bil), blood glucose (Glucose), blood urea nitrogen (BUN), total cholesterol (T. Chol) and calcium (Ca). The globulin concentration was obtained by subtracting the albumin concentration from the total protein concentration and albumin/globulin ratio was found from the value. The serum concentrations of sodium (Na) and potassium (K) were measured. Bilirubin was measured by the alkaline azobilirubin method at the time of the examination in Week 26 and 52 of administration and at the time of the examination in Week 78 and 104 of administration by stabilizing diazonium salt method.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: Week 26, 52, 78 and 104
- Metabolism cages used for collection of urine: No, the urine collected by pressing the groin
- Animals fasted: Not specified
- Parameters checked: specific gravity, pH, protein, glucose and occult blood

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The last necropsy in this study was performed on Days 728 (Week 104), and 729 and 730 (Week 105) for males and on Days 728 (Week 104), and 730 and 731 (Week 105) for females. The dead animals were necropsied immediately after they were found dead and the animals killed by moribundity were necropsied immediately after they were killed during the study period. Samples of all the tissues listed below (under histopathology) from all animals were preserved.
After necropsy, the following organ weights were measured in all the animals killed according the killing schedule and in all the surviving animals at the end of the study: brain, pituitary gland, thyroid (including parathyroid gland), thymus, heart, liver, spleen, kidney, adrenal, gonad (testis and ovary), skeletal muscle (crural triceps). However, since the thymus is physiologically retracted severely with ageing, it was not weighed from Week 52 of administration and collected for histological observation only.

HISTOPATHOLOGY: Yes
In histopathological examination, in addition to the above organs (including the thymus), spinal cord (cervical part, chest and lumbar region), sciatic nerve, bone/bone marrow (sternum, femur and knee joint and spine), lymph node (cervical and mesenteric), aorta, tongue, pharynx, salivary gland, oesophagus, stomach, pancreas, duodenum, jejunum, ileum, caecum, colon, head (nasal cavity accessory nasal sinus and middle ear), trachea, pharynx, lung, diaphragm, urinary bladder, epididymis, prostate, seminal vesicle and coagulation gland, uterus, eye ball and accessory gland, skin (dorsolumber part), mammary gland (abdomen) and other organs showing microscopic abnormalities were removed as far as possible from all the animals used and fixed in 10% buffered neutral formalin.
Thereafter the fixed organs and tissues were embedded in paraffin in the usual manner, stained with haematoxylin and eosin and examined histopathologically. The liver of the part of animals killed in week 52 of administration according to the killing schedule was examined by electron microscopy. The removed hepatic tissues were pre-fixed in 3% glutaraldehyde solution and post-fixed in 2% osmic acid solution and embedded in epon in the usual manner. The ultrathin sections were prepared with a ultramicrotome and pre-fixed in uranyl acetate and post-fixed in lead citrate and examined with an electron microscope.

Statistics:

The test of significance was analyzed between the control and treated groups in each test values by Student's t-test. The frequency of histological lesions was analyzed by Fisher's direct probability calculation method.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Abnormal changes in the hindlimbs were observed in 3/56 males of the 2000 ppm group at the terminal stage of the study. In the hindlimbs of these animals, loss of vigor, especially from the knee joint to the crural part and paralytic-like signs were observed. Since changes in hindlimbs were only observed at the highest dose level in one sex and only in 3/56 animals, this observation cannot clearly be related to the treatment with the test substance.
No further treatment-related clinical signs were noted in any of the treated groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The cumulative death rates were as follows:
Control: 15/56 males and 21/56 females
20 ppm: 12/56 males and 17/56 females
200 ppm: 15/56 males and 23/55 femlaes
2000 ppm: 22/56 males and 22/56 females
The final cumulative death rate (39%) was slightly increased in males of the 2000 ppm group when compared to that of the control group (27%). No noticable differences were observed for the female rats between the cumulative death rate of the high dose group and the control group. The increase in mortality of males was mainly due to the increase in dead animals due to foreign body pneumonia (and rhinitis) when compared to the control group. There were no differences in the mortality rates between males and females given 200 ppm or less and those of the control group.

For details, please refer to attachment 1 under "Overall remarks, attachments".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: There were no treatment-related changes in body weight between males and females and those of the control group.
200 ppm: A significant suppression of body weight gain or a tendency to suppress the body weight gain was observed in females from Week 11 to 68 of administration, but thereafter they recovered. No treatment-related changes in body weight were observed in males, when compared to the control group.
2000 ppm: A clear suppression of body weight gain was observed in males and females throughout the study period when compared to the control groups.

For details, please refer to attachment 2 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

20 ppm: No difference in food consumption was observed in male and female rats relative to the control animals.
200 ppm: A statistically significant decrease in food consumption was sporadically observed in females in the early stage of the study period. Afterwards they recovered and no noticeable differences in food consumption relative to the control group were observed.
2000 ppm: A decrease in food consumption was observed in males and females almost throughout the entire study period and the total mean values decreased by 9% for males and by 17% for females when compared with the respective control group.

For details, please refer to attachment 3 under "Overall remarks, attachments".

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: There were no treatment-related differences in food efficiency compared to the control group.
200 ppm: There were no treatment-related differences in food efficiency compared to the control group.
2000 ppm: The total mean value of food efficiency decreased by 11% for males and by 24% for females, as compared with those in the control group.

For details, please refer to attachment 3 under "Overall remarks, attachments".

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

effects observed, non-treatment-related

Description (incidence and severity):

20 ppm: There were no treatment-related differences in water consumption compared to the control group.
200 ppm: A statistically significant increase in water consumption of males was observed sporadically in some weeks during the study period. However, there were no treatment-related differences in the total mean value for water consumption in males and females when compared to the respective control groups.
2000 ppm: The total mean values for water consumption for male rats were increased about 13% relative to the control group. A statistically significant increase in water consumption was also observed in females from week 14 to 48 of administration, however, there were no statistically significant differences in the overall mean values for water consumption in either males or females compared to the respective control animals.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: There were no treatment-related changes in haematological parameters observed in males when comapred to the control group. A slight decrease in RBC count in Week 52 of administration and slight decreases in haematocrit and RBC count in Week 78 of administration were observed for female rats relative to the control group, but these changes were extremely slight and therefore not considered toxicologically relevant. There were no further treatment-related changes observed in female rats when compared to the control animals.
200 ppm: No treatment-related changes in haematological parameters were observed in males when compared to the respective control group. In females, decreases in haematocrit, haemoglobin (only week 78 of administration), decrease in RBC count, and increases in mean corpuscular volume and mean corpuscular haemoglobin were observed in week 52 and 78 of administration relative to the control group.
2000 ppm: Slight but statistical significant decreases in haematocrit, haemoglobin and RBC count were observed in males in Week 78 of administration and anemic tendency was also observed when compared to the control group. In females, statistically significant decreases in haematocrit, haemoglobin and RBC count were observed in week 52 and 78 of administration, whereas mean corpuscular volume, mean corpuscular haemoglobin and reticulocyte count were increased relative to the control group. However, most of the changes observed in females recovered almost completely in Week 104 of administration with exception of the decrease in RBC count and the increase in mean corpuscular volume. The increase in mean corpuscular volume observed in females was considered to be due to the increase in regenerated immature erythrocytes.

For details, please refer to attachment 5 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: Statistically significant increases in GOT and GPT were observed in male rats after 52 Weeks of treatment when compared to the control group. These changes were neither observed in females nor at the other examination time points. Therefore, there was no clear correlation between these changes and the treatment with the test article.
200 ppm: Statistically significant increases in GOT and GPT were observed in male and female rats 26 and 52 weeks after treatment and additionally in males 78 weeks after treatment when compared with the control animals. However, there was no clear dose-dependency as these changes were more marked in the 200 ppm group than in the high dose group, thus there was no clear correlation between these changes and the test article administration.
2000 ppm: Blood glucose, total cholesterol, and calcium were decreased in males at each examination point from Week 52 of administration and in females at each examination point from Week 26 of administration until the end of the treatment period, as compared with those in the control group. This reduction reached statistical significance in most cases. The statistically significant reduction of calcium correlated with observed histopathological findings in the thyroid and bone and was therefore considered to be related to the treatment with the test material. In contrast, the decreases in blood glucose and total cholesterol observed in males and females were considered to reflect the low nutrient state of animals, which showed a marked suppression of body weight gain.

Statistically significant changes were also observed in other examination parameters, but they were not considered to be related to the treatment with the test substance.
For details, please refer to attachment 6 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: No treatment-related differences were observed in males or females when compared to the respective control group.
200 ppm: No treatment-related differences were observed in males or females when compared to the respective control group.
2000 ppm: A statistically significant decrease in urinary specific gravity was observed in males from Week 52 to 104 and in females in Week 26 and 52 of administration when compared to the control animals.

For details, please refer to attachment 4 under "Overall remarks, attachments".

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: No treatment-related changes were observed in male or female rats during the study.
200 ppm: Statistically significant decreases in the absolute and relative weights of the crural muscle were observed in males from Week 52 of administration and in females from Week 78 of administration when compared to the control animals. Furthermore, statistically significant increases in the absolute and relative weights of the spleen were observed in males in Week 52 and in females in Weeks 52 and 78 of administration when compared to the control animals. However, the absolute and relative spleen weights of male and female rats recovered and were similar to those of the control animals at Week 104 after treatment, but it is important to note that the standard deviations of the absolute spleen weight for female rats were very high at this examination time point.
2000 ppm: Statistically significant increase in the absolute and relative weights of the thyroid were observed in females from Week 52 of administration relative to the control group. A statistically significant decrease in the absolute and relative weights of the crural muscle was noted for males and females from Week 52 when compared to the control animals. The relative weight of the thyroid of males and the absolute weight and/or relative weight of the liver and spleen of males and females statistically significantly increased at each examination point when compared to the control group.

In addition to the changes noted above, further statistically significant changes were observed, but they were not considered to be related to the treatment with the test substance.

For details, please refer to attachment 7 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: No treatment-related gross pathological findings were observed in male or female rats.
200 ppm: No treatment-related gross pathological findings were observed in male or female rats.
2000 ppm: Hypertrophy of the thyroid and atrophy of the crural muscle were observed with a high frequency in males and females at sacrifice schedule in Week 52 and 104 of administration as compared with those in the control group. In males of the same group, proximal arcuation of the tibia and extension deficiency of the knee joint were observed sporadically in some of the animals at the end of the examination. In the soft X-ray examination of the diseased limbs of animals with severe changes, clear extension of the tibia, proximal arcuation of the knee joint, and dilatation of the epiphysis were observed.

For details, please refer to attachment 8 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

20 ppm: There were no treatment-related histopathological findings in males and females with regards to non-neoplastic lesions.
200 ppm: The total frequency of follicular hypertrophy of the thyroid (females only), the mucosal cornification of the anterior stomach and the frequency (classified by period) of atrophy of the crural muscle statistically significantly increased from Week 78 of administration when compared to the control group.
2000 ppm: There were statistically significant increases in frequency of various non-neoplastic lesions in the thyroid, knee-joint, crural muscle, sciatic nerve, stomach, bone marrow, spleen, liver, adrenal, nasal cavity, lung and esophagus when compared to the control group. In the thyroid, retention of colloid substance and follicular hypertrophy characterized by squamatization of the epithelial cells increased or tended to increase from Week 26 of administration. The total incidence of these changes were 80% in males and females. Inner bronchial groove remnant of the thyroid also increased statistically significantly, but was not considered to be related to the treatment with the test article. In the knee-joint, excessive remnant of chrondrial plate of the epiphysis (delay in closure of chondrial plate of epiphysis), which is usually closed with aging were observed in males and females. The same lesions were observed mainly in the tibia of males and in both the tibia and femur. Furthermore, chondrial plate of epiphysis of the tibia was hypertrophied in part of the males because of hyperplasia of cartilage cells and hyperplasia of neo-bone trabecula was observed. In the crural muscle, focal muscular atrophy was observed from Week 52 of administration and in the sciatic nerve, nerve fiber degeneration was observed in males and females with a high frequency in Week 104 of administration. In the stomach, increased mucosal cornification of the anterior stomach increased or tended to increase in males and females from Week 26 of administration. In bone marrow, hematogenesis tended to increase in males and females of the dead animals and animals sacrificed due to moribundity from Week 79 of administration and in females extramedullary hematopoiesis of the spleen increased significantly in some periods. In the liver, frequency of small granuloma increased in males, but there were no further noticeable histological changes and no treatment-related histoptahological changes were observed in hepatic cells using an electron microscope. Therefore, the changes in the liver were not considered to be related to the treatment with the test substance. Frequency (classified by period) of focal sinus dilatation (males and females) associated with blood retention in the adrenal cortex, foreign body rhinitis (males and females) and pneumonia (males) were statistically significantly increased from Week 79 of administration. In males, dilatation of the esophageal cavity, which was considered to be correlated to onset of the above foreign body nasal catarrh and pneumonia were statistically significantly increased in the same period.
In total, follicular hypertrophy of the thyroid, abnormal knee-joint, atrophy of the crural muscle, degeneration of the sciatic nerve, increased mucosal cornification of the anterior stomach and increased hematogenesis of the spleen and bone marrow observed in the high dose groups were considered to be changes caused by the treatment with the test article.

For details, please refer to attachment 9 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No neoplastic lesions were observed which were considered to be related to the treatment with the test substance.

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: corresponding to 0.70 mg/kg bw/day for males and 0.83 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

16 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose level

Remarks on result:

other: the NOAEL of 0.7 mg/kg bw/day (20 ppm) was converted using a correction factor of 20% based on the stability of the test substance in the diet. Consequently this corresponded to 0.56 and 0.66 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: additionally, changes in body weight, food consumption and efficiency, haematology, clinical biochemistry, urinalysis, and gross pathology were observed in both sexes at the higher dose level.

Remarks on result:

other: corresponding to 6.9 mg/kg bw/day for males and 8.5 mg/kg bw/day for females

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

160 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: additionally, changes in body weight, food consumption and efficiency, haematology, clinical biochemistry, urinalysis, and gross pathology were observed in both sexes at the higher dose level.

Remarks on result:

other: the LOAEL of 6.9 and 8.5 mg/kg bw/day was converted using a correction factor of 20% based on the assumed instability of the test substance in the diet. Consequently this corresponded to 5.5 and 6.8 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no tumorigenic effects observed at the highest dose level

Remarks on result:

other: corresponding to 74 mg/kg bw/day for males and 91 mg/kg bw/day for females

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 ppm

System:

other: haematopoetic, musculoskeletal, nervous and endocrine system

Organ:

blood

thyroid gland

other: crural muscle, sciatic nerve / nervous tissue

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The present study was conducted to assess the chronic toxicity and carcinogenicity of the test substance in rats when given for a time frame of approximately 104 weeks. The study was similar to the OECD guideline 453 and was performed under GLP conditions. Fischer 334 rats were given the test substance as a diet admixture at doses levels of 0, 20, 200 or 2000 ppm for 24 months (104 weeks of administration). These dose level corresponded to 0.7, 6.9 and 74 mg/kg bw/day for male and to 0.83, 8.5 and 91 mg/kg bw/day for female rats. As shown by subsequent repeated-dose studies, the low- and mid-dose level needed to be converted using a correction factor of 20% based on the insufficient stability of the test substance in the diet. Retrospective recalculation of the dose levels 20 and 200 ppm corresponded therefore to achieved doses of 0.56 and 5.52 mg/kg bw/day for males and 0.66 and 6.8 mg/kg bw/day for females.
In the males and females of the 2000 ppm group, marked suppression of body weight gain, decrease in food consumption, decrease in food efficiency, anemia, decrease in serum calcium, increases in the absolute and relative weights of the thyroid, and decreases in the absolute and relative weights of the crural muscle were observed. In histopathological examination, the frequency of follicular hypertrophy of the thyroid, delay in closure of chondrial plate of the tibial and femoral epiphysis and atrophy of the crural muscle increased significantly. In the 200 ppm group, decreases in absolute and relative weights of the crural muscle were observed in the males and females and atrophic changes in the crural muscle were observed histologically with a high frequency, as compared with those in the control group. Furthermore, anemia was observed in females. The frequency of follicular hypertrophy of the thyroid increased significantly. In the 20 ppm group, no abnormal changes related to the treatment with the test article were observed in males or females. According to these results, the NOAEL for general systemic toxicity was set to 20 ppm, which equals a dose of 16 ppm using a correction factor of 20% (corresponding to 0.56 and 0.66 mg/kg bw/day in male and female rats, respectively).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.56 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1 and 2) and consistent studies, and is thus sufficient to fulfil the standard information requirement set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

System:

other: haematopoietic, hepatobiliary, musculoskeletal, nervous and endocrine system

Organ:

blood

liver

spleen

thyroid gland

other: nervous system and skeletal muscle

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun - 13 Sep 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted in 1981

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

No ophthalmological examination, body weight only recorded weekly, not all organs histopathologically examined

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kent, UK
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 312 - 402 g (males) and 200 - 252 g (females)
- Housing: Groups of 5 animals of the same sex were housed in suspended stainless steel cages fitted with mesh tops and floors.
- Diet: Rat and Mouse No. 1 (E) SQC maintenance diet, ad libitum (no access to food during each 6-h inhalation exposure)
- Water: Tap water, ad libitum (no access to food during each 6-h inhalation exposure)
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21.5
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.8 - <= 2 µm

Remarks on MMAD:

MMAD / GSD: 1.8 - 2.0 / 2.76 - 2.87

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system comprised a snout-only inhalation chamber, rat restraining tubes, a Wright Dust Feed mechanism, an electrical power interrupter unit, polymethyl methacrylate pre-chamber, a diluent air control system and an exhaust system incorporating in-line particulate filters.
- Method of holding animals in test chamber: rat restraining tubes
- Source and rate of air: filtered air, aerosol was generated into a calibrated airflow of 7 L/min, which was diluted by an additional airflow of 50 L/min
- System of generating particulates/aerosols: Wight Dust Feed (WDF) particulate aerosol generator
- Temperature, humidity, pressure in air chamber: 20.1 - 22.3 °C, humidity and pressure were not specified
- Method of particle size determination: Size distribution in each test chamber was determined twice during each week of treatment using a cascade impactor operated at an airflow of 3 L/min. The collection substrates for the device were stored pending extraction of the test substance collected and spectrophotometric analysis of the mass of test substance on each stage of the device.
- Treatment of exhaust air: The extract air passed through a trapping system compromising polypropylene fiber depth filters followed by a glass filter.

TEST ATMOSPHERE
- Brief description of analytical method used: Three samples were taken daily per 6 h exposure after approximately 1, 3, 5 h. The samples were collected onto previously weighed glass fiber filters in an open face holder. The atmosphere samples were taken by drawing a previously selected volume of chamber air through the filters at a calibrated flow of 4 L/min. The air volume of each sample collected was measured (in-line wet type gas meter). The test substance collected on each filter was extracted and the extract was analysed to determine the mass of the test substance collected (spectrophotometric assessment)
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The achieved chamber concentrations are presented in terms of µg/L air and were derived from the analytically determined mass of test material collected on each filter and the measured volume of chamber air sampled. Acetone was used to extract the test material from the collection substrate. the resulting solutions of approximate concentrations of 10 to 0.5 µg/mL were quantified by spectrophotometry.

Duration of treatment / exposure:

28 days

Frequency of treatment:

5 days/week for 6 h/day

Dose / conc.:

0.1 mg/m³ air (nominal)

Remarks:

corresponding to 0.095 mg/m³ air (achieved concentration)

Dose / conc.:

0.3 mg/m³ air (nominal)

Remarks:

corresponding to 0.318 mg/m³ air (achieved concentration)

Dose / conc.:

1 mg/m³ air (nominal)

Remarks:

corresponding to 1.153 mg/m³ air (achieved concentration)

Dose / conc.:

3 mg/m³ air (nominal)

Remarks:

corresponding to 2.898 mg/m³ air (achieved concentration)

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Details on study design:

The study consists of two study groups, one main group and one recovery group.
Main group: Groups of 5 male and 5 female rats, which were exposed to either the negative control (air only) or to the test substance at target concentrations of 0.1,0.3, 1.0 and 3.0 µg/L air for 6 h/day, 5 days a week for a period of 4 weeks.
Recovery group: Groups of 5 male and 5 female rats, which were exposed to either the negative control (air only) or to the test substance at the target concentration of 3.0 µg/L air for 6 h/day, 5 days a week for a period of 4 weeks. The recovery group was retained untreated for a further period of 4 weeks, thus serving to assess reversibility of any effects.
For details on test concentrations, please refer to attachment 1 under "Overall remarks, attachments".

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day (mortality) and once per day (clinical signs)
- Cage side observations checked: mortality and signs of ill-health and behavioural changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly interval with special attention on audible respiratory noise

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, starting 7 days before exposures

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was calculated as g food per rat per week.
Food consumed by each cage of rats was recorded daily, commencing 7 d before the start of exposures. Food intake was determined based on total amounts of food given to and left by each cage in each group and the number of rats surviving in each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 4
- Anaesthetic used for blood collection: Yes (identity not indicated)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: Haematocrit (HCT), haemoglobin (HB), red cell count (RBC), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean cell haemoglobin (MCH), total white cell count (WBC), differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells (LUC)) were determined as well as cell morphology, common morphological changes (anisocytosis, micro/macrocytosis, hypo/hyperchromasia), platelet count (Pit), reticulocyte count (Retic), prothrombin time (PT), and activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 4
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked; Total protein (Total Prot), Albumin (Alb), globulin (Glob Total), urea, creatinine (Creat), sodium (Na), potassium (K), calcium (Ca Total), inorganic phosphorus (Phos), chloride (CI), total cholesterol (Choi Total), alkaline phosphatase (Alk. Phos), total bilirubin (Bili. Total), glucose (Gluc) hexokinase mediated, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma Glutamyl transferase (gGT) and creatinine phosphokinase (CPK Total) [creatinine kinase].

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Following 4 weeks of exposure all rats were sacrificed on the first two days of Week 5. Exposures were continued until the day prior sacrifice. The recovery groups were sacrificed following 4 weeks of exposure and a 4 week withdrawal period on the first day of Week 9. All rats were sacrificed by intraperitoneal injection of pentobarbitone sodium followed by exsanguination from the brachial blood vessels.
At scheduled necropsy, adrenals, kidneys, liver, lungs, spleen, testes and epididymides of all animals (males, females) were dissected and weighed. Adrenals and kidneys were weighed together and testes and epididymides separated.
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 midline 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. Any abnormalities in the appearance and size of the thoracic viscera were recorded. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. Gastrointestinal tract (GIT) 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 illumination. 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. Gross lesions were examined from all animals and the following organs were collected: adrenals, alimentary tract (oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum), aorta, eyes, epididymides, femur (with joint), heart, kidney, larynx, liver, lungs (all lobes, mainstream bronchi), lymph nodes (cervical, mesenteric, tracheobronchial), mammary gland, nasal passages, optic nerve, ovaries, oviduct, pancreas, pharynx, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal column, spinal cord (cervical, thoracic, lumbar), spleen, sternum (bone and marrow), testes, thymus, thyroid (with parathyroids), tongue, trachea (including bifurcation), ureter, urinary bladder, uterus (corpus and cervix) and vagina.

HISTOPATHOLOGY: Yes
Larynx, lungs (all lobes, mainstream bronchi), trachea (including bifurcation), and any macroscopic abnormalities were examined in all animals (males, females) using a light microscope. In the animals (males, females) of control groups and high-dose groups also adrenals, epididymides, heart, kidneys and liver were examined using a light microscope.

Statistics:

Statistical analyses were carried out separately for each sex. Data relating to food 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. Bw data were analysed using weight gain. Food consumption data to Week 4 were analysed using cumulative cage totals.
Statistical tests used for body weight, food consumption, organ weight and clinical pathology data were frequency, parametric and/or non-parametric analysis. For details, please refer to "Any other information on materials and methods incl. tables".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical observations that were considered to be as a result of treatment with the test substance. The type and frequency of clinical signs observed during the exposure and recovery were those routinely seen in this laboratory in this age and strain of animals.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the course of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no conclusive evidence of a reaction to treatment, with body weight gains being essentially comparable in all groups including the control group.
Body weight gain was statistically significantly higher in the high dose group than concurrent controls during the 4 week reversibility period. It was a minor change, which was not considered to be of toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Values for all treated group animals were similar to that of the control animals during the exposure and recovery periods.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on the haematological parameters. Occasional differences from control values attained statistical significance, but in the absence of any correlation across the sexes these are all considered to be of no toxicological importance.
For details, please refer to attachment 2 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on the biochemical parameters. Occasional differences from control values attained statistical significance, most notably group mean glucose values of high and high intermediate dose males, but in the absence of any correlation across the sexes, and the absence of effects on any other parameters, these are all considered to be of no toxicological importance.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

An increase in lung weights (body weight adjusted) was observed in animals (males, females) of the high dose groups compared to the control animals. For both sexes this was a reversible finding (as evident by the recovery groups). Increased lung weights (body weight adjusted) were also noted for females of the high intermediate dose group compared to the control animals. Females of the recovery high dose group (previously exposed to 3.0 µg/L) showed slightly but statistically significantly lower kidney weights. Since no similar finding was noted at termination of animals following 4 weeks exposure, this change is of no biological significance.
No other test substance-related effects on organ weights were noted.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no lesions evident at either the terminal kills or recovery kills that were considered to be attributable to treatment with the test substance. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological changes were observed for larynx, lungs and trachea and tracheal bifurcation.

Larynx:
Main group:
Changes of the larynx were seen at all dose levels except the low-dose groups. In the high dose and high intermediate dose groups those changes included squamous metaplasia of the ventrolateral epithelium, hyperplasia of the ventral epithelium, necrosis of the ventral cartilage, subepithelial inflammatory infiltration and epithelial hyperplasia (ventral pouch, arytenoid cartilage). The changes seen were similar in character and degree. In the low intermediate dose groups squamous metaplasia of the ventrolateral epithelium, hyperplasia of the ventral epithelium, necrosis of the ventral cartilage and subepithelial inflammatory infiltration were observed.
Recovery group:
Following the 28 day reversibility period, histopathology revealed changes of the larynx in the high-dose groups. There was some evidence of recovery. No lesions were observed in the ventral pouch and arytenoids and no evidence of vesiculation in the ventrolateral epithelium. Squamous metaplasia was still present in ventrolateral epithelium of males and females accompanied by ventral epithelial hyperplasia in affected males. The severity of these changes was reduced. Subepithelial inflammatory infiltration was decreased when compared with animals killed at the end of the treatment period. There was no evidence of recovery in the ventral cartilage in investigated animals; necrosis was still present in all animals.

Lungs:
Main group:
In rats from high dose and high intermediate dose groups also changes of the lungs were observed when compared with the controls. Those changes were fibrosis, granulomatous inflammation, bronchiolar metaplasia in the alveolar ducts, prominent goblet cells in the bronchioles and terminal bronchioles, mucous/cellular debris in the airways, epithelial hyperplasia in the bronchioles and terminal bronchioles, bronchiolitis and an increased incidence of foamy alveolar macrophages and extravasation of eosinophils. In the low intermediate and low dose groups the changes of the lungs were similar to the controls.
Recovery group:
In lungs, there was evidence of a degree of recovery. No granulomatous inflammation, bronchiolar metaplasia in alveolar duct and changes in bronchioles were observed. No treatment-related effect on the incidence or severity of foamy alveolar macrophages or in the extravasation of eosinophils occurred. Minimal degree of fibrosis was still present in alveolar ducts. All other findings in the respiratory tracts of rats killed after the reversibility period were considered to be incidental.

Trachea and tracheal bifurcation:
Main group:
In the high-dose and the high intermediate dose-groups changes in trachea and tracheal bifurcation were present. Epithelial hyperplasia was seen at the tracheal bifurcation. The incidence of this finding was dose-related. Treatment-related epithelial hyperplasia was also observed in trachea of 2 females from the high dose group.
Recovery groups:
A complete recovery of the trachea and tracheal bifurcation lesions was observed in the high-dose recovery groups.

Other findings:
Main group:
In the nasal turbinates, there was low incidence of changes in the respiratory and olfactory epithelium throughout all main dose groups. The nature of these findings was inconsistent and they were considered to be of no toxicological significance.
Recovery group:
There were no lesions evident at the recovery kills that were considered to be attributable to treatment with the test substance. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic findings.

For details, please refer to attachment 5 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEC

Remarks:

general systemic toxicity

Effect level:

3 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse systemic effects observed at the highest concentration tested.

Key result

Dose descriptor:

NOAEC

Remarks:

respiratory system

Effect level:

0.1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at the respiratory system at this concentration.

Key result

Dose descriptor:

LOAEC

Remarks:

respiratory system

Effect level:

0.3 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.3 mg/m³ air (nominal)

System:

other: respiratory system

Organ:

larynx

lungs

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The present subacute inhalation toxicity study was conducted according to the OECD guideline 412 and GLP conditions. Male and female rats were exposed to the test item as aerosol at nominal concentrations of 0.1, 0.3, 1.0 and 2.0 mg/m³ air via snout-only exposure conditions. The aerosol was highly respirable to rats, with mass median aerodynamic diameter (MMAD) of 1.8 - 2.0 µm. There was no clear evidence for general toxicity in male and female rats. Histological changes included inflammatory and proliferative lesions in the respiratory tract of rats from the high, high intermediate and low intermediate dose groups sacrificed after 28 d of exposure. Noted increase in lung weight in high-dose group animals and females of the high intermediate dose group was regarded as attributable to these lesions. A complete recovery of the trachea and tracheal bifurcation lesions, partial recovery of the lung lesions; and some evidence of recovery of the laryngeal lesions was observed in the high-dose recovery groups. No treatment-related histological changes were noted in all low-dose group rats. Therefore, the NOAEC regarding the respiratory system was set to 0.1 mg/m³ air. As no clear evidence for systemic effects was observed, the NOAEC for general toxicity was set to 3 mg/m³ air.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

3 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirement set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun - 13 Sep 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted in 1981

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

No ophthalmological examination, body weight only recorded weekly, not all organs histopathologically examined

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kent, UK
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 312 - 402 g (males) and 200 - 252 g (females)
- Housing: Groups of 5 animals of the same sex were housed in suspended stainless steel cages fitted with mesh tops and floors.
- Diet: Rat and Mouse No. 1 (E) SQC maintenance diet, ad libitum (no access to food during each 6-h inhalation exposure)
- Water: Tap water, ad libitum (no access to food during each 6-h inhalation exposure)
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21.5
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.8 - <= 2 µm

Remarks on MMAD:

MMAD / GSD: 1.8 - 2.0 / 2.76 - 2.87

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system comprised a snout-only inhalation chamber, rat restraining tubes, a Wright Dust Feed mechanism, an electrical power interrupter unit, polymethyl methacrylate pre-chamber, a diluent air control system and an exhaust system incorporating in-line particulate filters.
- Method of holding animals in test chamber: rat restraining tubes
- Source and rate of air: filtered air, aerosol was generated into a calibrated airflow of 7 L/min, which was diluted by an additional airflow of 50 L/min
- System of generating particulates/aerosols: Wight Dust Feed (WDF) particulate aerosol generator
- Temperature, humidity, pressure in air chamber: 20.1 - 22.3 °C, humidity and pressure were not specified
- Method of particle size determination: Size distribution in each test chamber was determined twice during each week of treatment using a cascade impactor operated at an airflow of 3 L/min. The collection substrates for the device were stored pending extraction of the test substance collected and spectrophotometric analysis of the mass of test substance on each stage of the device.
- Treatment of exhaust air: The extract air passed through a trapping system compromising polypropylene fiber depth filters followed by a glass filter.

TEST ATMOSPHERE
- Brief description of analytical method used: Three samples were taken daily per 6 h exposure after approximately 1, 3, 5 h. The samples were collected onto previously weighed glass fiber filters in an open face holder. The atmosphere samples were taken by drawing a previously selected volume of chamber air through the filters at a calibrated flow of 4 L/min. The air volume of each sample collected was measured (in-line wet type gas meter). The test substance collected on each filter was extracted and the extract was analysed to determine the mass of the test substance collected (spectrophotometric assessment)
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The achieved chamber concentrations are presented in terms of µg/L air and were derived from the analytically determined mass of test material collected on each filter and the measured volume of chamber air sampled. Acetone was used to extract the test material from the collection substrate. the resulting solutions of approximate concentrations of 10 to 0.5 µg/mL were quantified by spectrophotometry.

Duration of treatment / exposure:

28 days

Frequency of treatment:

5 days/week for 6 h/day

Dose / conc.:

0.1 mg/m³ air (nominal)

Remarks:

corresponding to 0.095 mg/m³ air (achieved concentration)

Dose / conc.:

0.3 mg/m³ air (nominal)

Remarks:

corresponding to 0.318 mg/m³ air (achieved concentration)

Dose / conc.:

1 mg/m³ air (nominal)

Remarks:

corresponding to 1.153 mg/m³ air (achieved concentration)

Dose / conc.:

3 mg/m³ air (nominal)

Remarks:

corresponding to 2.898 mg/m³ air (achieved concentration)

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Details on study design:

The study consists of two study groups, one main group and one recovery group.
Main group: Groups of 5 male and 5 female rats, which were exposed to either the negative control (air only) or to the test substance at target concentrations of 0.1,0.3, 1.0 and 3.0 µg/L air for 6 h/day, 5 days a week for a period of 4 weeks.
Recovery group: Groups of 5 male and 5 female rats, which were exposed to either the negative control (air only) or to the test substance at the target concentration of 3.0 µg/L air for 6 h/day, 5 days a week for a period of 4 weeks. The recovery group was retained untreated for a further period of 4 weeks, thus serving to assess reversibility of any effects.
For details on test concentrations, please refer to attachment 1 under "Overall remarks, attachments".

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day (mortality) and once per day (clinical signs)
- Cage side observations checked: mortality and signs of ill-health and behavioural changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly interval with special attention on audible respiratory noise

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, starting 7 days before exposures

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was calculated as g food per rat per week.
Food consumed by each cage of rats was recorded daily, commencing 7 d before the start of exposures. Food intake was determined based on total amounts of food given to and left by each cage in each group and the number of rats surviving in each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 4
- Anaesthetic used for blood collection: Yes (identity not indicated)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: Haematocrit (HCT), haemoglobin (HB), red cell count (RBC), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean cell haemoglobin (MCH), total white cell count (WBC), differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells (LUC)) were determined as well as cell morphology, common morphological changes (anisocytosis, micro/macrocytosis, hypo/hyperchromasia), platelet count (Pit), reticulocyte count (Retic), prothrombin time (PT), and activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 4
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked; Total protein (Total Prot), Albumin (Alb), globulin (Glob Total), urea, creatinine (Creat), sodium (Na), potassium (K), calcium (Ca Total), inorganic phosphorus (Phos), chloride (CI), total cholesterol (Choi Total), alkaline phosphatase (Alk. Phos), total bilirubin (Bili. Total), glucose (Gluc) hexokinase mediated, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma Glutamyl transferase (gGT) and creatinine phosphokinase (CPK Total) [creatinine kinase].

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Following 4 weeks of exposure all rats were sacrificed on the first two days of Week 5. Exposures were continued until the day prior sacrifice. The recovery groups were sacrificed following 4 weeks of exposure and a 4 week withdrawal period on the first day of Week 9. All rats were sacrificed by intraperitoneal injection of pentobarbitone sodium followed by exsanguination from the brachial blood vessels.
At scheduled necropsy, adrenals, kidneys, liver, lungs, spleen, testes and epididymides of all animals (males, females) were dissected and weighed. Adrenals and kidneys were weighed together and testes and epididymides separated.
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 midline 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. Any abnormalities in the appearance and size of the thoracic viscera were recorded. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. Gastrointestinal tract (GIT) 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 illumination. 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. Gross lesions were examined from all animals and the following organs were collected: adrenals, alimentary tract (oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum), aorta, eyes, epididymides, femur (with joint), heart, kidney, larynx, liver, lungs (all lobes, mainstream bronchi), lymph nodes (cervical, mesenteric, tracheobronchial), mammary gland, nasal passages, optic nerve, ovaries, oviduct, pancreas, pharynx, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal column, spinal cord (cervical, thoracic, lumbar), spleen, sternum (bone and marrow), testes, thymus, thyroid (with parathyroids), tongue, trachea (including bifurcation), ureter, urinary bladder, uterus (corpus and cervix) and vagina.

HISTOPATHOLOGY: Yes
Larynx, lungs (all lobes, mainstream bronchi), trachea (including bifurcation), and any macroscopic abnormalities were examined in all animals (males, females) using a light microscope. In the animals (males, females) of control groups and high-dose groups also adrenals, epididymides, heart, kidneys and liver were examined using a light microscope.

Statistics:

Statistical analyses were carried out separately for each sex. Data relating to food 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. Bw data were analysed using weight gain. Food consumption data to Week 4 were analysed using cumulative cage totals.
Statistical tests used for body weight, food consumption, organ weight and clinical pathology data were frequency, parametric and/or non-parametric analysis. For details, please refer to "Any other information on materials and methods incl. tables".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical observations that were considered to be as a result of treatment with the test substance. The type and frequency of clinical signs observed during the exposure and recovery were those routinely seen in this laboratory in this age and strain of animals.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the course of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no conclusive evidence of a reaction to treatment, with body weight gains being essentially comparable in all groups including the control group.
Body weight gain was statistically significantly higher in the high dose group than concurrent controls during the 4 week reversibility period. It was a minor change, which was not considered to be of toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Values for all treated group animals were similar to that of the control animals during the exposure and recovery periods.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on the haematological parameters. Occasional differences from control values attained statistical significance, but in the absence of any correlation across the sexes these are all considered to be of no toxicological importance.
For details, please refer to attachment 2 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on the biochemical parameters. Occasional differences from control values attained statistical significance, most notably group mean glucose values of high and high intermediate dose males, but in the absence of any correlation across the sexes, and the absence of effects on any other parameters, these are all considered to be of no toxicological importance.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

An increase in lung weights (body weight adjusted) was observed in animals (males, females) of the high dose groups compared to the control animals. For both sexes this was a reversible finding (as evident by the recovery groups). Increased lung weights (body weight adjusted) were also noted for females of the high intermediate dose group compared to the control animals. Females of the recovery high dose group (previously exposed to 3.0 µg/L) showed slightly but statistically significantly lower kidney weights. Since no similar finding was noted at termination of animals following 4 weeks exposure, this change is of no biological significance.
No other test substance-related effects on organ weights were noted.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no lesions evident at either the terminal kills or recovery kills that were considered to be attributable to treatment with the test substance. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological changes were observed for larynx, lungs and trachea and tracheal bifurcation.

Larynx:
Main group:
Changes of the larynx were seen at all dose levels except the low-dose groups. In the high dose and high intermediate dose groups those changes included squamous metaplasia of the ventrolateral epithelium, hyperplasia of the ventral epithelium, necrosis of the ventral cartilage, subepithelial inflammatory infiltration and epithelial hyperplasia (ventral pouch, arytenoid cartilage). The changes seen were similar in character and degree. In the low intermediate dose groups squamous metaplasia of the ventrolateral epithelium, hyperplasia of the ventral epithelium, necrosis of the ventral cartilage and subepithelial inflammatory infiltration were observed.
Recovery group:
Following the 28 day reversibility period, histopathology revealed changes of the larynx in the high-dose groups. There was some evidence of recovery. No lesions were observed in the ventral pouch and arytenoids and no evidence of vesiculation in the ventrolateral epithelium. Squamous metaplasia was still present in ventrolateral epithelium of males and females accompanied by ventral epithelial hyperplasia in affected males. The severity of these changes was reduced. Subepithelial inflammatory infiltration was decreased when compared with animals killed at the end of the treatment period. There was no evidence of recovery in the ventral cartilage in investigated animals; necrosis was still present in all animals.

Lungs:
Main group:
In rats from high dose and high intermediate dose groups also changes of the lungs were observed when compared with the controls. Those changes were fibrosis, granulomatous inflammation, bronchiolar metaplasia in the alveolar ducts, prominent goblet cells in the bronchioles and terminal bronchioles, mucous/cellular debris in the airways, epithelial hyperplasia in the bronchioles and terminal bronchioles, bronchiolitis and an increased incidence of foamy alveolar macrophages and extravasation of eosinophils. In the low intermediate and low dose groups the changes of the lungs were similar to the controls.
Recovery group:
In lungs, there was evidence of a degree of recovery. No granulomatous inflammation, bronchiolar metaplasia in alveolar duct and changes in bronchioles were observed. No treatment-related effect on the incidence or severity of foamy alveolar macrophages or in the extravasation of eosinophils occurred. Minimal degree of fibrosis was still present in alveolar ducts. All other findings in the respiratory tracts of rats killed after the reversibility period were considered to be incidental.

Trachea and tracheal bifurcation:
Main group:
In the high-dose and the high intermediate dose-groups changes in trachea and tracheal bifurcation were present. Epithelial hyperplasia was seen at the tracheal bifurcation. The incidence of this finding was dose-related. Treatment-related epithelial hyperplasia was also observed in trachea of 2 females from the high dose group.
Recovery groups:
A complete recovery of the trachea and tracheal bifurcation lesions was observed in the high-dose recovery groups.

Other findings:
Main group:
In the nasal turbinates, there was low incidence of changes in the respiratory and olfactory epithelium throughout all main dose groups. The nature of these findings was inconsistent and they were considered to be of no toxicological significance.
Recovery group:
There were no lesions evident at the recovery kills that were considered to be attributable to treatment with the test substance. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic findings.

For details, please refer to attachment 5 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEC

Remarks:

general systemic toxicity

Effect level:

3 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse systemic effects observed at the highest concentration tested.

Key result

Dose descriptor:

NOAEC

Remarks:

respiratory system

Effect level:

0.1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at the respiratory system at this concentration.

Key result

Dose descriptor:

LOAEC

Remarks:

respiratory system

Effect level:

0.3 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.3 mg/m³ air (nominal)

System:

other: respiratory system

Organ:

larynx

lungs

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The present subacute inhalation toxicity study was conducted according to the OECD guideline 412 and GLP conditions. Male and female rats were exposed to the test item as aerosol at nominal concentrations of 0.1, 0.3, 1.0 and 2.0 mg/m³ air via snout-only exposure conditions. The aerosol was highly respirable to rats, with mass median aerodynamic diameter (MMAD) of 1.8 - 2.0 µm. There was no clear evidence for general toxicity in male and female rats. Histological changes included inflammatory and proliferative lesions in the respiratory tract of rats from the high, high intermediate and low intermediate dose groups sacrificed after 28 d of exposure. Noted increase in lung weight in high-dose group animals and females of the high intermediate dose group was regarded as attributable to these lesions. A complete recovery of the trachea and tracheal bifurcation lesions, partial recovery of the lung lesions; and some evidence of recovery of the laryngeal lesions was observed in the high-dose recovery groups. No treatment-related histological changes were noted in all low-dose group rats. Therefore, the NOAEC regarding the respiratory system was set to 0.1 mg/m³ air. As no clear evidence for systemic effects was observed, the NOAEC for general toxicity was set to 3 mg/m³ air.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.1 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirement set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Feb - 03 March 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

adopted in 1981

Deviations:

yes

Remarks:

Occlusive instead of semi-occlusive conditions

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna U.K. Ltd., Cambridgeshire, UK
- Age at study initiation: 10 - 12 weeks on arrival
- Weight at study initiation: within a weight range of 2.2 - 2.6 kg on arrival
- Housing: All rabbits were housed individually.
- Diet: SQC Rabbit Diet, ad libitum; 10 g of autoclaved hay was given to occasional animals showing a loss of condition.
- Water: Tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 21.7
- Humidity (%): 45.6 - 50.8
- Air changes (per hr): approximately 19
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: mid-dorsal region
- % coverage: approximately 10% of total body surface
- Type of wrap if used: Treatment site was then covered with an impervious bandage consisting of gauze covered with ‘Elastoplast’ elastic adhesive dressing backed with impervious 'Sleek' plaster.
- Time intervals for shavings or clipplings: Hair clipping was carried out approximately 24 h prior to the first application of the test substance.

REMOVAL OF TEST SUBSTANCE
- Washing: with warm tap water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 2, 3, and 4 mL for the low, mid and high dose groups; the vehicle control animals received 4 mL of distilled water.
- Constant volume or concentration used: no
- For solids, paste formed: yes, powder was moistened with water

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

21 days for 6 h/day

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for ill health, behavioural changes or signs of reaction to treatment; twice daily for mortality

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: immediately prior to the first daily application of the test substance and subsequently daily
Local dermal reactions (erythema; edema) resulting from treatment were assessed on a numerical basis according to a modified Draize scoring system.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing and once a week thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
Food consumption of all animals was recorded at weekly intervals throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination on Day 20; for specified animals procedure was repeated on Day 22
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: packed cell volume (PCV), haemoglobin (Hb), red blood cell count (RBC), platelet count (Pits), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), total white blood cell count (WBC), differential white blood cell count (Neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M)) and thrombotest (TT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination on Day 20; for specified animals procedure was repeated on Day 22
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), alkaline phosphatase (AP), total bilirubin, cholesterol (Chol), urea nitrogen (Urea Nitr), total protein, albumin (Alb), globulin (Glob), albumin/globulin ratio (A/G), sodium (Na), potassium (K), calcium (Ca), chloride (CI), inorganic phosphorus (P) and creatinine

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After 21 days of treatment (Days 22 or 23), all animals were finally killed by i.v. administration of an overdose of pentobarbitone sodium. A complete autopsy was performed on all animals. Adrenals, kidneys, liver, ovaries and testes with epididymides of all animals (males, females) were dissected free of fat and weighed.

HISTOPATHOLOGY: Yes
Histopathological evaluation of the following tissues was carried out in animals (males, females) of the high dose group and control group: kidneys, liver, skin and abnormal tissues.
Tissues were preserved in 10% buffered formalin.
Fixed tissue samples required for microscopic examination were embedded in paraffin wax (m. p. 56 °C), sections were cut at 4 µm and stained with haematoxylin and eosin.

Statistics:

All analyses were carried out separately for male and female rabbits.
The following tests were used for food and water consumption, bodyweight, relative organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:
- 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 a Student’s ‘t’ test and Williams’ test for a dose-related response, although only the one thought most appropriate for the response pattern observed has been reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate for organ weight data, analysis of covariance was used in place of analysis of variance.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of reaction to treatment were observed.

Dermal irritation:

no effects observed

Description (incidence and severity):

No dermal reactions to treatment were observed amongst rabbits of the control and test groups. Infrequent, transient cases of staining (light brown) of the dose site were recorded amongst animals of the high dose group (1000 mg/kg bw/day).

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related effects on mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight losses or reduced body weight gain were recorded for female rabbits of the high-dose group during the dosing period. Compared to the female controls, differences were statistically significant for all three weeks. No other significant differences in body weight between animals receiving the test substance and the controls were noted.
For details, please refer to attachment 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in food consumption during study Week 1 was recorded for female rabbits in the high-dose group in comparison with the female controls. Food consumption for these animals was also reduced (but did not achieve statistical significance) during study weeks 2 and 3. For all other treated animals food consumption was similar to that of the control animals.
For details, please refer to attachment 2 under "Overall remarks, attachments".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A treatment-related lower lymphocyte count was observed for animals treated with 1000 mg/kg bw/day, reaching statistical significance in female rabbits, but not in male rabbits. There were no other statistically significant differences between rabbits treated with the test substance and rabbits of the respective control group.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increases in the liver enzymes GPT and GOT were observed in males and females treated with 300 and 1000 mg/kg bw/day. The increase was statistically significant for GPT in females treated with 1000 mg/kg bw/day and for GOT in females treated with 300 and 1000 mg/kg bw/day. Females treated with 1000 mg/kg bw/day also showed statistically significantly increased levels of bilirubin. Statistically significantly increased levels of cholesterol were observed for male and female animals treated with 1000 mg/kg bw/day.
Overall, a clear effect on biochemical parameters was observed amongst rabbits of the high-dose groups. Amongst females of the mid-dose group, the occurrence of a real effect on liver enzymes was further supported by individual data. Individual values above the laboratory control range were observed for GOT in 3/5 females and for GPT in 1/5 females (Laboratory 99 percentiles for female rabbits: GOT, 42 mU/mL; GPT, 99 mU/mL).
For details, please refer to attachment 3 and 6 under "Overall remarks, attachments".

Endocrine findings:

not examined

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):

There were no statistically significant differences between animals treated with the test substance and the controls. For details, please refer to attachment 4 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed at termination revealed no changes that were attributable to treatment with the test substance.
An increased incidence of irregular cortical scarring of the kidneys was recorded amongst rabbits of all groups. However, this was not considered to be related to treatment with the test substance.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In microscopic pathology, no changes were revealed that were attributable to treatment with the test substance.
One female rabbit of the high-dose group showed localised area of ulceration and minimal acanthosis was considered most probably due to physical trauma, since similar changes were not observed throughout this treated site.
Microscopic changes seen in other tissues examined were considered to be spontaneous in origin and therefore of no toxicological importance.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

other: additionally, changes in body weight (females), food consumption (females) and haematological parameters (males and females) were observed at 1000 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No dermal irritation observed at the highest dose level tested.

Remarks on result:

other: corresponding to a NOAEL for local effects of 11.81 mg/cm².

Remarks:

The local NOAEL of 11.81 mg/cm² was calculated retrospectively according to Derelanko, M. J., The toxicologist’s pocket handbook, CRC Press, 2008 and is based on a body surface area of 2540 cm² for a 3 kg rabbit. The local NOAEL of 1000 mg/kg bw/day corresponds to an amount of 3000 mg test substance for a rabbit (3 kg) and divided by 10% of the body surface area (254 cm²), this results in a local NOAEL of 11.81 mg/cm².

Key result

Critical effects observed:

no

Conclusions:

The study was conducted similar to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused no dermal irritation when applied dermally for three weeks to the intact skin of male and female rabbits up to and including 1000 mg/kg bw/day. Treatment-related disturbances in body weight, food consumption and various haematological and biochemical parameters were observed for rabbits receiving the test substance at 1000 mg/kg bw/day although, in the main, statistical significance was only achieved for females. Amongst animals receiving 300 mg/kg bw/day, females showed increased enzyme levels, in particular GOT. Thus, 1000 mg/kg bw/day was regarded as the NOAEL for local skin effects and the NOAEL for systemic effects was 100 mg/kg bw/day for males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

42

Species:

rabbit

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistend study, and is thus sufficient to fulfil the standard information requirement set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Feb - 03 March 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

adopted in 1981

Deviations:

yes

Remarks:

Occlusive instead of semi-occlusive conditions

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna U.K. Ltd., Cambridgeshire, UK
- Age at study initiation: 10 - 12 weeks on arrival
- Weight at study initiation: within a weight range of 2.2 - 2.6 kg on arrival
- Housing: All rabbits were housed individually.
- Diet: SQC Rabbit Diet, ad libitum; 10 g of autoclaved hay was given to occasional animals showing a loss of condition.
- Water: Tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 21.7
- Humidity (%): 45.6 - 50.8
- Air changes (per hr): approximately 19
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: mid-dorsal region
- % coverage: approximately 10% of total body surface
- Type of wrap if used: Treatment site was then covered with an impervious bandage consisting of gauze covered with ‘Elastoplast’ elastic adhesive dressing backed with impervious 'Sleek' plaster.
- Time intervals for shavings or clipplings: Hair clipping was carried out approximately 24 h prior to the first application of the test substance.

REMOVAL OF TEST SUBSTANCE
- Washing: with warm tap water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 2, 3, and 4 mL for the low, mid and high dose groups; the vehicle control animals received 4 mL of distilled water.
- Constant volume or concentration used: no
- For solids, paste formed: yes, powder was moistened with water

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

21 days for 6 h/day

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for ill health, behavioural changes or signs of reaction to treatment; twice daily for mortality

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: immediately prior to the first daily application of the test substance and subsequently daily
Local dermal reactions (erythema; edema) resulting from treatment were assessed on a numerical basis according to a modified Draize scoring system.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing and once a week thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
Food consumption of all animals was recorded at weekly intervals throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination on Day 20; for specified animals procedure was repeated on Day 22
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: packed cell volume (PCV), haemoglobin (Hb), red blood cell count (RBC), platelet count (Pits), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), total white blood cell count (WBC), differential white blood cell count (Neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M)) and thrombotest (TT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination on Day 20; for specified animals procedure was repeated on Day 22
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), alkaline phosphatase (AP), total bilirubin, cholesterol (Chol), urea nitrogen (Urea Nitr), total protein, albumin (Alb), globulin (Glob), albumin/globulin ratio (A/G), sodium (Na), potassium (K), calcium (Ca), chloride (CI), inorganic phosphorus (P) and creatinine

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After 21 days of treatment (Days 22 or 23), all animals were finally killed by i.v. administration of an overdose of pentobarbitone sodium. A complete autopsy was performed on all animals. Adrenals, kidneys, liver, ovaries and testes with epididymides of all animals (males, females) were dissected free of fat and weighed.

HISTOPATHOLOGY: Yes
Histopathological evaluation of the following tissues was carried out in animals (males, females) of the high dose group and control group: kidneys, liver, skin and abnormal tissues.
Tissues were preserved in 10% buffered formalin.
Fixed tissue samples required for microscopic examination were embedded in paraffin wax (m. p. 56 °C), sections were cut at 4 µm and stained with haematoxylin and eosin.

Statistics:

All analyses were carried out separately for male and female rabbits.
The following tests were used for food and water consumption, bodyweight, relative organ weight and clinical pathology data:
- If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with values different from the mode was analysed by appropriate methods. Otherwise:
- 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 a Student’s ‘t’ test and Williams’ test for a dose-related response, although only the one thought most appropriate for the response pattern observed has been reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirleys’ test).
Where appropriate for organ weight data, analysis of covariance was used in place of analysis of variance.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of reaction to treatment were observed.

Dermal irritation:

no effects observed

Description (incidence and severity):

No dermal reactions to treatment were observed amongst rabbits of the control and test groups. Infrequent, transient cases of staining (light brown) of the dose site were recorded amongst animals of the high dose group (1000 mg/kg bw/day).

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related effects on mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight losses or reduced body weight gain were recorded for female rabbits of the high-dose group during the dosing period. Compared to the female controls, differences were statistically significant for all three weeks. No other significant differences in body weight between animals receiving the test substance and the controls were noted.
For details, please refer to attachment 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in food consumption during study Week 1 was recorded for female rabbits in the high-dose group in comparison with the female controls. Food consumption for these animals was also reduced (but did not achieve statistical significance) during study weeks 2 and 3. For all other treated animals food consumption was similar to that of the control animals.
For details, please refer to attachment 2 under "Overall remarks, attachments".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A treatment-related lower lymphocyte count was observed for animals treated with 1000 mg/kg bw/day, reaching statistical significance in female rabbits, but not in male rabbits. There were no other statistically significant differences between rabbits treated with the test substance and rabbits of the respective control group.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increases in the liver enzymes GPT and GOT were observed in males and females treated with 300 and 1000 mg/kg bw/day. The increase was statistically significant for GPT in females treated with 1000 mg/kg bw/day and for GOT in females treated with 300 and 1000 mg/kg bw/day. Females treated with 1000 mg/kg bw/day also showed statistically significantly increased levels of bilirubin. Statistically significantly increased levels of cholesterol were observed for male and female animals treated with 1000 mg/kg bw/day.
Overall, a clear effect on biochemical parameters was observed amongst rabbits of the high-dose groups. Amongst females of the mid-dose group, the occurrence of a real effect on liver enzymes was further supported by individual data. Individual values above the laboratory control range were observed for GOT in 3/5 females and for GPT in 1/5 females (Laboratory 99 percentiles for female rabbits: GOT, 42 mU/mL; GPT, 99 mU/mL).
For details, please refer to attachment 3 and 6 under "Overall remarks, attachments".

Endocrine findings:

not examined

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):

There were no statistically significant differences between animals treated with the test substance and the controls. For details, please refer to attachment 4 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed at termination revealed no changes that were attributable to treatment with the test substance.
An increased incidence of irregular cortical scarring of the kidneys was recorded amongst rabbits of all groups. However, this was not considered to be related to treatment with the test substance.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In microscopic pathology, no changes were revealed that were attributable to treatment with the test substance.
One female rabbit of the high-dose group showed localised area of ulceration and minimal acanthosis was considered most probably due to physical trauma, since similar changes were not observed throughout this treated site.
Microscopic changes seen in other tissues examined were considered to be spontaneous in origin and therefore of no toxicological importance.
For details, please refer to attachment 5 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

other: additionally, changes in body weight (females), food consumption (females) and haematological parameters (males and females) were observed at 1000 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No dermal irritation observed at the highest dose level tested.

Remarks on result:

other: corresponding to a NOAEL for local effects of 11.81 mg/cm².

Remarks:

The local NOAEL of 11.81 mg/cm² was calculated retrospectively according to Derelanko, M. J., The toxicologist’s pocket handbook, CRC Press, 2008 and is based on a body surface area of 2540 cm² for a 3 kg rabbit. The local NOAEL of 1000 mg/kg bw/day corresponds to an amount of 3000 mg test substance for a rabbit (3 kg) and divided by 10% of the body surface area (254 cm²), this results in a local NOAEL of 11.81 mg/cm².

Key result

Critical effects observed:

no

Conclusions:

The study was conducted similar to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused no dermal irritation when applied dermally for three weeks to the intact skin of male and female rabbits up to and including 1000 mg/kg bw/day. Treatment-related disturbances in body weight, food consumption and various haematological and biochemical parameters were observed for rabbits receiving the test substance at 1000 mg/kg bw/day although, in the main, statistical significance was only achieved for females. Amongst animals receiving 300 mg/kg bw/day, females showed increased enzyme levels, in particular GOT. Thus, 1000 mg/kg bw/day was regarded as the NOAEL for local skin effects and the NOAEL for systemic effects was 100 mg/kg bw/day for males and females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

11.81 mg/cm²

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirement set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Additional information

Repeated dose studies with the test substance have been conducted in rats, mice, dogs and rabbits.

 

Repeated dose toxicity: oral administration

Data on repeated dose toxicity are available after exposure for 28 days, 90 days, 52 weeks (dog) and 2 years to characterize the potency of the test substance to induce toxicity after short-term and subchronic exposure.

Rats

Two subacute (Report number: 2-4-132-80 and ZIR 2/89719), one subchronic (Report number: ZIR 5/901840), one combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (Report number: 615-1-04-24768) and two combined chronic/carcinogenicity studies (Report number: ZIR 9/942098 and 83-5121) are available, which are either considered as supporting (subacute) or as key (subchronic and chronic) studies. All studies were conducted similar to OECD guidelines and under GLP conditions.

In all of these repeated dose toxicity studies, the test substance caused decreased body weight gain, food intake and food efficiency. Further, changes in haematological and clinical biochemistry parameters were observed. T4 serum level after four weeks showed a dose-related reduction, T3 and TSH were not significantly affected. Moreover, minor, but consistent, changes were observed for total protein, albumin, calcium, mean red blood cell (RBC), urea nitrogen, creatinine and mean corpuscular haemoglobin concentration (MCHC) values. The decrease of erythrocytes parameters were suggestive of anemia.

Based on the subchronic and combined chronic/carcinogenicity rat studies, the test substance affected several target organs with the following treatment-related tissue changes: Haemosiderin deposits in spleen and liver, cortical degeneration of adrenals (males), increased prominence of ultimobranchial cysts in the thyroid and C-cell hyperplasia (females), degenerative/atrophic skeletal muscles (males), axonal degeneration in spinal cord and sciatic nerves, adipose replacement of exocrine pancreas (males), lipofuschin accumulation in kidneys (females), bile duct hyperplasia in the liver, and hyperplastic and erosive lesions of the nonglandular epithelium in the stomach (males) were observed. These histopathological findings were accompanied by organ weight changes (adrenals, thyroid, liver and kidney) and gross pathological findings (stomach, adrenals and skeletal muscle).

The principal effect caused by the test substance is haemolysis indicated by a decrease in red blood cells and haemoglobin and by an increase in the mean corpuscular volume and mean corpuscular haemoglobin concentration accompanied by subsequent effects on spleen, kidneys and liver. Among these effects are increased pigmentation of Kupffer cells, haemosiderosis and extramedullary haematopoiesis.

The carcinogenicity as well as the reproduction/developmental toxicity of the test item is discussed in the corresponding sections.

In total, the lowest oral NOAEL in rats was 16 ppm (corresponding to 0.56 mg/kg bw/day for males and 0.66 mg/kg bw/day for females) from the 2 years combined chronic/carcinogenicity study.

 

Mice

One subacute and one subchronic study are available addressing the toxicity of the test substance in mice, which were both considered as supporting studies. Both studies (Report number: ZIR 3/89720 and ZIR 11/901841) were conducted similar to the respective OECD guidelines and under GLP conditions. Moreover, a carcinogenicity study is available, the results of which are presented in detail in the respective robust study entry and are summarised in the endpoint summary under chapter 7.7.

In the subacute and subchronic toxicity studies, the test substance caused decreased body weight gain, food intake and impaired food efficiency. The toxic effects were more diffuse and no specific target organ could be determined in either study.

In total, based on both the subacute and subchronic toxicity study in mice, a NOAEL could not be set (< 100 ppm, corresponding to < 15 mg/kg bw/day for males and < 19 mg/kg bw/day for females).

 

Dogs

Three oral repeated dose toxicity studies in dogs are available, covering different study durations including a 28-day supporting study (Report number: ZIR 1-G/89637), a 90-day key study (Report number: ZIR 8-G/901813) and a 52-week (Report number: ZIR 10/920533) study. All three studies were conducted similar to the respective OECD guidelines and under GLP conditions.

Dogs showed a subdued behaviour and/or unsteady gait, with slight alterations in body weight and food efficiency and increase in liver weight. They vomited and had diarrhoea. The target organs were liver and spleen, with at the start increased pigmentation of Kupffer cells and/or macrophages with Perls’ positive material, possibly reflecting increased iron metabolism and turnover. A slight lowering of red cell indices, acute inflammatory cell infiltration in the lungs, decreases in heart weight, likely related to convulsions were also observed.

In total, the lowest most relevant oral NOAEL was 50 ppm (corresponding to 1.6 mg/kg bw/day for males and 1.9 mg/kg bw/day for females) from the 52-weeks dog study.

 

Repeated dose toxicity: inhalation exposure (dust)

Two subacute inhalation toxicity studies with rats are available. The key study (Report number: UCB 709/003932) was conducted according to OECD 412 and GLP, while the supporting study (Report number: UCB 708/002245) was a dose-range finder. In the key study, male and female rats were exposed to the test item as aerosol (dust) at nominal concentrations of 0.1, 0.3, 1.0 and 2.0 mg/m³ air via snout-only exposure conditions. The aerosol was highly respirable to rats, with mass median aerodynamic diameter (MMAD) of 1.8 - 2.0 µm. No clear evidence for systemic toxicity was observed in this study in rats. Histopathological findings were observed in the larynx, lungs and tracheal bifurcation. This was considered to be a local effect, which was also observed and verified in the supporting study. The most sensitive adverse effect was observed at 0.3 mg/m³, considering the histopathological findings in the larynx. There was a partial to complete recovery in the 28 day recovery period.

In total, the lowest most relevant inhalative NOAEC (respiratory system) was 0.1 mg/m³ air from the 28-day inhalation study conducted with rats.

 

Repeated dose toxicity: dermal application

A 21-day percutaneous study on rabbits (Report number: ZIR 4/89689) was conducted similar to OECD 410 and under GLP conditions. The test substance caused no dermal irritation when applied dermally under occlusive conditions for three weeks to the intact skin of male and female rabbits up to and including 1000 mg/kg bw/day. Treatment-related disturbances in body weight, food consumption and various haematological and biochemical parameters were observed for rabbits receiving the test substance at 1000 mg/kg bw/day although, statistical significance was only achieved for females. Amongst animals receiving 300 mg/kg bw/day, females showed increased enzyme levels, in particular glutamic-oxaloacetic transaminase (GOT). Thus, 1000 mg/kg bw/day was regarded as the NOAEL for local skin effects (corresponding to approximately 11.81 mg/cm²) and the NOAEL for systemic effects was 100 mg/kg bw/day for males and females.

 

Conclusion

Overall, the lowest most relevant oral NOAEL was 16 ppm (corresponding to 0.56 mg/kg bw/day for males and 0.66 mg/kg bw/day for females) from the chronic rat study. The lowest dermal NOAEL was 1000 mg/kg bw/day in regards to local effects and 100 mg/kg bw/day for systemic effects in rabbits, while the lowest inhalation NOAEC was 0.1 mg/m³ air regarding local effects on the respiratory system and 3 mg/m³ air with respect to systemic toxicity.

Further, it was concluded that the test substance affected the target organs/systems liver, blood and musculoskeletal, nervous and endocrine system (all animals) as well as the spleen (rat and mouse). Only in one of the two combined chronic/carcinogenicity studies in rats could a NOAEL be determined (Study number 83-5121). The respective NOAEL of 0.56 mg/kg bw/day is below the relevant guidance value for classification as STOT RE 1 of 1.25 mg/kg bw/day (2 years). The corresponding LOAEL from the same study was with 6.9 mg/kg bw/day well above the guidance value for STOT RE 1. The LOAEL from the second chronic study (Study number: ZIR 9/942098) in rats was 2.5 mg/kg bw/day. Based on these data, classification as STOT RE 1 is not warranted. This is supported by the results from the 90-days study in rats (Study number: ZIR 5/901840) in which at the lowest dose level of 7.4 mg/kg bw/day (LOAEL) haematological and biochemistry changes were observed which in regard to severity do not support a classification as STOT RE 1 (Guidance on the Application of the CLP Criteria, Version 5.0, July 2017, Chapter 3.9.2.5.2 Hematotoxicity).

Taking all the available data into consideration and applying a weight-of-evidence approach, the classification criteria for Specific Target Organ Toxicity after Repeated Exposure (STOT RE), Category 2, H373 (Regulation (EC) No 1272/2008) are met which is in line with the harmonised classification of the substance according to Annex VI of Regulation (EC) No 1272/2008 (Index No 006-012-00-2).

An assessment on endocrine disrupting potential is ongoing during the renewal (AIR) process under regulation(EC) No 1107/2009, a final outcome is currently not available.

Justification for classification or non-classification

Based on the observations of systemic toxic effects (significant changes in liver, blood, spleen and musculoskeletal, nervous and endocroone system) in the repeated dose toxicity studies at relevant dose levels, classification criteria for Specific Target Organ Toxicity after Repeated Exposure (STOT RE), Category 2, H373 (Regulation (EC) No 1272/2008) are met. This is in line with the harmonised classification of the test substance (Annex VI of Regulation (EC) 1272/2008, index number 006-012-00-2).