2,6-dibromo-4-cyanophenyl octanoate

Endpoint summary

Description of key information

Oral subchronic toxicity

Key, M-166147-01-1; subchronic (90 d, rat, similar to OECD 408, GLP):

NOAEL (systemic): 150 ppm (corresponding to 1.6 mg/kg bw/day in males and 1.8 in mg/kg bw/day females, respectively)

LOAEL (systemic): 600 ppm (corresponding to 6.5 mg/kg bw/day in males and 7.8 in mg/kg bw/day females, respectively)

 

Key, M-227060-01-1; subchronic (90 d, dog, similar to OECD 409, GLP):

NOAEL (systemic): 1.43 mg/kg bw/day (males and females)

LOAEL (systemic): 7.14 mg/kg bw/day (males and females)

 

Oral chronic toxicity

Key, source, RA-A, CAS 1689-84-5, M-240-237-03-1; chronic (52 wks, dog, similar to OECD 452, GLP):

NOAEL (systemic): 0.3 mg/kg bw/day (males and females)

LOAEL (systemic): 1.5 mg/kg bw/day (males and females)

 

Dermal subacute toxicity

Key, M-227015-01-2; subacute (21 d, rabbit, OECD 410, GLP):

NOAEL (systemic): 1000 mg/kg bw/day (males and females)

NOAEL (local): 30 mg/kg bw/day (males and females)

LOAEL (local): 300 mg/kg bw/day (males and females)

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:

9 Sep - 10 Dec 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 1998

Deviations:

yes

Remarks:

only 2 dogs per sex per dose used, no detailed clinical observations noted, liver was weighed without gall bladder, ornithite decarboxylase was not determined, microscopic exmination of limited organs/tissues of control animals

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Details on species / strain selection:

The beagle dog is a commonly used non-rodent species for toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna UK Ltd., Huntingdon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 19 - 21 weeks
- Weight at study initiation: 6.4 - 10.0 kg
- Fasting period before study: no
- Housing: kennels, floor area of 4.5 m², with graded whitewood sawdust used as litter, up to 2 animals per kennel (same sex and dose group)
- Diet: standard dry diet (Diet A: Special Diets Services Ltd.), 400 g per animal per 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): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 9 Sep 1992 To: 10 Dec 1992

Route of administration:

oral: capsule

Vehicle:

other: gelatine capsules

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Once each week, after the animals had been weighed the dose to be given to individual animals was calculated according to body weight. The required quantities of test substance were weighed out, and inserted into gelatine capsules, (Parke-Davis, profit gelatine capsules size 000) for the next week. Animals of the control group received gelatine capsules without the test substance.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

0.43 mg/kg bw/day (actual dose received)

Dose / conc.:

1.43 mg/kg bw/day (actual dose received)

Dose / conc.:

7.14 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

2

Control animals:

yes

Details on study design:

- Dose selection rationale: The dosages were chosen by the Sponsor with reference to available toxicological information on the test susbtance and with reference to previous studies with the read across source substance.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: regularly throughout the day

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the beginning of treatment, once weekly during the treatment period and before termination

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of treatment and during Week 13
- Dose groups that were examined: all animals
- Procedure: 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 ("Mydriacyl").

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once before dosing commenced and then during Weeks 6 and 13.
- Anesthetic used for blood collection: No
- Animals fasted: Yes (overnight)
- How many animals: all animals of all groups
- Parameters checked: Packed cell volume (PCV), hemoglobin (Hb), red cell count (RBC), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), total white cell count (WBC Total), platelet count (Plts), reticulocyte count (Retic), differential WBC count (including counts of neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M)), methemoglobin (MetHb), prothrombin time (PT), and activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once before dosing commenced and then during Weeks 6 and 13.
- Animals fasted: Yes (overnight)
- How many animals: all animals of all groups
- Parameters checked: total protein, albumin (Alb), globulin (Glob), urea nitrogen (Urea nitr), creatinine (Creat), sodium (Na), potassium (K), calcium (Ca), inorganic phosphorus (P), chloride (Cl), glucose, alkaline phosphatase (AP), alanine aminotransferase (ALAT, in tables GPT), aspartate aminotransferase (ASAT, in tables GOT), total bilirubin, and cholesterol (Chol).

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: once before dosing commenced and then during Weeks 6 and 13.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (food overnight, water 5 h prior to the start of collection)
- Parameters checked: Volume (Vol), pH, specific gravity (SG), protein (Prot), glucose, ketones, bile pigments, urobilinogen, hem pigments, microscopic examinations (epithelial cells (E), polymorphonuclear leucocytes (P), mononuclear leucocytes (M), erythrocytes (R), organisms (O), renal tube casts (C), and other abnormal constituents (A)).

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Rectal temperatures
- Time schedule: twice before dosing commenced and then at 2, 4, 6 and 24 h after dosing on Days 1, 2 and 3 of dosing and on a suitable day during Weeks 5, 9 and 13 of dosing.

Bone marrow:
Prior to autopsy, bone marrow was obtained from each animal where practicable by sternal puncture and a smear prepared for qualitative examination.

Sacrifice and pathology:

Surviving animals were sacrificed by exsanguination under pentobarbitone anesthesia. The animals were starved overnight prior to post mortem examination.

GROSS PATHOLOGY: Yes
Organs weighed (paired organs being weighed separately): adrenals, brain, heart, kidneys, liver, lungs, pancreas, pituitary, spleen, testes (with epididymides) or ovaries, thymus, thyroids, and uterus or prostate.

HISTOPATHOLOGY: Yes
- fixed tissues: adrenals, alimentary tract (esophagus, stomach body and antrum, duodenum, jejunum, ileum, cecum, colon, rectum), aorta (arch and abdominal), brain (cerebral cortex, thalamic nuclei, mid-brain, medulla and cerebellum), eyes, femur (with articular surface), gall bladder, heart, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary gland, ovaries, pancreas, pituitary, prostate, salivary gland (submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar regions), spleen, sternum (with marrow), testes (and epididymides), thymus, thyroids (and parathyroids), tongue, trachea, urinary bladder, uterus, vagina, and macroscopically abnormal tissues.
Tissues were fixed in 10% buffered formalin. The eyes were preserved in Davidson's fixative whilst additional pieces of liver and kidney were placed in formol calcium. A small piece of the liver was fresh frozen in hexane cooled in an acetone bath containing solid carbon dioxide.
- embedding media: paraffin wax
- thickness of sections: 4 µm
- staining: hematoxylin and eosin (all tissues), Periodic acid Schiff (PAS, fresh frozen liver sections to analyze glycogen)

For cryocuts:
- tissues: liver fixed in formol calcium
- thickness of sections: 12 µm
- staining: Oil red O

Animals investigated:
- 7.14 mg/kg bw/day: all tissues listed above
- all animals of all other dose groups and the control: kidneys, liver, thyroids (and parathyroids) and macroscopically abnormal tissues.

Optional endpoint(s):

None

Other examinations:

None

Statistics:

Data from male and female animals were analyzed both together and separately.

If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 95%), the proportion of animals with values different from the mode were analyzed by Fisher's exact test followed by Mantel's test for a trend with proportion, if appropriate.

Otherwise the Bartlett's test was used 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, Williams' test for a dose-related response was used. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the t-test and Williams' test (Shirley's test).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were recorded that could be attributable to treatment. A low incidence of liquid feces seen in treated animals was similar to that in the controls and was therefore considered not relevant.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 0.43 and 1.43 mg/kg bw/day: No difference regarding body weight and body weight gain between control and treatment groups up to and including 1.43 mg/kg bw/day.
- 7.14 mg/kg bw/day: statistically significantly decreased mean body weight gain from Week 10 onwards compared to animals of the control group. The terminal body weight was also statistically significantly decreased compared to the control group.

Observations that were not statistically significant:
Mean body weight gain at Week 13 for animals receiving 0.43 and 1.43 mg/kg bw/day were lower than for the controls, the differences were not statistically significant and, at 0.43 mg/kg bw/day the reduced mean body weight gain was due to a single animal.

Summarized results can be found in Attachment 1 in the attached background material.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related differences regarding food consumption were observed between control and treatment groups.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related differences regarding ophthalmological findings were observed between control and treatment groups. Incidental findings observed were consistent with the age and strain of animal used.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 0.43 and 1.43 mg/kg bw/day: No treatment-related differences regarding hematology were observed between control and treatment groups up to and including 1.43 mg/kg bw/day.
- 7.14 mg/kg bw/day: mean values for red cell parameters (PCV, Hb and RBC) were decreased in Weeks 6 and 13 but the difference did not reach statistical significance.

Summarized results can be found in Attachment 2 in the attached background material.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences regarding clinical chemistry were observed between control and treatment groups. In Week 6 and 13, mean phosphorus value were decreased in the mid and high dose group, reaching statistical significance in Week 6. However, since the differences were only slight and as individual values were within the background range this finding was considered of no toxicological importance. Other statistical significant intergroup differences were altered glucose and calcium levels at Week 13, but the changes were either slight or reflected trends already present pre-dose and these findings were considered to be of no toxicological importance.

Summarized results can be found in Attachment 3 in the attached background material.

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related differences regarding urinalysis were observed between control and treatment groups.

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 0.43 and 1.43 mg/kg bw/day: No treatment-related differences regarding organ weights were observed between control and treatment groups up to and including 1.43 mg/kg bw/day.
- 7.14 mg/kg bw/day: statistically significantly increased absolute liver weight, individual relative liver weights for all animals were in excess of the concurrent control range, and for 3 of the animals were near to or in excess of the laboratory's normal upper limit of 4% body weight.

Not considered treatment-related:
- 0.43 mg/kg bw/day: statistically significant increase in relative brain weight compared to the control group (considered an anomaly and in part due to lower body weights)
- 1.45 mg/kg bw/day: mean relative liver weight was statistically significantly increased compared to the control group (not considered treatment-related since there was no dosage relationship, and no histopathological lesions or blood biochemical changes to account for this weight difference), statistically significant increase in relative brain weight compared to the control group (considered an anomaly and in part due to lower body weights).
- 7.14 mg/kg bw/day: mean relative kidney weight was statistically significantly increased compared to the control group (not considered treatment related since there was no dose-relationship, and no histopathological lesions or blood biochemical changes to account for this weight difference), statistically significant increase in relative brain weight compared to the control group (considered an anomaly and in part due to lower body weights).

Summarized results can be found in Attachment 4 in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings which could be attributed to treatment.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 0.43 and 1.43 mg/kg bw/day: No treatment-related differences regarding histopathology were observed between control and treatment groups up to and including 1.43 mg/kg bw/day.
- 7.14 mg/kg bw/day: Minimal centrilobular hepatocyte enlargement (1/2 male dogs).

The minimal spermatogenesis in the testes and corresponding reduced spermatozoa in the epididymides (2/2 male of the high dose group) were considered to represent incomplete normal sexual maturation and to be consistent with the age of the dogs.

Summarized results can be found in Attachment 5 in the attached background material.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

no effects observed

Description (incidence and severity):

Rectal temperature
No treatment-related differences regarding rectal temperature were observed between control and treatment groups. In the high dose group, statistically significant increase in rectal temperature was observed in Week 5 compared to the control group but this could not be attributed to the test substance. Additionally, no change of rectal temperature was observed at any other time point.

Summarized results can be found in Attachment 6 in the attached background material.

Bone marrow smears
There were no abnormalities of cellularity, distribution or morphology in any animal.

Details on results:

Dose Groups as described in some of the tables:
Group 1: control
Group 2: 0.43 mg/kg bw/day
Group 3: 1.43 mg/kg bw/day
Group 4: 7.14 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

1.43 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at this dose level.

Key result

Dose descriptor:

LOAEL

Effect level:

7.14 mg/kg bw/day (actual dose received)

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

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

7.14 mg/kg bw/day (actual dose received)

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 effects of the test substance on Beagle dogs when given for a time frame of approximately 13 weeks. The study was similar to the OECD guideline 409 (dated 1981) and was performed under GLP conditions. Beagle dogs received the test substance via gelatin capsules at doses of 0.43, 1.43 and 7.14 mg/kg bw/day.

Under the conditions of the test, the test substance caused reduced body weight gain and changes in hematology in treated animals compared to controls at 7.14 mg/kg bw/day. Increased liver weights were observed in the 7.14 mg/kg bw/day group and minimal centrilobular hepatocyte enlargement in a single animal of this group.
Based on the described effects the NOAEL could be set at 1.43 mg/kg bw/day for male and female Beagle dogs.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 May - 25 Jun 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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 2018

Deviations:

yes

Remarks:

Sensory reactivity was not assessed, lipid analysis in blood, endocrine parameters, thyroid parameters (T3, T4, TSH) and terminal vaginal cytology were not investigated, the estrus cycle was not determined at necropsy, limited number of organs weighed.

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is frequently used in safety evaluation studies as a representative of a rodent species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 5 weeks
- Weight at study initiation: 126.1 - 164.0 g
- Fasting period before study: no
- Housing: individually in stainless steel, screen-bottom cages
- Diet: Certified Rodent Chow® #5002 meal (Purina Mills, Inc.), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1 May 1991 To: 6 Aug 1991

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): once weekly for Weeks 1 and 2, twice weekly for Weeks 3 and 4, and once weekly thereafter. The diets were fed to the animals once weekly during Weeks 1 and 2. Due to instability of the test material in the diet, the feeding regimen was changed to twice weekly during Week 3 and four times/week (i.e., Wednesday, Friday, Sunday, and Tuesday) starting with Week 4 and continuing throughout the study.
- Mixing appropriate amounts with (Type of food): Certified Rodent Chow® #5002 meal. A specified amount of the diet was weighed into a mixing bowl. A specified amount of the test material was added to acetone in a beaker and placed in a water bath set to maintain approximately 50 °C
and the contents were stirred until the test material was completely dissolved in the acetone. This mixture was then added to the basal diet and was mixed for approximately 30 min. Each dose level was prepared independently in sequential order of increasing concentration.
- Storage temperature of food: The diets were stored at room temperature for Weeks 1, 2, and 3 (first mix). Beginning with Week 3 (second mix), dose preparations were stored in a freezer until dispensed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity was determined for the lowest and highest concentrations. Samples taken from the top, bottom, and two opposing sides of the mix were assayed in duplicate for test material content. Homogeneity ranged from 96% to 107% and 94% to 102% of the theoretical levels for diets mixed at 150 and 2100 ppm, respectively.

Stability of the test material in the diet was measured in samples taken before initiation of treatment from the lowest and highest test material concentrations. Therefore, 4 sets of samples were taken and analyzed on the day of mixing, after storage at room temperature for 10 days and after being stored in a freezer for 2 or 5 weeks. Analyses of the samples stored in the freezer determined the test substance concentration to be 92 - 100%% of the target concentration.

Due to instability of the test diets after 10 days at room temperature (76% and 77% of theoretical for the 150- and 2,100-ppm dose levels, respectively), additional stability samples were taken and assayed. One set of samples was taken from the low- and mid-high dose levels mixed for Week 3 (first mix), stored at room temperature for 3 days, and analyzed. These assays resulted in values that were 87% of theoretical for the 150 and 1100 ppm dose levels.
Two additional sets of samples were also taken from the low- and mid-high dose levels mixed for Week 3 (second mix). One set was stored at room temperature for 3 days, and analyzed for the read across source substance to determine whether the test substance was hydrolyzed.
The remaining set was stored in a freezer for 1 day, kept at room temperature for 2 days, and analyzed for the test substance. These assays resulted in values that were 91% and 90% of theoretical for the 150- and 1100-ppm dose levels, respectively. The amount of the read across substance did not completely account for the loss of the test substance that would have been expected to occur in those samples.

All diets were assayed each week for the first 4 weeks (except for the high-dose level diets that were mixed and assayed for the first 2 weeks only). Each week thereafter, one test material dietary concentration was selected sequentially for analysis. These samples and a sample of the control diet were analyzed as a group approximately every 4 weeks. Mean results were 91 - 105%, (150 ppm), 92 - 97% (600 ppm), 85 - 100% (1100 ppm) and 99 - 101% (2100 ppm) of the theoretical levels.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously via the diet

Dose / conc.:

150 ppm

Remarks:

actual ingested dose of 1.6 mg/kg bw/day for males and 1.8 mg/kg bw/day for females.

Dose / conc.:

600 ppm

Remarks:

actual ingested dose of 6.5 mg/kg bw/day for males and 7.8 mg/kg bw/day for females.

Dose / conc.:

1 100 ppm

Remarks:

actual ingested dose of 12.6 mg/kg bw/day for males and 15.8 mg/kg bw/day for females.

Dose / conc.:

2 100 ppm

Remarks:

Because of the high mortality, no actual ingested dose could be calculated.

No. of animals per sex per dose:

20 (150, 600, 1100 ppm)
30 (control and 2100 ppm group)

Control animals:

yes, plain diet

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Satellite groups: 10/sex/group of the control and 2100 ppm group were designated as recovery animals; however, because of mortality in the 2100 ppm group during the first week of the study, the remaining high-dose animals were sacrificed during Week 2. The 10 rats/sex designated as recovery animals in the control group were sacrificed during Week 14.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once before initiation of treatment, on the first day of treatment, weekly thereafter, and on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, calculated as weekly consumption
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: weekly during Week 12 and 13

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and during Week 13
- Dose groups that were examined: all animals of all dose groups
- procedure: pupils were dilated with 0.5% Mydriacyl®, and the eyes were examined with an indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before initiation of treatment, Week 2, 3 and 13
- Anesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes (overnight)
- How many animals: 10 males, 9 females before initiation of treatment, remaining 2100 ppm group animals (Week 2), and all animals in Week 3 and 13
- Parameters checked: Red blood cell count (RBC), hemoglobin (Hem), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, prothrombin time (after Weeks 4 and 13 only), white blood cell count, differential blood cell count, blood cell morphology, and reticulocyte count smears were made.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before initiation of treatment, Week 2, 3 and 13
- Anesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes (overnight)
- How many animals: 10 males, 9 females before initiation of treatment, remaining 2100 ppm group animals (Week 2), and all animals in Week 3 and 13
- Parameters checked: glucose, urea nitrogen, creatinine, total protein, albumin, globulin, total bilirubin, cholesterol, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (APh), calcium, inorganic phosphorus, sodium, potassium, and chloride.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: Week 2, 3 and 13 over a period of 16 h (from the animals described for hematology and clinical chemistry)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (overnight)
- Parameters checked: volume, appearance, pH, specific gravity, glucose, bilirubin, protein, ketones, blood, urobilinogen, and microscopic examination of sediment.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

A necropsy was done on each high-dose animal (2100 ppm) that died or was sacrificed before the scheduled necropsy. Animals that were sacrificed were fasted overnight, then anesthetized with methoxyflurane, weighed, exsanguinated, and necropsied.

During Week 14, animals in control and treatment groups (150, 600 and 1100 ppm) were fasted overnight, then anesthetized with methoxyflurane, weighed, exsanguinated, and necropsied.

GROSS PATHOLOGY: Yes
Necropsy included macroscopic examination of the external surface of the body; all orifices; the cranial cavity; the external surfaces of the brain and spinal cord (cut surfaces were examined when tissues were trimmed); the nasal cavity and paranasal sinuses; and the thoracic, abdominal, and pelvic cavities viscera.
Organs weighed: Adrenals, brain, kidneys, liver, ovaries, and testes.

HISTOPATHOLOGY: Yes
- tissues collected: adrenals, aorta, brain, cecum, cervix, colon, duodenum, epididymides, esophagus, eyes (preserved in Zenker's solution for scheduled sacrifice animals), femur with bone marrow (articular surface of the distal end), heart, ileum, jejunum, kidneys, lacrimal gland (exorbital), lesions, liver, lungs, lymph nodes (mesenteric), mammary gland (females only), muscle (thigh), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submandibular), sciatic nerve, seminal vesicles, skin, spinal cord (cervical, mid-thoracic, and lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, and vagina.
The tissues were preserved in 10% phosphate-buffered formalin.
- embedding media: paraffin
- staining: hematoxylin and eosin
- all tissues examined for: all animals in the control group, the 1100 ppm group, and from the animals that died or were sacrificed at an unscheduled interval, including animals in the 2100 ppm group
- macroscopic lesions, lungs, liver, and kidneys were examined for all animals.
- heart, femur with bone marrow (articular surface of the distal end), lymph node (mesenteric), spleen, thymus and sternum with bone marrow were evaluated in the 150 and 600 ppm group.

Bone marrow smears from the femur of each animal at the scheduled sacrifice were prepared, stained with Wright's stain, and retained for possible examination.

Optional endpoint(s):

None

Other examinations:

None

Statistics:

Levene's test was done to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, transformations were used to stabilize the variance.
Analysis of variance (ANOVA) was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t-test was used for pairwise comparisons between treated and control groups.
One-way ANOVA was used to analyze body weights; cumulative body weight gains; food consumption; clinical chemistry and hematology values (except red blood cell morphology); urine pH, volume, and specific gravity; organ weights; organ-to-body weight percentages; and organ-to-brain weight ratios.
Group comparisons were evaluated at the 5.0% two-tailed probability level.

All differences cited are based on comparisons with the control group.

The dose level of 2,100 ppm clearly exceeded the maximum tolerated dose, as evidenced by the excessive mortality; therefore, statistical findings for this group were not be discussed in the result section of the study report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 150, 600 and 1100 ppm: No clinical signs occurred.
- 2100 ppm: thin, hunched, and yellow hair coat in the genital area.

Summarized results can be found in Attachment 1 in the attached background material.

Mortality:

mortality observed, treatment-related

Description (incidence):

- 150, 600 and 1100 ppm: No mortality occurred.
- 2100 ppm: 9/30 males and 26/30 females were found dead during Week 1. Therefore, all surviving animals of this dose group were sacrificed in Week 2.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 150 ppm: No differences regarding body weight were observed between the control and treatment group.
- 600 ppm: The body weight gain was statistically significantly decreased during Weeks 2 through 14 compared to the controls (females), terminal body weights of females were 90% of those of the controls, cumulative body weight gains were statistically significantly reduced during Weeks 1 and 2 (males) and during Weeks 1 through 13 (females).
- 1100 ppm: The body weight gain was statistically significantly decreased during Weeks 2 through 14 compared to the controls (males and females), terminal body weights of females were 78% of those of the controls, terminal body weights of males were 87% of those of the controls, cumulative body weight gains were statistically significantly reduced during Weeks 1 through 13 (males and females).

Summarized results can be found in Attachment 2 and 3 (selected results) in the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 150 ppm: No differences regarding food consumption were observed between the control and treatment group.
- 600 ppm: statistically significantly reduced during Week 1 (males and females).
- 1100 ppm: statistically significantly reduced during Week 1 (males and females).
As the reduced food consumption was only observed during Week 1, this is not considered toxicologically relevant.
Summarized results can be found in Attachment 4 in the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

no effects observed

Description (incidence and severity):

No differences were observed between control and treatment groups regarding water consumption up to and including the highest dose level of 1100 ppm.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No differences were observed between control and treatment groups regarding ophthalmological findings up to and including the highest dose level of 1100 ppm.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 150 and 600 ppm: No differences were observed between control and treatment groups regarding hematology up to and including 600 ppm.
- 1100 ppm: Slightly but statistically significantly higher red blood cell count, hemoglobin and hematocrit values were observed in females at Week 5 when compared to the control group. Statistically significantly lower platelat counts were observed in females at Week 5 and statistically significantly increased absolute lymphocyte count in females at Week 13 compared to the control group (a trend was also seen in Week 5 but the difference did not reach statistical significance).
Since the hematologic findings were observed mainly at week 5, occurred only in females, and the observed changes were minor, the abovementioned changes were not considered toxicologically relevant.

In the 2100 ppm group, lower platelet count, absolute lymphocyte count, and higher red blood cell count, hemoglobin and hematocrit were observed in Week 1 compared to the control.

Summarized results can be found in Attachment 5 in the attached background material.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 150 ppm: No differences were observed regarding clinical chemistry between the control and treatment group.
- 600 ppm: slightly, but statistically significantly lower total bilirubin (females, Week 5) and chloride (females, Week 5), all compared to controls.
- 1100 ppm: statistically significantly decreased total protein (females Week 5 and 13, males Week 5, in Week 13, a decrease was observed but the difference to controls did not reach statistical significance), statistically significantly decreased globulin (males and females, Week 5 and 13) and slightly but statistically significantly higher alkaline phosphatase (males and females, Week 13), lower total bilirubin and chloride (females, Week 5), statistically significantly lower potassium levels (females, Week 5), all compared to controls. The effect were considered to be toxicologically relevant.
In the 2100 ppm group, lower globulin and higher urea nitrogen, aspartate aminotransferase, and alanine aminotransferase activities were observed in Week 1 compared to the control.

Not seen as toxicologically relevant:
Albumin was significantly higher for males given 600 ppm and for females given 150 or 600 ppm in Week 5. However, since this was not seen in the 1100 ppm group and not in Week 14, it is not considered toxicologically relevant.

Summarized results can be found in Attachment 6 in the attached background material.

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- 150 and 600 ppm: No differences regarding urinalysis were observed between control and treatment groups up to and including 600 ppm.
- 1100 ppm: statistically significantly higher urine volume (males, Week 5), statistically significantly lower urine gravity (females, Week 5) compared to the control group.

Summarized results can be found in Attachment 7 in the attached background material.

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 150 ppm: No differences regarding organ weights were observed between control and the treatment group.
- 600 ppm: Statistically significantly increased absolute liver weights and liver-to-body weight ratios (females).
- 1100 ppm: Statistically significantly increased absolute liver weights and liver-to-body weight ratios (females), statistically significantly increased liver-to-body weight ratios (males).

Other significant organ weight changes seen in treated animals were consistent with the overall reduction in body weights; numerous organ-to-body weight percentage increases (for adrenal, kidney, liver, brain, testis and ovary) suggest the lower body weights may have resulted more from reduced musculoskeletal mass rather than reduced parenchymal organ mass.

Summarized results can be found in Attachment 8 and 10 (selected results) in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences were observed between control and treatment groups. All findings were of low incidence and randomly scattered across all treatment groups. None of these findings were considered to be significant test material-related effects.

Macroscopic findings in animals that died unscheduled:
2100 ppm: 12/26 females had diffusely red lungs. 13/26 and 2/9 males showed dark or red foci or areas in the stomach, principally involving the glandular mucosa.

Macroscopic findings in animals after interim sacrifice:
- 2100 ppm: 20/21 males and 4/4 females showed changes in the stomach including dark or red foci or areas in the glandular mucosa, red foci or areas in the nonglandular mucosa, or diffuse redness. In 5/21 males and 2/4 females the mesenteric lymph nodes were mottled and the adrenals and liver were described as diffusely dark in four and three males, respectively.

Macroscopic findings in animals after terminal sacrifice:
- Control: In 1/30 males dark or red foci or areas were observed in the stomach.
- 150 ppm: In 2/20 males dark or red foci or areas were observed in the stomach.
- 600 ppm: In 3/20 males dark or red foci or areas were observed in the stomach. 1/20 females had red foci in the thymus or areas macroscopically described.
- 1100 ppm: 1/20 males and 1/20 females had liver findings described as hepatic anomalies. Microscopically, these were composed of normal liver
tissue and were not considered adverse. In the stomach, dark or red foci or areas were seen in 1/20 males and 2/20 females. 2/20 females had red foci in the thymus or areas macroscopically described.
These findings and several other findings in terminal sacrifice animals were of low incidence and randomly scattered across all treatment groups. None of these findings were considered to be significant test material-related effects.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings in animals that died unscheduled:
- 2100 ppm: 23/26 females and 9/9 males had microscopic congestion in the lung. 3/26 females showed microscopic hemorrhage within the lung. The congestion and hemorrhage seen in these animals correlated with the diffuse redness described macroscopically in the lungs of these animals. 15/26 females showed minimal to slight individual hepatocyte necrosis in the centrilobular areas of the liver. 6/26 females and 2/9 males showed areas of glandular mucosal erosion, congestion, or hemorrhage in the stomach. These gastric changes correlated with the dark and red areas described macroscopically in many of these animals. 14/26 females and 8/9 males showed minimal to moderately severe lymphocyte (thymocyte) necrosis in the thymus. 17/26 females and 5/9 males had microscopic hemorrhage in the thymuses. Other microscopic findings in the unscheduled deaths were of low incidence and not considered to be test material-related.

Microscopic findings in animals after interim sacrifice:
- 2100 ppm: 20/21 males and 4/4 females showed minimal to moderate lymphocyte necrosis in the spleen and all animals in and in the mesenteric lymph nodes. This was characterized by pyknotic and karyolytic nuclear debris scattered among the periarteriolar lymphoid sheath lymphocytes in the spleen or among paracortical lymphocytes in the lymph node. All animals showed minimal to moderately severe lymphocyte (thymocyte) necrosis in the thymus. This was similarly characterized by pyknotic and karyolytic nuclear debris scattered predominantly in the thymic cortex. In 12/21 males and 3/4 females lymphocyte depletion was diagnosed in the spleen and in the mesenteric lymph node for 2/21 males, and in the thymus for 9/21 males and 3/4 females. Hematopoietic cell depletion was found in the femoral and sternal bone marrow for 11/21 and 13/21 males, respectively, and in the femoral and sternal bone marrow for all females. It should be noted that these animals had lost 15-20% of their original body weight before death or moribund sacrifice. Thus, it is not known to what extent inanition may have contributed to these histopathological findings. All animals showed some areas of hemorrhage, congestion, or glandular mucosa erosions in the stomach. These gastric findings were minimal to moderate in severity, and their occurrence correlated with the diffuse redness and dark or red foci or areas noted macroscopically. Other microscopic findings noted in interim sacrifice animals were of low incidence and not considered to be test material-related.

Microscopic findings in animals after terminal sacrifice:
- 150 and 600 ppm: No differences regarding histopathology were observed between control and treatment groups up to and including 600 ppm.
- 1100 ppm: A slight increase in the incidence of lymphocyte necrosis in the thymus (males and females, 25% compared to 13% in controls) were observed. A slight increase in the incidence of myocardial degeneration and necrosis in the heart (males: 55% vs 16.7% in controls, females: 50% vs 10% in controls) and slightly higher incidence of lymphocyte necrosis in the spleen (females) were observed compared to controls. The observed macroscopic abnormalities in the liver were microscopically identified as protruding lobes of normal hepatic tissues and were diagnosed as accessory lobes. These were probably developmental aberrations and were therefore not test material-related lesions.

Summarized results can be found in Attachment 9 and 10 (selected results) in the attached background material.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Effect level:

150 ppm

Based on:

test mat.

Basis for effect level:

other: No adverse effects were observed at this dose level.

Remarks on result:

other: corresponding to an actually ingested dose of 1.6 and 1.8 mg/kg bw/day for males and females, respectively

Key result

Dose descriptor:

LOAEL

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: corresponding to an actually ingested dose of 6.5 and 7.8 mg/kg bw/day for males and females, respectively

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

600 ppm

Conclusions:

In this study, the toxicity of the test substance was investigated in rats during and after oral administration of dietary concentrations of the test substance of 150, 600, 1100 and 2100 ppm for 90 days. The study was conducted according to guideline EPA-OP 82-1 (similar to OECD guideline 408, 2018) and under GLP conditions.
Due to high mortality in the 2100 ppm group during the first week, these animals were sacrificed during Week 2. Body weight, food consumption, and clinical chemistry parameters as well as liver weights (absolute and relative) were altered after treatment with the test substance at 600 ppm and above. Therefore, the NOAEL was set at 150 ppm for males and females (1.6 and 1.8 mg/kg bw/day respectively).

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to the analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

0.3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to this dose level.

Remarks on result:

other: Source: CAS 1689-84-5, 1987, dog

Key result

Dose descriptor:

LOAEL

Effect level:

1.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: Source: CAS 1689-84-5, 1987, dog

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1.5 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.3 mg/kg bw/day

Study duration:

chronic

Species:

dog

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006. The study with the lowest dose descriptor was selected for endpoint conclusion.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

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:

13 Jan - 7 Feb 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

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: Hazleton Research Products Inc, Michigan, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 17 - 18 weeks
- Weight at study initiation: 2.447 - 2.999 kg (males), 2.457 - 2.840 kg (females)
- Fasting period before study: no
- Housing: individually in stainless steel, screen-bottom cages.
- Diet: Certified High Fiber Rabbit Chow5 #5325 (Purina Mills, Inc.), ca 180 g/day and animal
- Water: tap water, ad libitum
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Jan 1992 To: 7 Feb 1992

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Remarks:

the test material was moistened with deionized water (2.0 mL)

Details on exposure:

TEST SITE
- Area of exposure: dorsal trunk
- % coverage: ca. 10%
- Type of wrap if used: gauze patch, fixed with Coban self-adhesive wrap and secured with elastic tape
- Time intervals for shavings or clippings: before initiation of treatment and as needed thereafter

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the test site was wiped with a clean towel moistened with tap water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the appropriate amount of the test material was spread on a gauze patch, moistened with 2.0 mL of deionized water and applied to the skin
- For solids, paste formed: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (flexible plastic collar)

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Dose analysis was not done because the test material was applied neat. Homogeneity, stability, and dose confirmation analyses were the responsibility of the Sponsor.

Duration of treatment / exposure:

6 h per treatment
The treatment period was 3 weeks

Frequency of treatment:

daily, 5 days/week

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for mortality and moribundity twice daily. Once daily, the animals were removed from the cage and examined for signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: immediately before each application of the test or control material (except on Day 1) and on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment (Day 1), weekly thereafter, and at necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to study start (Week -2) and before scheduled necropsy (Week 4)
- Anesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals of all dose groups
- Parameters checked: Red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell count, differential blood cell count and blood cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre study and before scheduled necropsy
- Animals fasted: No
- How many animals:
- Parameters checked: Glucose, urea nitrogen, creatinine, total protein, albumin, globulin, total bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, calcium, inorganic phosphorous, sodium, potassium and chloride.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

After 3 weeks of treatment, all animals were anesthetized with sodium pentobarbital, weighed, exsanguinated, and necropsied.

GROSS PATHOLOGY: Yes
The necropsy of all animals included macroscopic examination of the external surface of the body; all orifices; the cranial cavity; the external surfaces of the brain and spinal cord; the nasal cavity and paranasal sinuses; and the abdominal, thoracic, and pelvic cavities and viscera. Cut surfaces of the brain and spinal cord were examined when tissues were trimmed.
Organs weighed (paired organs weighed separatly): Adrenals, brain, kidneys, liver, ovaries, and testes.

HISTOPATHOLOGY: Yes
- fixed tissues: adrenals, aorta, brain, cecum, colon, duodenum, epididymides, esophagus, eyes, femur with bone marrow (with articular surface), gall bladder, heart, ileum, jejunum, kidneys, lacrimal gland, lesions, liver, lungs, lymph nodes (mesenteric), mammary gland, muscle (thigh), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submandibular), sciatic nerve, seminal vesicle, skin (treated and untreated), spinal cord (cervical, thoracic and lumbar regions), spleen, sternum with bone marrow, stomach, testes, thymus, thyroids (and parathyroids), trachea, urinary bladder and uterus.
Tissues were fixed in 10% phosphate-buffered formalin. The eyes were preserved in Zenker's solution.
- embedding media: paraffin wax
- staining: hematoxylin and eosin
- animals investigated: control and high-dose animals

Other examinations:

Bone marrow smears were taken from the sternum at scheduled sacrifice but not examined.

Statistics:

Levene's test was done to test for variance homogeneity. In the case of heterogeneity of variance at p ≤ 0.05, transformations were used to stabilize the variance.
Analysis of variance (ANOVA) was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t-test was used for pairwise comparisons between treated and control groups.
One-way ANOVA was used to analyze initial body weights, cumulative body weight gains, food consumption, clinical chemistry and hematology values (except red blood cell morphology), organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios.
One-way analysis of covariance (ANCOVA) was used to analyze body weights, with initial body weights as the covariate. Although Levene's test for variance homogeneity was done (see above), no transformations were used because covariance adjustment removes extraneous heterogeneity. If the ANCOVA was significant, Dunnett's t-test was used for pairwise comparisons between treated and control groups.
Group comparisons were evaluated at the 5.0% two-tailed probability level.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test material-related observations during the study period.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

- 0 mg/kg bw/day: slight erythema (2/10 males, 1/10 females)
- 30 mg/kg bw/day: slight erythema (5/10 males, 1/10 females)
- 300 mg/kg bw/day: slight (10/10 males, 8/10 females) to moderate erythema (1/10 males) and slight desquamation (4/10 males, 4/10 females) and edema (1/10 females)
- 1000 mg/kg bw/day: slight (9/10 males, 10/10 females) to moderate erythema (1/10 males, 1/10 females), slight desquamation (8/10 males, 6/10 females), moderate desquamation (1/10 males), slight fissuring (5/10 males, 3/10 females), moderate fissuring (1/10 males), slight edema (1/10 males, 4/10 females), moderate edema (1/10 males, 2/10 females) and slight atonia (1/10 males)

The observations did not increase in severity during treatment.

Summarized results can be found in Attachment 1 in the attached background material.

Mortality:

no mortality observed

Description (incidence):

All animals survived to scheduled sacrifice.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences were observed regarding body weight and cumulative body weight gains between control and treatment groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in food consumption for females. A significant increase in food consumption was present in Week 3 for males given 30 mg/kg bw/day; however, this difference was not considered to be toxicologically significant, since there was no dose dependency.

Summarized results can be found in Attachment 2 in the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences were observed regarding hematology between control and treatment groups.

Statistically significant differences for red blood cell count and urea nitrogen were considered normal biological variation. The difference for red blood cell count was small; the differences for urea nitrogen were small, occurred in intermediate-dose males only, and were similar to findings observed before treatment was initiated.

Summarized results can be found in Attachment 3 in the attached background material.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No differences were observed regarding clinical chemistry between control and treatment groups.

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences were observed regarding organ weights between control and treatment groups.

Females of the 1000 mg/kg bw/day group showed increased absolute and relative kidney weights (< 15%), but these fell within the biological variation. Additionally, there were no macroscopic or microscopic findings in the kidneys of these animals correlating with the higher organ weights.

Summarized results can be found in Attachment 4 in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences were observed at necropsy between control and treatment groups.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences were observed regarding histopathology between control and treatment groups.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The dermal irritation at this dose level was comparable to the vehicle control.

Key result

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to the highest dose level.

Key result

Critical effects observed:

no

The codes and abbreviations used in the tables are explained in Attachment 5 in the attached background material. 

Conclusions:

The study was conducted similar to OECD guideline 410 (1981) and under GLP. Under the conditions of the test, the test substance caused dermal irritation when applied dermally for three weeks to the intact skin of male and female New Zealand White rabbits, starting at 300 mg/kg bw/day. No systemic toxicity was observed. Thus, 30 mg/kg bw/day is regarded as the NOAEL for local skin effects and the NOAEL for systemic effects is 1000 mg/kg bw/day for males and females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rabbit

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006. The study with the lowest dose descriptor was selected for endpoint conclusion.

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:

13 Jan - 7 Feb 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

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: Hazleton Research Products Inc, Michigan, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 17 - 18 weeks
- Weight at study initiation: 2.447 - 2.999 kg (males), 2.457 - 2.840 kg (females)
- Fasting period before study: no
- Housing: individually in stainless steel, screen-bottom cages.
- Diet: Certified High Fiber Rabbit Chow5 #5325 (Purina Mills, Inc.), ca 180 g/day and animal
- Water: tap water, ad libitum
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Jan 1992 To: 7 Feb 1992

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Remarks:

the test material was moistened with deionized water (2.0 mL)

Details on exposure:

TEST SITE
- Area of exposure: dorsal trunk
- % coverage: ca. 10%
- Type of wrap if used: gauze patch, fixed with Coban self-adhesive wrap and secured with elastic tape
- Time intervals for shavings or clippings: before initiation of treatment and as needed thereafter

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the test site was wiped with a clean towel moistened with tap water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the appropriate amount of the test material was spread on a gauze patch, moistened with 2.0 mL of deionized water and applied to the skin
- For solids, paste formed: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (flexible plastic collar)

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Dose analysis was not done because the test material was applied neat. Homogeneity, stability, and dose confirmation analyses were the responsibility of the Sponsor.

Duration of treatment / exposure:

6 h per treatment
The treatment period was 3 weeks

Frequency of treatment:

daily, 5 days/week

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for mortality and moribundity twice daily. Once daily, the animals were removed from the cage and examined for signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: immediately before each application of the test or control material (except on Day 1) and on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment (Day 1), weekly thereafter, and at necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to study start (Week -2) and before scheduled necropsy (Week 4)
- Anesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals of all dose groups
- Parameters checked: Red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell count, differential blood cell count and blood cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre study and before scheduled necropsy
- Animals fasted: No
- How many animals:
- Parameters checked: Glucose, urea nitrogen, creatinine, total protein, albumin, globulin, total bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, calcium, inorganic phosphorous, sodium, potassium and chloride.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

After 3 weeks of treatment, all animals were anesthetized with sodium pentobarbital, weighed, exsanguinated, and necropsied.

GROSS PATHOLOGY: Yes
The necropsy of all animals included macroscopic examination of the external surface of the body; all orifices; the cranial cavity; the external surfaces of the brain and spinal cord; the nasal cavity and paranasal sinuses; and the abdominal, thoracic, and pelvic cavities and viscera. Cut surfaces of the brain and spinal cord were examined when tissues were trimmed.
Organs weighed (paired organs weighed separatly): Adrenals, brain, kidneys, liver, ovaries, and testes.

HISTOPATHOLOGY: Yes
- fixed tissues: adrenals, aorta, brain, cecum, colon, duodenum, epididymides, esophagus, eyes, femur with bone marrow (with articular surface), gall bladder, heart, ileum, jejunum, kidneys, lacrimal gland, lesions, liver, lungs, lymph nodes (mesenteric), mammary gland, muscle (thigh), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submandibular), sciatic nerve, seminal vesicle, skin (treated and untreated), spinal cord (cervical, thoracic and lumbar regions), spleen, sternum with bone marrow, stomach, testes, thymus, thyroids (and parathyroids), trachea, urinary bladder and uterus.
Tissues were fixed in 10% phosphate-buffered formalin. The eyes were preserved in Zenker's solution.
- embedding media: paraffin wax
- staining: hematoxylin and eosin
- animals investigated: control and high-dose animals

Other examinations:

Bone marrow smears were taken from the sternum at scheduled sacrifice but not examined.

Statistics:

Levene's test was done to test for variance homogeneity. In the case of heterogeneity of variance at p ≤ 0.05, transformations were used to stabilize the variance.
Analysis of variance (ANOVA) was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t-test was used for pairwise comparisons between treated and control groups.
One-way ANOVA was used to analyze initial body weights, cumulative body weight gains, food consumption, clinical chemistry and hematology values (except red blood cell morphology), organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios.
One-way analysis of covariance (ANCOVA) was used to analyze body weights, with initial body weights as the covariate. Although Levene's test for variance homogeneity was done (see above), no transformations were used because covariance adjustment removes extraneous heterogeneity. If the ANCOVA was significant, Dunnett's t-test was used for pairwise comparisons between treated and control groups.
Group comparisons were evaluated at the 5.0% two-tailed probability level.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test material-related observations during the study period.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

- 0 mg/kg bw/day: slight erythema (2/10 males, 1/10 females)
- 30 mg/kg bw/day: slight erythema (5/10 males, 1/10 females)
- 300 mg/kg bw/day: slight (10/10 males, 8/10 females) to moderate erythema (1/10 males) and slight desquamation (4/10 males, 4/10 females) and edema (1/10 females)
- 1000 mg/kg bw/day: slight (9/10 males, 10/10 females) to moderate erythema (1/10 males, 1/10 females), slight desquamation (8/10 males, 6/10 females), moderate desquamation (1/10 males), slight fissuring (5/10 males, 3/10 females), moderate fissuring (1/10 males), slight edema (1/10 males, 4/10 females), moderate edema (1/10 males, 2/10 females) and slight atonia (1/10 males)

The observations did not increase in severity during treatment.

Summarized results can be found in Attachment 1 in the attached background material.

Mortality:

no mortality observed

Description (incidence):

All animals survived to scheduled sacrifice.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences were observed regarding body weight and cumulative body weight gains between control and treatment groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in food consumption for females. A significant increase in food consumption was present in Week 3 for males given 30 mg/kg bw/day; however, this difference was not considered to be toxicologically significant, since there was no dose dependency.

Summarized results can be found in Attachment 2 in the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences were observed regarding hematology between control and treatment groups.

Statistically significant differences for red blood cell count and urea nitrogen were considered normal biological variation. The difference for red blood cell count was small; the differences for urea nitrogen were small, occurred in intermediate-dose males only, and were similar to findings observed before treatment was initiated.

Summarized results can be found in Attachment 3 in the attached background material.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No differences were observed regarding clinical chemistry between control and treatment groups.

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences were observed regarding organ weights between control and treatment groups.

Females of the 1000 mg/kg bw/day group showed increased absolute and relative kidney weights (< 15%), but these fell within the biological variation. Additionally, there were no macroscopic or microscopic findings in the kidneys of these animals correlating with the higher organ weights.

Summarized results can be found in Attachment 4 in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences were observed at necropsy between control and treatment groups.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences were observed regarding histopathology between control and treatment groups.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The dermal irritation at this dose level was comparable to the vehicle control.

Key result

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to the highest dose level.

Key result

Critical effects observed:

no

The codes and abbreviations used in the tables are explained in Attachment 5 in the attached background material. 

Conclusions:

The study was conducted similar to OECD guideline 410 (1981) and under GLP. Under the conditions of the test, the test substance caused dermal irritation when applied dermally for three weeks to the intact skin of male and female New Zealand White rabbits, starting at 300 mg/kg bw/day. No systemic toxicity was observed. Thus, 30 mg/kg bw/day is regarded as the NOAEL for local skin effects and the NOAEL for systemic effects is 1000 mg/kg bw/day for males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.39 mg/cm²

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006. The study with the lowest dose descriptor was selected for endpoint conclusion.

Mode of Action Analysis / Human Relevance Framework

Please refer to the information provided in the endpoint summary. 

Additional information

Repeated dose toxicity: oral administration

Data on repeated dose toxicity are available for the test substance to characterize the potency to induce toxicity after (sub)chronic oral exposure in rodents and non-rodents. These are two key studies addressing the subchronic toxicity of the test substance in the species rat and dog.

No data on repeated dose toxicity of the target substance (CAS 1689-99-2) is available for chronic oral exposure. Therefore, a read-across approach from the analogue substance (CAS 1689-84-5) was performed from a key 52-week oral toxicity study in Beagle dogs. As discussed in the chapter on toxicokinetics, the target substance is converted to the source substance and hence, the read-across is based on (bio) transformation to common compound(s), which thus have the same biological target(s) and therefore cause the same type of effects. A detailed justification for read-across is provided in the technical dossier (see IUCLID section 13.2).

In the first key study (M-166147-01-1, 1992) five groups of 20 Sprague-Dawley rats/sex/dose were treated orally for at least 13 weeks with the test substance (CAS 1689-99-2) by admixture in the diet at constant nominal concentrations of 150, 600 and 1100 ppm. Two additional group of 30 rats/sex received either the plain diet or the test substance in the diet at nominal concentrations of 2100 ppm. The study was conducted according to EPA OPP 82-1 (similar to OECD guideline 408, 2018) and under GLP conditions. There were major deviations to the current OECD guideline, in particular missing examinations like sensory reactivity, lipid analysis and endocrine parameters, but the study is considered valid. Mean achieved dose levels were 1.6, 6.5 and 12.6 mg/kg bw/day (males) and 1.8, 7.8and 15.8 mg/kg bw/day (females) in the 150, 600 and 1100 ppm group, respectively. 10/sex/group of the control and 2100 ppm group were designated as recovery animals; however, because of mortality in the 2100 ppm group during the first week of the study (9/30 males and 26/30 females), the remaining high-dose animals were sacrificed during Week 2. The 10 rats/sex designated as recovery animals in the control group were sacrificed during Week 14. No other deaths occurred during the study. Clinical signs observed in the animals in the 2100 ppm group included, thin, hunched and yellow hair-coat in the genital area. No clinical signs of ill-health were observed for any of the treatment groups. The body weight gains were statistically significantly reduced for males and females of the 600 ppm group (males: Week 1 and 2, females: Week 1 through 13), and the terminal body weights of females were 90% of those of the controls. The body weight gains were statistically significantly reduced for males and females (Week 1 through 13) in the 1100 ppm group. The terminal body weights were significantly reduced for male and female rats of the 600 and 1100 ppm group compared with the control. The food consumption was statistically significantly decreased in Week 1 for males and females of the 600 and 1100 ppm group. As the effects was transient, it is not considered to be toxicologically relevant. No effects on water consumption or ophthalmological findings were observed at any dose level. Following the urinalysis, statistically significantly higher urine volume were noted for male rats in Week 5 and statistically significantly lower urine gravity were noted for female rats in Week 5 compared to the control group. Animals of the 1100 ppm group showed alterations in hematology and clinical chemistry: from Week 5 on, the lymphocyte count in female rats was increased, reaching statistical significance in Week 13. Additionally, slightly but statistically significantly higher red blood cell count, hemoglobin, and hematocrit was observed in female rats in Week 5 but not in Week 13. In Week 13, lower total protein and globulin and slightly higher alkaline phosphatase levels were observed in males and females. At sacrifice, female rats of the 600 and 1100 ppm group had increased liver weights and liver-to-body weight ratios, while the liver-to-body weight ratio was statistically significantly increased in males of the 1100 ppm group. Other significant organ weight changes seen in treated animals were consistent with the overall reduction in body weights. Histopathological analysis did not show any abnormalities of the liver, but the incidences of lymphocyte necrosis in the thymus and myocardial degeneration and necrosis in the heart in males and females was slightly increased in male and female rats of the 1100 ppm group. Animals of the 2100 ppm group had lower platelet count and absolute lymphocyte count and higher red blood cell count, hemoglobin and hematocrit as well as lower globulin and higher urea nitrogen, aspartate aminotransferase and alanine aminotransferase levels in Week 2 compared to animals of the control group. 12/26 females given 2100 ppm that died before sacrifice had diffusely red lungs. There were instances of macroscopic changes in stomach, mottled mesenteric lymph nodes and diffusely dark adrenals and liver. In animals given 2100 ppm, there was lymphohematopoietic necrosis and depletion in the thymus, spleen, mesenteric lymph node and bone marrow. These animals had suffered severe losses in bodyweight before death and it is not known to what extent inanition may have contributed to these findings.

Taken together, the body weight gain, clinical chemistry parameters and liver weight were altered after treatment with the test substance at 600 ppm and above. Therefore, the NOAEL for general toxicity was set at 150 ppm, corresponding to 1.6 and 1.8 mg/kg bw/day for males and females, respectively.

 

The potential immunotoxicity of the test substance was assessed in male C57BL mice according to an U.S. EPA test guideline, under GLP conditions. A summary of the study was included in the endpoint summary for toxicity to reproduction endpoint as it is considered a supporting study with useful data (M-410294-01-1, 2011, Section 7.9.2 Immunotoxicity).

Groups of 10 C57BL mice rats/per dose were exposed daily via the diet to 30, 100 and 300 ppm test item (CAS 1689-99-2), corresponding to 5, 16 and 49 mg/kg bw/day (mean ratios) of the test substance for at least 28 days. The dosing solutions were prepared by mixing the appropriate amounts of the test substance with the diet. The control group received the plain diet only. One group of rats received 7.5 mg/kg bw Cyclophosphamide as a positive control for immunosuppression. Cyclophosphamide was administered solubilized in deionized water via gavage. Both the concentration and homogeneity of the test substance in the diet and the positive control in the vehicle were analyzed and considered valid. Clinical signs were recorded daily and body weight and food consumption were measured weekly. A detailed physical examination was performed weekly throughout the study. On Day 26, rats were injected in the tail vein with a 0.1 mL preparation of Sheep Red Blood Cells (SRBC), containing 1 x 10^9 cells/mL. Four days after SRBC immunization, blood was taken from all animals (non-fasted) and analyzed for anti SRBC-IgM using an ELISA kit. When animals were sacrificed on Day 30, the gross necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded but not sampled. The organ weight was recorded for spleen and thymus. No mortality was observed in the study. There were no treatment-related signs observed in the treatment groups and positive control group. Body weight and feed consumption were not affected by treatment with the test substance or the positive control as compared to the vehicle control. One animal in the 30 ppm group showed body weight loss between study Days 22 and 29. This was considered not to be treatment-related as it was not found in any other animal. No differences regarding gross pathology or organ weights were found between treatment and control groups at necropsy. The SRBC response was unchanged in treatment groups compared to control groups. In the positive control group, the concentration of anti-SRBC IgM was 76% lower than the controls. Spleen and thymus were smaller and lighter than in vehicle control animals and atrophic/small spleen and/or thymus were noted (7/10 and 8/10 animals, respectively). With these results, the positive control cyclophosphamide confirmed the sensitivity of the test system. No immunotoxic properties were noted in male mice at doses of test item up to and including 300 ppm, corresponding to 49 mg/kg bw/day.

Under the conditions of the study, the NOAEL for immunotoxicity and systemic toxicity was set to 49 mg/kg bw/day (the highest administered dose).

 

A second subchronic study (M-344551-01-1, 1984) was conducted with CD rats. For this study no robust study summary was included in the IUCLID data set. However, it has been described and discussed here for purpose of data completeness. In this study, CD rats (20/sex/group) were fed with the test substance (CAS 1689-99-2) at doses of 0, 150, 400 and 950 ppm continuously via the diet. All animals were observed for a 13-14 week period (13 weeks for females, 14 weeks for males). Mean daily intake levels were 10.4, 28.5 and 75.2 mg/kg bw/day for male rats and 12.7, 35.9 and 99.9 mg/kg bw/day for female rats. A female of the high dose group was found dead in the cage during Week 7 of treatment. One female from each of the control, intermediate and low dose groups died during the bleeding process. No conclusive evidence of treatment-related effects on mortality was found. Generally, hair loss and failure to groom were more prevalent in treatment groups than in the control group. Body weight gain was reduced in all treated male groups and in the females in the 400 and 950 ppm dose groups. There was no consistent difference in food consumption between the various groups. The packed cell volume, hemoglobin concentration and erythrocyte numbers were increased and platelet count reduced in the mid- and high dose females. In the males, a reduced platelet count was noted in Week 6 in the mid-dose group (significant) and high dose group (not significant), and in Week 12 in the high dose group (significant). The clinical chemistry parameters showed raised serum alkaline phosphatase, elevated blood urea and altered electrolyte balance in both sexes, as well as increased ALT in males. Urine alkalinity was increased in females of the high dose group after Week 11. The absolute and relative liver weight was increased in males administered 950 ppm and in females administered 400 and 950 ppm. The increase in relative weight of brains, hearts, kidneys and testes may be considered as stemming from decreased body weight. Changes in the absolute and/or relative weight of ovaries, adrenal glands and thyroid glands may implicate these as targets of test substance toxicity. With regard to ovarian weight, absolute weight (more relevant parameter for reproductive organs) was not reduced at the low dose level and the reduction at the mid and high dose level was not associated with any histopathological changes. Histopathological changes were seen mainly in females of the mid- and high-dose group. These included a reduction in cellularity of the bone marrow and atrophy of the uterus. Changes in the liver indicative of enzyme induction were observed in all females groups and in males of the 950 ppm group. The observed liver weight increase and an elevation in serum alkaline phosphatase levels was probably due to cholestasis resulting from the swelling of hepatocytes. The absence of additional histopathological lesions in the liver indicates that the elevation of serum alanine aminotransferase (ALT) in high dosage males and the depression in plasma globulins found in high dose males and females probably reflect secondary hepatic changes associated with enzyme induction (seen as an adaptive response).

Under the conditions of the test, the NOAEL of the test substances was set at 150 ppm, corresponding to 0.4 mg/kg bw/day for male and 12.7 mg/kg bw/day for female rats.

 

In the second key study (M-227060-01-1, 1993), groups of 2 Beagle dogs/sex/dose were treated orally with the test substance (CAS 1689-99-2) via gelatin capsules. The study was conducted similar to the OECD guideline 409 (dated 1981) and under GLP conditions. Therefore, the study was considered valid and reliable. The administered doses were 0.43, 1.43 and 7.14 mg/kg bw/day. Animals of the control groups received empty gelatin capsules. No unscheduled deaths occurred during the course of the study and no clinical signs were recorded that could be attributable to treatment. A low incidence of liquid feces seen in treated animals was similar to that in the controls and was therefore considered of no importance. Body weight was unaffected in animals in the low- and mid dose group. Animals in the high dose group showed statistically significantly reduced body weight gain from Week 10 onwards compared to the control group. The terminal body weight was also significantly lower than the control group. This effect was not seen in food consumption, which was similar across all groups. No treatment-related adverse effects were observed in any of the treatment groups for the ophthalmological examination, clinical chemistry parameters and urinalysis. In the high dose group, mean values for red cell parameters (PCV, Hb and RBC) were increased in Weeks 6 and 13, although the difference did not reach statistical significance. Statistically significant difference of rectal temperatures noted in Week 5, 24 h following dosing in animals receiving 7.14 mg/kg bw/day. However, no clear treatment effect was demonstrated. No abnormality was detected at Week 1, 9 and 13. Bone marrow examination revealed no abnormalities of cellularity, distribution or morphology. The absolute and relative liver weight for animals receiving 7.14 mg/kg bw/day were increased. The mean liver weights for animal receiving 7.14 and 1.43 mg/kg bw/day were significantly higher than for the controls. There were no treatment-related macroscopic findings. Minimal centrilobular hepatocyte enlargement in 1/2 male dogs of the 7.14 mg/kg bw/day group was associated with elevated liver weight compared to control. No treatment-related findings were observed in any of the female dogs. Minimal spermatogenesis in the testes and corresponding reduced spermatozoa in the epididymides of 2/2 male dogs of the high dose group were considered to represent incomplete normal sexual maturation and to be consistent with age of the dogs.

Based on the described effects the NOAEL was set at 1.43 mg/kg bw/day for male and female Beagle dogs.

 

The third key study in dogs was performed under GLP conditions and similar to OECD guideline 452 (M-240237-03-1, 1988) with the analogue substance (CAS 1689-84-5). Groups of 6 male and 6 female Beagle dogs received the test substance via gelatin capsules for 52 weeks at doses of 0.1, 0.3, 1.5 and 7.5 mg/kg bw/day, diluted 1:25 in lactose. Animals of the control group received lactose only. All animals were checked daily for signs of ill health. The body weight, feed consumption and water consumption were measured weekly. Before beginning of treatment and in Week 52, ophthalmoscopic examinations were performed. Blood was withdrawn before the study and in weeks 13, 26 and 52 to assess hematology and clinical chemistry parameters. At the same intervals, urine was collected for urinalysis. At sacrifice, animals were subjected to gross necropsy, several organs were weighed and histopathology was performed. No treatment-related mortality occurred during the study. One male of the control group was sacrificed in Week 38 of the study due to repeated seizures. The animal showed signs of ill-health two days before sacrifice. Clinical signs were restricted mainly to panting, salivation, liquid feces and pale gums. Salivation, liquid feces were seen throughout all control and treated groups but were predominantly observed for dogs receiving 1.5 and 7.5 mg/kg bw/day and was considered to be of toxicological relevance in this dose groups. The group mean body weight gain for males receiving 1.5 and 7.5 mg/kg bw/day and for females receiving 7.5 mg/kg bw/day was statistically significantly lower than that seen for controls. The majority of dogs on study consumed almost all the offered feed. Female animals in the control group and those receiving 0.1 or 0.3 mg/kg bw/day showed a tendency towards lower food consumption throughout the dosing period than that seen for females from other treated group. Food consumption was considered to be affected in a toxicological relevant matter at 7.5 mg/kg bw/day (both sexes). Group mean hematocrit, hemoglobin and red blood cells values were statistically significantly decreased in the group receiving 7.5 mg/kg bw/day during Weeks 13, 26 and 52. In the 1.5 mg/kg bw/day group, these values were also decreased throughout the study but the difference to the control group did not reach statistical significance and the finding was therefore not considered relevant. A significant increase in urea level was seen in males and females receiving 7.5 mg/kg bw/day during Week 13, 26 and 52. Significantly decreased alkaline phosphatase levels were seen in males and females receiving 7.5 mg/kg bw/day during Week 13 and 26 and males only during Week 52. In the treatment groups receiving lower doses, no relevant findings were observed regarding clinical chemistry. Ophthalmoscopic and bone marrow investigations did not show any differences between control and treatment groups, no changes were seen in the urinalysis and there was no macroscopic finding which could be attributed to treatment. Weights of specific organs were altered at 1.5 and 7.5 mg/kg bw/day in both sexes: Increased liver weights were recorded in males and females receiving 1.5 or 7.5 mg/kg bw/day. A significantly lower spleen weights was also noted in males receiving 7.5 mg/kg bw/day. In addition, prostate weights in males receiving 1.5 or 7.5 mg/kg bw/day were greater than control values although the differences did not attain statistical significance. There were no histopathological changes considered to be directly related to the administration of the test substance.

Taken together, the test substance induced reduced body weight or body weight gain in treated animals compared to controls at 1.5 mg/kg bw/day and higher. The liver weight was increased in the 1.5 and 7.5 mg/kg bw/day group. Clinical signs (panting) were observed at 1.5 mg/kg bw/day. Hematology and clinical chemistry was altered at 7.5 mg/kg bw/day and above.

Based on decreased body weight gain, panting and increased liver weights at 1.5 mg/kg bw/day, the NOAEL of the test substance was 0.3 mg/kg bw/day for dietary administration of the test substance to the male and female Beagle dog over 52 weeks. The study was conducted under GLP conditions according to OECD guideline 452 and is considered valid.

 

Repeated dose toxicity: dermal administration

A key study assessing the potential of the test substance (CAS 1689-99-2) to cause toxicity following repeated dermal application is available (M-277015-01-2, 1992). The study was conducted according to EPA OPP 82-2 (similar to OECD 410, 1981) and under GLP. Ten New Zealand White rabbits/sex/dose received 30, 300 and 1000 mg/kg bw test substance/day on 5 days/week for at least 3 weeks. The test substance was moistened with water and applied to over 10% of the rabbit’s skin for 6 h/day. During this time, animals wore a plastic collar to prevent them from ingesting the test item. The control group was exposed to deionized water only. No mortality occurred during the course of the study and treated animals did not show any treatment-related clinical signs. Dermal irritation was observed in all groups, which increased in severity and number of affected animals with increasing dose. Slight irritation was noted for animals given 0 or 30 mg/kg bw/day. Slight to moderate irritation was observed for animals given 300 or 1000 mg/kg bw/day. In the 300 mg/kg bw/day group slight to moderate erythema and slight desquamation was observed in males, and slight erythema, edema and desquamation for females. In the 1000 mg/kg bw/day group slight to moderate erythema, desquamation, fissuring and edema and slight atonia was observed in males, and slight to moderate erythema and edema and slight desquamation and fissuring in females. These observations did not increase in severity within a dose group during the treatment period. The body weight and food consumption of the treated animals was not significantly affected during the treatment period. The results of the hematology and clinical chemistry parameters were comparable between control and treatment groups. No test material-related changes were observed in terminal body weights, organ weight data, and macroscopic and microscopic pathology findings. 

Under the conditions of the test, the test substance caused dermal irritation when applied dermally for 3 weeks to the intact skin of male and female New Zealand White rabbits, starting at 300 mg/kg bw/day. No systemic toxicity was observed. Therefore, 30 mg/kg bw/day is regarded as the NOAEL for local skin effects and the NOAEL for systemic effects is 1000 mg/kg bw/day for males and females.

 

Conclusion:

In summary, subchronic oral dose toxicity studies were performed with the test substance in Sprague-Dawley rats and Beagle dogs, respectively. Additionally, one 52-week study in Beagle dogs was available with the read-across source substance (CAS 1689-84-5). The test material caused alterations of the liver in all species, however, to a lesser extend in dogs. Hematological and clinical parameters were altered after treatment with the test substance in rats and in dogs. Body weight was affected in all studies and was the most sensitive parameter in the 52-week toxicity dog study. The lowest relevant NOAEL for repeated oral exposure was 0.3 mg/kg bw/day in the 52-week study in dogs. Since the effect in liver in all studies is seen as an adaptive response to treatment and – especially in dogs – showed no functional disturbance or morphological changes, which were toxicologically relevant, no classification is needed. The dermal application of the test substance for three weeks did not lead to systemic effects, which is in line with the lower dermal absorption of the test material.

Justification for classification or non-classification

An extensive data base exists to assess repeated dose toxicity following the oral and dermal route. Based on the available data on subacute, subchronic and chronic exposure, the source substance does not meet the classification criteria in regard to STOT-RE according to the CLP regulation (EC) No. 1272/2008 which is in line with the harmonized classification of the source substance according to Annex VI of the CLP Regulation (EC) No. 1272/2008 (Index no. 608-017-00-0).