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Administrative data

Description of key information

Key study (sub-acute): A Repeated dose 28-Day Oral toxicity Study in Rodents for the test substance was performed in rats. The No-observed-effect-level of the test substance was at 20 mg per kg body weight and day in both sexes.

Key study (sub-chronic): Based on the read-across approach from the analogue substance OS2200, the sub-chronic NOAEL for the test substance was determined to be 13.22 mg/kg bw/day for general systemic toxicity.

Key study (sub-acute): A short term inhalation repeated dose toxicity study is available for the degradation product methanol performed with a method similar to OECD Guideline 412. Rats exposed to up to 5010 ppm (6 hr/d, 5d/wk for 4 weeks) showed no treatment-related histopathological effects. Inhalation exposure only resulted in some slight treatment-related signs.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 8, 2004 - February 23, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
There are no deviations from the recommended guideline. Meets the requirements of GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Fischer, F344/NHsd, SPF.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman
- Age at study initiation: about 7 weeks
- Weight at study initiation: males about 155 g; females about 110 g. All the animals were within ±20% of the mean weight per sex at the time of allocation groups.
- Housing: group caging, Makrolon cages type IV
- Diet (e.g. ad libitum): ad libitum. Exception: feed was withdrawn the day before blood sampling
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): average of 21.9 ºC
- Humidity (%): average of 45.4 ºC
- Air changes (per hr): 12 per hour
- Photoperiod (hrs dark / hrs light): artificial light from 6 a.m. to 6 p.m.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was blended with the vehicle (corn oil). Preparations of the test substance were made freshly every day shortly before the administration to the animals. Appropriate preparations were made to allow a uniform dose volume for all groups.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Altromin 1324 forte, gamma irradiated with 25kGy60Co

VEHICLE
- Justification for use and choice of vehicle (if other than water): as the test substance is known to react immediately with water, corn oil was chosen as solvent.
- Amount of vehicle (if gavage): 5 mL per kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determinations of the test substance in some selected preparations for stability, concentration and homogeneity were performed by gas chromatography with flame ionisation detection (GC/FID).
Concentration test: A deviation of the mean of the 3 samples from the target concentration of at most ± 10 % was tolerated.
Homogeneity test: A deviation of the individual samples from the mean of at most ± 10 % was tolerated.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Once a day for 28 consecutive days.
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
63 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Low dose (20 mg/kg/day): 10 animals (5 males and 5 females).
Mid dose (63 mg/kg/day): 10 animals (5 males and 5 females).
High dose (200 mg/kg/day): 10 animals (5 males and 5 females) and an additional group of 10 animals (5 males and 5 females) for observation of reversibility of substance effects.
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
the doses were derived from the results of the dose range findings study.
Groups of 5 male and 5 female rats were given doses of 100 mg or 316 mg or 1000 mg test substance per kg body weight for 7 consecutive days. Investigations: Body weights, feed consumption, animal observation, gross pathology.
Results: 1 high dosed female died early, with the death related to the test substance. Gross necropsy findings gave some indication for hepatotoxicity.Body weights of the mid and high dosed males and high dosed females were significantly reduced. Feed consumption was reduced in parallel.
Therefore 200 mg test substance per kg body weight, interpolated between low and mid dose of the dose range finding study, are chosen as high dose for the main study. 20 mg test substance per kg, i.e. 10 % of the high dose, is chosen as the low dose, where no toxic effects are anticipated. The middose is interpolated geometrically.

- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals, once a week

BODY WEIGHT: Yes
- Time schedule for examinations: all animals, once a week

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: all the animals groups on day 29, except the dose satellite group and the control satellite group that the blood was collected 14 days after (on day 43).
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 30 animals
- Parameters checked in table [No.3] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: all the animals groups on day 29, except the dose satellite group and the control satellite group that the blood was collected 14 days after (on day 43).
- Animals fasted: Yes
- How many animals: 30 animals
- Parameters checked in table [No.9] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: organ weights (see table 4, summary data)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Analysis of variance followed by the Scheffé-test, t-test and H-test of Kruskal and Wallis followed by the test of Nemenyi were used
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One single animal (contral satellite graup) was found to suffer fram an eye lesion. This is not related to the test substance.
Mortality:
no mortality observed
Description (incidence):
No test substance related death occurred during the study. All animals survived until their scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dosed satellite group males had a significantly lower body weight than the controls in the last week of dosing and in the first week of the recovery period. Feed consumption of the high dosed males was marginally impaired.
The body weight gain of the high dosed satellite group males, body weight gain was significantly reduced in the first week and the fourth week of dosing and significantly elevated in the first week of the recovery period.
There were no significant differences in the body weight gain of the females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the males, the high dosed and the high dosed satellite group had a slightly lower feed consumption during the dosing period, followed by a slightly higher feed consumption at the end of the recovery period, both when compared with the controls. The differences were not markedly pronounced and are given only borderline relevance.
There were no noteworthy differences or dose related trends noted in the feed consumption of the females.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The test substance causes damage to mature erythrocytes in the peripheral blood, resulting in alterations in virtually all erythrocyte-related parameters at the haematological examination. In parallel to this damage there is a marked repair by ongoing by an increased new production of young erythrocytes, notable in an increased proportion of polychromatic erythrocytes in the peripheral blood and also in haematopoiesis in the spleen, inducing even a significant weight increase of this organ, and - less pronounced - a hypercellularity of the bone marrow. The repair was efficient enough to allow the animals to live without physical behavioural abnormalities. There was no indication for a negative effect to the blood cell production in the bone marrow found.
Effects were similarly noted in males and females at the same doses, no sex difference in the response to the test substance can be derived from the results.
Effects were only partly reversible during the recovery period, though there was a clear tendency towards a return to normal.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Some haematological changes in neutrophil and lymphocyte counts and some clinical biochemical changes and the increased liver weights remain without a clear interpretation. The haematological changes may be secondary to the erythrocyte alterations, the biochemical ones may indicate a borderline effect to the liver.
Effects were similarly noted in males and females at the same doses, no sex difference in the response to the test substance can be derived from the results.
Effects were only partly reversible during the recovery period, though there was a clear tendency towards a return to normal.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
All observations made are within anormal behavioural pattern of rats of the strain and age used. Reactivity, reflexes and grip strength were also adequate.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The liver and the spleen weight differences were attributed to the test substance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In all high dosed animals a large spleen was noted. This alteration is attributed to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologically, extramedullary haematopoiesis was found in the spleens of all high dosed animals and most mid dosed animals. Hypercellularity of the bone marrow may be also due to the test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat. (dissolved fraction)
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Mean body weight gain (Table 1)

Data are presented by mean, standard deviation (sd) and number of animals (n). Significant differences to the control group are indicated by an underline number.

Table1: Males

group

body weight gain (g/day per animal) for Days

 

-5-1

1-8

8-15

15-22

22-28 28-36

36-42

K mean

4.90

3.94

3.77

3.03

2.47

 

-

sd

0.62

0.54

0.22

0.31

0.76

 

 

n

5

5

5

5

5

 

 

KS mean

5.17

4.17

3.74

3.09

2.83

2.08

1.30

sd

0.58

0.59

0.47

0.59

0.47

0.52

0.68

n

5

5

5

5

5

5

5

A mean

4.47

4.06

3.29

2.77

1.90

-

-

sd

0.66

0.62

0.54

0.30

0.25

 

 

n

5

5

5

5

5

 

 

B mean

4.47

3.60

3.17

3.03

2.90

-

-

sd

0.59

0.31

0.65

0.59

0.38

 

 

n

5

5

5

5

5

 

 

C mean

4.97

3.49

3.03

2.49

1.93

-

-

sd

0.84

0.81

0.75

0.61

0.45

 

 

n

5

5

5

5

5

 

 

CS mean

4.67

3.31

3.37

2.74

1.70

2.83

1.97

sd

0.42

0.37

0.44

0.41

0.61

0.17

0.49

n

5

5

5

5

5

5

5

Table 1 cont.: Females

group

body weight gain (g/day per animal) for Days

 

 

-5-1

1-8

8-15

15-22

22-28

28-36

36-42

K

mean

2.77

1.71

1.63

1.34

0.78

-

-

 

sd

0.62

0.30

0.16

0.46

0.19

 

 

 

n

5

5

5

5

5

 

 

KS

mean

2.90

1.66

2.00

1.34

0.95

0.58

0.03

 

sd

0.56

0.48

0.29

0.26

0.53

0.17

0.52

 

n

5

5

5

5

5

5

5

A

mean

2.57

1.74

2.00

1.31

0.88

-

-

 

sd

0.58

0.31

0.27

0.48

0.54

 

 

 

n

5

5

5

5

5

 

 

B

mean

2.97

2.06

1.77

1.23

1.23

-

-

 

sd

0.25

0.33

0.19

0.33

0.55

 

 

 

n

5

5

5

5

5

 

 

C

mean

2.30

1.54

1.89

1.54

0.83

 

-

 

sd

0.40

0.51

0.46

0.49

0.46

 

 

 

n

5

5

5

5

5

 

 

CS

mean

2.37

1.57

2.00

1.20

0.10

1.78

0.50

 

sd

0.75

0.17

0.39

0.16

1.39

1.03

0.60

 

 

5

5

5

5

5

5

5

Clinical Biochemistry Data (Table 2)

parameter (sex)

low dose

mid dose

high dose

high dose
after recovery

albumin (males)

-

-

x

-

cholesterol (males)

-

-

x

-

total protein (males)

-

-

x

-

urea (males)

-

-

-

+

alanin aminotransferase (females)

-

-

x

-

alkaline phosphatase (females)

-

-

-

+

calcium (females)

-

-

-

x

glucose (females)

-

-

-

x

key to the symbols:
-: no significant difference to the control, +
: significant increase, x: significant decrease.

All significant differences in clinical biochemistry are attributed to the test substance.

Haematology (Table 3)

parameter (sex)

low dose

mid dose

high dose

high dose
after recovery

red blood cell count (males)

-

X

X

X

red blood cell count (females)

-

X

X

X

haemoglobin concentration (males)

-

X

X

+

haemoglobin concentration (females)

-

X

X

-

haematocrit (males)

-

X

X

+

haematocrit (females)

-

-

X

-

mean corpuscular haemoglobin, pg (males)

-

-

+

+

mean corpuscular haemoglobin, pg (females)

-

+

+

+

mean corpuscular haemoglobin, g/dL (males)

-

-

X

-

mean corpuscular haemoglobin, g/dL (females)

-

-

X

-

mean cell volume (males)

-

-

+

+

mean cell volume (females)

-

+

+

+

white blood cell count (males)

-

-

+

-

white blood cell count (females)

-

-

+

-

monocytes, % (males)

-

-

-

+

eosinophils, % (males)

 

 

 

X

lymphocytes, n (males)

-

-

+

-

lymphocytes, n (females)

-

-

+

-

prothrombin time (males)

-

-

-

X

neutrophils, n (males)

-

-

+

-

neutrophils, n (females)

-

-

-

+

monocytes, n (males)

-

-

+

+

eosinophils, % (males)

-

-

-

X

platelet count (females)

-

-

+

-

polychromatic erythrocytes (%, males)

-

-

+

-

polychromatic erythrocytes (%, females)

-

-

+

-

key to the symbols:
-: no significant difference to the control, +: significant increase, x: significant decrease

 

All significant differences in haematology are attributed to the test substance.

Necropsy findings

In all high dosed animals a large spleen was noted. This alteration is attributed to the test substance.

Organ weights(Table 4)

Survey of the results:

parameter
(sex)

low dose

mid dose

high dose

high dose
after recovery

spleen (males, absolute weight)

-

-

+

-

spleen (females, absolute weight)

-

-

+

+

liver (females, absolute weight)

-

-

-

+

liver (males, organ weight/body weight ratio)

-

-

+

+

spleen (males, organ weight/body weight ratio)

-

-

+

+

liver (females, organ weight/body weight ratio)

-

-

-

+

spleen (females, organ weight/body weight ratio)

-

-

+

+

spleen (males, organ weight/brain weight ratio)

-

-

+

-

liver (females, organ weight/brain weight ratio)

-

-

-

+

spleen (females, organ weight/body weight ratio)

-

-

+

+

key to the symbols:
-: no significant difference to the control, +: significant increase, x: significant decrease

The liver and spleen weight differences are attributed to the test substance.

Histopathology

The presence of extramedullary haematopoiesis in the spleens of all high dosed and almost all mid dosed animals of both sexes is related to the test substance. A minor pronounced effect, similar to that mentioned before, is hypercellularity in the bone marrow, noted in a few animals without gaining statistical significance.

All other histopathological findings are not related to the test substance and considered as spontaneous alterations without toxicological relevance.


Conclusions:
The No-observed-effect-level of the test substance was at 20 mg per kg body weight and day in both sexes.
Executive summary:

The Repeated dose 28-Day Oral toxicity Study in Rodents for the test substance was performed in F344 rats. The test material was administered blended with corn oil, given orally by gavage to 3 groups of 5 male and 5 female rats, once a day for 28 consecutive days. The dose used were 20, 63, and 200 mg/kg/day. A negative control group (treated with the vehicle) was included. In addition, 2 groups of 5 males and 5 females each (i.e. one high dose satellite group and one control satellite group) were treated in the same way as their corresponding groups, but were kept for further 14 days without test substance administration in an attempt to observe the reversibility or persistence of test substance induced lesions.

Investigations:

Clinical observations and body weights: all animals, once a week.

Functional observations: all animals, once, in the last week of the dosing period.

Haematology, clinical biochemistry,necropsy with gross pathological examinationand organ weight determination: all animals on Day 29 and the satellite group animals on day 43.

Histopathological examination: all animals of high dose group and control group, and the spleens of all animals.

The effects noted at a dose of 200 mg/kg and below were mainly adaptations to damage to peripheral erythrocytes without an impairment of the blood cell production in the bone marrow. All other effects noted were only borderline ones.

The No-observed-effect-level of the test substance was at 20 mg per kg body weight and day in both sexes.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 April 2003 to 16 September 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 44 to 48 days.
- Weight at study initiation: 196 to 275 g for males and 150 to 216 g for females.
- Fasting period before study: no
- Housing: 5 of one sex per cage (stainless steel body with a stainless steel, mesh lid and floor and were suspended above absorbent paper) in a barriered rodent facility.
- Diet (e.g. ad libitum): Ad libitum, standard rodent diet.
- Water (e.g. ad libitum): Ad libitum, tap water.
- Acclimation period: 12 days before treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 ºC, on rare occasions deviations outside this range occurred resulting in an overall range of 21-26ºC.
- Humidity (%): 40 to 70%, on rare occasions deviations outside this range occurred resulting in an overall range of 24-71%.
- Photoperiod: 12 hr light/ 12 h dark.

IN-LIFE DATES: From20 September 2004 (treatment commenced) to 22 December 2004 (necropsy completed, main study) or to 18 January 2005 (necropsy completed, recovery study).
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amounts of test substance were measured out into the final formulation bottles for each dosage group. A sufficient amount of vehicle was supplied in a separate bottle. Within the animal facility just before daily dose administration, starting with the lowest concentration, the required amount of vehicle was withdrawn from the bottle using a syringe and the contents added to the respective final formulation bottle. The contents of the formulation bottle were then stirred with a spatula to remove any test substance adhering to the bottle walls, then mixed with a whirlimixer for approximately one minute to aid dissolution. Each subsequent dose concentration was formulated similarly, in ascending order. Formulation was stirred continuously using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: All formulations were shown to be homogenous in the vehicle and stable at ambient temperature for 2 days and for 15 days following refrigerated storage (approximately 4ºC).
- Concentration in vehicle: 0, 2.5, 7.5, 25 mg test substance/mL
- Amount of vehicle (if gavage): 2 mL/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Due to the rapid hydrolysis of OS2200, the hydrolysis product MIBKO was used to assess the concentration of OS2200. At week 1 and 12, MIBKO concentrations were analyzed in the OS2200 formulations giving at the most -9.2 and + 4.2% deviations from nominal concentration.
Duration of treatment / exposure:
13 weeks.
Frequency of treatment:
Once daily.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 rats/sex/dose for the main study (13 weeks treatment).
5 rats/sex/dose for the recovery study (13 weeks treatment +4 weeks recovery).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were selected in conjunction with previous work with the compound performed in the same laboratories with the Huntingdon Life Sciences report numbers ALS 66/95011 and ALS 77/952298. Results from these studies indicated that a level of 150 mg/kg/day or above would not be tolerated over 13-weeks of treatment and it was predicted that treatment at 50 mg/kg/day for 13 weeks would result in tolerable toxicity.
- Rationale for selecting satellite groups: 3 groups of 5 males and 5 females were treated with 0 (control), 5, 15, 50 mg/kg bw/day for 13 weeks, followed by a 4 week period without treatment to assess recovery from any treatment related effect
- Post-exposure recovery period in satellite groups: 4 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health. During the acclimatisation period and recovery Week 1, observations of the animals and their cages were recorded at least once per day and during recovery Week 2 onwards, at least once each week.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily throughout the treatment period, detailed observations were performed immediately before dosing and between one and two hours after completion of dosing of all groups. A further observation was performed as late as possible in the working day during the first week of treatment. In addition, a more detailed weekly physical examination was performed on each animal to monitor general health.Animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.

BODY WEIGHT: Yes
- Time schedule: The weight of each rat was recorded one week before treatment commenced, on the day that treatment commenced, weekly throughout the treatment and recovery periods before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: The weight of food supplied to each cage, that remaining and a estimate of any spilled was recorded for the week before treatment started, and each week throughout the treatment and recovery periods, from these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

FOOD EFFICIENCY: Yes
- Overall group mean values were calculated as the overall group mean bodyweight gain (bodyweight table), divided by the total mean food consumption (calculated from individual cage values), the resultant fraction expressed as a percentage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced, the eyes of all animals allocated to the study (including spare animals) were examined by means of a binocular indirect ophthalmoscope. During Week 12 of treatment the eyes of all animals of control and 50 mg/kg bw/day groups were examined. No ophthalmic examination was undertaken at the end of the recovery phase because no treatment related changes were observed at the Week 12 examination. Prior to each examination, the pupils of each animal were dilated using 0.5% tropicamide ophthalmic solution (Mydriacyl, Alcon Laboratories Ltd.). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: Yes (peripheral blood)
- Time schedule for collection of blood: during week 5 and 13 of treatment (before dosing on each occasion).
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes, overnight.
- How many animals: All animals.
- Parameters checked: Haematocrit (Hct), Haemoglobin (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (PLT), Abnormal morphology, Prothrhombin time (PT), Activated partial thromboplastin time (APTT).
Relating the recovery groups (based on 13 week haematology investigations): all red and white blood cell parameters were assessed for the control group, whereas only red cell parameterswere examined at treatment groups.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 5 and 13 of treatment (before dosing on each occasion).
- Animals fasted: Yes, overnight.
- How many animals: all animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb), Albumin/globulin ratio (A/G Ratio).
Relating to the recovery groups (based on based on 13 week clinical chemistry investigations): Glucose for both sexes and bilirubin and potassium for females.

URINALYSIS: Yes
- Time schedule for collection of urine: during week 13 of treatment and week 4 of recovery.
- Metabolism cages used for collection of urine: Yes .
- Animals fasted: Yes, the afternoon prior to sampling.
- How many animals: all animals.
- Parameters checked: Appearance, Volume (Vol), pH, Specific gravity (SG), Protein (Prot), Glucose (Gluc), ketones (Keto), bile pigments (Bili), haem pigments (Blood) by Multistix, Microscopic examination of the urine sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 5 and 12 (+ recovery animals on week 4 of recovery).
- Dose groups that were examined: Sensory reactivity and grip strength: All recovery phase animals and the first five surviving main study animals from all groups. Motor activity: All recovery phase animals during week 4 of recovery.
- Battery of functions tested: sensory reactivity, grip strength and motor activity assessment.
Sacrifice and pathology:
NECROPSY: The animal killed during the recovery period and those surviving until the end of the scheduled treatment or recovery period were killed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy.
Full macroscopic examination of the tissues.
All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.
The requisite organs were weighed and external and cut surfaces of the organs and tissues were examined as appropriate.
Organ weights: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus with cervix.
FIxation: Adrenals, Aorta-thoracic, Brain, Caecum, Colon,Duodenum, Epididymides, Eyes#, Femurs with joint, Head#, Heart, Ilum (including Peyer's patch), Jejunum, Kidneys, Liver, Lungs (including bronchi), Lymph nodes (mandibular, mesenteric, caudal), Mammary area, Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submandibular, sublingual), Sciatic nerves, Seminal vesicles, Skin, Spinal cord, Spleen, Sternum, Stomach, Testes, Thymus, Thyroid with parathyroid, Trachea, Urinary bladdes, Uterus und cervix, Any anormal tissue.
# Not process for examination.

HISTOPATHOLOGY: Yes
The relevant tissues were subject to histological processing for all animals.
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Femur with joint - longitudinal section including articular surface, epiphysial plate and bone marrow
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Liver - section from all main lobes
Lungs - section from two major lobes, to include bronchi
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Sternum - included bone marrow
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterus section separate from cervix section

PATHOLOGY: Light microscopy:
All tissues preserved were examinated in all animals from control and 50 mg/kg bw/day. Tissues reported at macroscopic examination as being abnormal were examinated for all main and recovery animals.
Statistics:
All statistical analyses were carried out separately for males and females. The test substance was analysed in relation to the control group. Analyses were carried out using the individual animal as the basic experiment unit. Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no unscheduled deaths during the treatment period. However, a single male rat receiving 15 mg/kg/day was killed in extremis due to poor clinical condition during the second week of the recovery period. Clinical signs observed before death comprised aggressive behaviour, a twisted left hind paw with skin encrustation on the digits, piloerection, partially closed eyelids, pale teeth, red discharge from the nose, elevated abnormal gait with a hunched posture, irregular respiration and slight muscle tremors. Macroscopic examination at the post-mortem of this animal also revealed dark adrenal glands, a small and dark thymus, gaseous distension of the gastrointestinal tract, congested lungs and bronchi. The histopathological investigation of this animal did not reveal a conclusive cause of death but ulcerative pododermatitis in the left hind paw was considered to be a contributory factor; thus the death of this animal is not considered to be associated with previous treatment. There were no post dose signs observed during the 13 week treatment period or 4 week recovery period that were considered to be attributable to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths during the treatment period. However, a single male rat receiving 15 mg/kg/day was killed in extremis due to poor clinical condition during the second week of the recovery period. Clinical signs observed before death comprised aggressive behaviour, a twisted left hind paw with skin encrustation on the digits, piloerection, partially closed eyelids, pale teeth, red discharge from the nose, elevated abnormal gait with a hunched posture, irregular respiration and slight muscle tremors. Macroscopic examination at the post-mortem of this animal also revealed dark adrenal glands, a small and dark thymus, gaseous distension of the gastrointestinal tract, congested lungs and bronchi. The histopathological investigation of this animal did not reveal a conclusive cause of death but ulcerative pododermatitis in the left hind paw was considered to be a contributory factor; thus the death of this animal is not considered to be associated with previous treatment. There were no post dose signs observed during the 13 week treatment period or 4 week recovery period that were considered to be attributable to treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the 13 week treatment period, bodyweight gains although variable showed no clear dosage related trend in either sex and it was considered not to have been affected by treatment. During the 4 week recovery period, the overall bodyweight gain was superior or similar to that of control among previously treated male and female groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The overall (Weeks 1 to 13) food consumption means among treated female groups were slightly, but statistically significantly, higher than control; although the mean values did not follow a dosage related trend and the differences from control were minor. The overall food consumption for the male treated groups was essentially similar to control, during the 13 week treatment period. During the 4 week recovery period food consumption was marginally higher than control for both sexes previously treated at 50 mg/kg/day and for males previously treated at 15 mg/kg/day. The food intake of the remaining recovery animals was similar to that of their respective controls.
Food efficiency:
no effects observed
Description (incidence and severity):
Overall, food conversion efficiency ratios for all treated groups were considered essentially similar to control. The food conversion efficiency recorded during the 4 week recovery period was variable, resulting in overall values that were superior to that of control for all male treated groups and were inferior to control for females previously treated at 15 mg/kg/day. The food conversion efficiency ratios for the remaining treated groups were considered to be essentially similar to that of their respective controls. The pattern of the overall ratios tended to follow a similar pattern to that of the recovery bodyweight gains. In the absence of any previous effect of treatment on food conversion efficiency these changes were considered not to be related to the previous treatment.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
At the Week 12 ophthalmic examination there was no effect of treatment observed in the eyes of animals treated with OS2200. As such further ophthalmic investigations were not undertaken at the end of the 4 week recovery period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were clear effects of treatment on red blood cell parameters for both sexes. These were confined to animals receiving 50 mg/kg/day and were observed mainly at the Week 13 investigation. Reversibility of these findings was evident after a 4-week period without treatment.
In Week 5 it was recorded a higher mean reticulocyte value recorded for females receiving 50 mg/kg/day. A similar difference was again noted for these females in Week 13, with the magnitude of differences from control being greater in Week 13 than in Week 5. A higher mean reticulocyte value was also recorded for males receiving 50 mg/kg/day in Week 13. In addition, in Week 13 both sexes treated at 50 mg/kg/day showed lower than control mean red blood cell values, with the males also showing lower than controls mean haemoglobin (Hb) and haematocrit (Hct) values. Associated with these effects on primary red blood cell parameters, in Week 13, were slightly higher than control mean cell haemoglobin (MCH) and mean cell volume (MCV) mean values recorded for females receiving 50 mg/kg/day. There were no treatment-related effects on blood film comments. Recovery from the red cell effects was clearly evident by the end of the 4-week period without treatment. At the end of the recovery period mean Hb and RBC values for females which had previously been treated at 50 mg/kg/day, were slightly higher than controls. This was considered to be due to an over compensation of red blood cell production following cessation of treatment, which was also reflected in the slightly lower than control mean reticulocyte value recorded for these females. At the recovery investigations females from other previously treated groups showed higher than control red cell parameters and lower reticulocyte values, of a similar magnitude to females previously treated at 50 mg/kg/day. In the absence of any effects in these groups during treatment, these differences were considered to be indicative of normal biological variation associated with the small group size rather than associated with previous treatment. There was considered to be no clear effect of treatment on clotting parameters for both sexes. In Week 13, females receiving 50 mg/kg/day also showed a higher than control mean platelet value, however, the degree of difference from controls was minor and individual values for these treated females were in the range of individual control values. Thus, this difference was not considered to be due to treatment. In Weeks 5 and 13 males receiving 50 mg/kg/day showed lower mean PT and APTT values, compared with controls. The magnitude of the difference from control for the PT values decreased with time, such that in Week 13 the mean was 96% of the control value and similar to the mean for males receiving 15 mg/kg/day. Consequently this difference from control was considered not to be of toxicological importance. Between Week 5 and 13, the degree of difference from control for mean APTT increased, however, the difference in Week 13 was considered to be mainly due to a low value for a single male (No. 69), unduly influencing the group mean; with the individual values for the remaining males at 50 mg/kg/day being similar to controls. There were no other differences from controls, including white blood cell parameters and other statistical significances, which were considered be treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In Week 13 only, females receiving 15 or 50 mg/kg/day showed higher than control mean bilirubin values. The magnitude of the difference from controls was similar for both groups. Full recovery in bilirubin values were seen by the end of the recovery period. Females receiving 50 mg/kg/day showed lower than control mean potassium values in Weeks 5 and 13, with the difference from control increasing over time. There was no difference from control for potassium at the end of the recovery period. In addition, in Week 5 only, these females also showed a slightly lower than control mean sodium value. All but one individual sodium value for these females was within the concurrent control range, thus this difference from control was considered not to be treatment-related. Both sexes receiving 50 mg/kg/day showed differences from controls for mean glucose values in Week 13. Males showed a higher than control mean value, whilst conversely females showed a lower than control mean value of similar magnitude. At the end of the recovery period similar differences from controls were again exhibited for these groups, with females which had previously received 15 mg/kg/day also showing a lower than control mean value. Due to the sexes showing opposite trends, the wide degree of variation in individual values and the mean value for females previously treated with 15 mg/kg/day showing a lower mean at recovery than females which had received 50 mg/kg/day in Week 13, the differences from controls in glucose values were considered not to be associated with treatment. In Week 5 slightly lower than control mean creatinine values were noted for all male treated groups, which attained statistical significance, with all male treated groups having the same mean value. The majority of individual creatinine values for the treated male animals were, however, within the range of the individual control values, thus this change was not considered to be of toxicological importance. In Week 13 slightly lower than control AST values were recorded for all treated male groups, but this change was considered to be fortuitous as the mean values did not follow a dosage related trend and all the individual values (except for one male receiving 50 mg/kg/day) were within the range for the individual control values, thus this change was not considered to be related to treatment.
There were no other differences from controls, including those that attained statistical significances, which were considered to be treatment related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no effects of treatment on urine parameters for males at the end of the treatment period. Females receiving 15 or 50 mg/kg/day did however show higher than control mean protein values both at the end of the treatment and recovery periods. These differences from controls did not follow a dosage relationship and the majority of individual values recorded for these females were within or close to the concurrent control ranges and thus an effect of treatment is considered to be doubtful. All other parameters are considered essentially similar to control.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At the week 5 and 12 assessments of sensor reactivity and grip strength, there were no effects of treatment observed in animals treated with OS2200, as no effects were observed, no further assessment was undertaken during the recovery period. Males treated at 50mg/kg/day showed higher low beam scores at several times during the week 12 assessment. Total scores for males receiving 50mg/kg/day were also significantly higher than control. However high beam scores were only marginally higher than control, no similar change was observed among treated females at this dose level and also no treatment related clinical signs were observed during the week 13 treatment period, thus these changes were considered to be of minor toxicological importance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Analysis of the organ weights for animals killed after completion of 13 weeks of treatment revealed a statistically significantly higher than control mean spleen weight (adjusted for bodyweight) for males receiving 50 mg/kg/day. Recovery was evident at the end of the 4 week recovery period with spleen weights for males previously treated at 50 mg/kg/day being comparable with control. Higher than control mean heart weights were recorded for females treated at 5 and 15 mg/kg/day, while heart weights for females treated at 50 mg/kg/day were comparable with control, thus, as no microscopic changes were observed in the heart, this change was not considered to be associated with treatment. Analysis of organ weights for animals killed at the end of the 4 week recovery period revealed a statistically significantly lower than control mean spleen weight (adjusted for bodyweight) for females previously treated at 50 mg/kg/day. In addition, slightly higher than control adrenal weights were recorded for previously treated male groups when compared with control. However, the mean values did not follow a dosage related trend; thus, in the absence of treatment related microscopic changes being observed in the adrenals, these differences from control are not considered to be associated with previous treatment. All other parameters are considered essentially similar to control.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination for animals killed after completion of 13 weeks of treatment with OS2200, or 4 weeks of recovery, did not reveal any macroscopic changes which could be attributed treatment. The incidence and distribution of all the findings were considered to fall within the background range of macroscopic changes.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings:
Kidney: increased severity and/or incidence of cortical tubules with hyaline droplets and cortical tubular basophilia were seen in males treated at 15 or 50 mg/kg/day of OS2200, when compared with controls. Increased incidence only of cortical tubular basophilia was seen in males treated at 5 mg/kg/day OS2200 when compared with controls. Increased incidence and severity of medullary tubular basophilia was seen in males treated at 50 mg/kg/day OS2200, when compared with controls. Tubular basophilia was unaffected in female rats treated with OS2200. Spleen: Increased incidence and severity of extramedullary haemopoiesis was seen in all OS2200 treated groups in both sexes, when compared with controls. Dosage-relationships were not evident. Increased incidence and severity of haemosiderosis was seen in males and females receiving 50 mg/kg/day of OS2200, when compared with controls.
Recovery phase:
Kidney: Cortical tubules with hyaline droplets were not present in any of recovery animals. Recovery had occurred for cortical tubular basophilia or medullary tubular basophilia in all previously treated groups. Spleen: Extramedullary haemopoiesis and haemosiderosis had broadly resolved in both males and females.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: (red blood cell rate of turnover resulting in associated changes in the spleen)
Remarks on result:
other: The NOAEL of 15.0 mg/kg-bw/day is the starting point for the read-across approach.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
erythrocyte development
spleen
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Histopathology: Treatment-related findings

Kidney:

Dose (mg/kg bw/day

Male

0

5

15

50

Cortical tubules with hyaline droplets

Minimal

1

0

4

6

Slight

0

0

0

3

Total

1

0

4

9b

Cortical tubular basophilia

Minimal

2

6

7

7

Slight

0

0

1

1

Total

2

6

8a

8a

Medullary tubular basophilia

Minimal

2

4

2

4

Slight

0

0

0

1

Total

2

4

2

5

Total number of kidneys examined

10

10

10

10

Dose (mg/kg bw/day

Male (recovery)

0

5

15

50

Cortical tubules with hyaline droplets

Minimal

3

5

3

4

Slight

0

0

0

0

Total

3

5

3

4

Medullary tubular basophilia

Minimal

3

1

2

1

Slight

0

0

0

0

Total

3

1

2

1

Total number of kidneys examined

5

5

4

5

Spleen:

Dose (mg/kg bw/day

Male

Female

0

5

15

50

0

5

15

50

Extramedullary haemopoiesis

Minimal

2

6

4

7

3

3

7

5

Slight

0

2

2

2

1

6

2

5

Moderate

0

0

0

1

0

0

0

0

Total

2

8a

6

10b

4

9

9

10a

Haemosiderosis

Minimal

3

2

4

8

3

5

4

1

Slight

0

2

0

2

1

0

3

4

Moderate

0

0

0

0

1

0

0

5

Total

3

4

4

10b

5

5

7

10a

Total number of spleens examined

10

10

10

10

10

10

10

10

Dose (mg/kg bw/day

Male (recovery)

Female (recovery)

0

5

15

50

0

5

15

50

Extramedullary haemopoiesis

Minimal

4

2

4

3

3

2

1

1

Slight

0

0

0

0

1

1

0

0

Total

4

2

4

3

4

3

1

1

Haemosiderosis

Minimal

1

0

1

1

4

2

3

3

Slight

2

2

0

3

1

2

1

2

Total

3

2

1

4

5

4

4

5

Total number of spleens examined

5

5

4

5

5

5

5

5

a-p<0.05, b-p<0.01 with Fisher’s Exact test, on total incidences only.

After oral administration of OS2200 to the rats for 13 weeks, changes were seen in the kidney and spleen. In the male kidney, cortical tubules with hyaline droplets and cortical tubular basophilia were identified with a severity and/or higher incidence in rats treated at 15 or 50 mg/kg/day OS2200. In the spleen, there were increased incidence and severity of extramedullary haemopoiesis in all treated groups of both sexes, compared with controls. Also increased incidence and severity of haemosiderosis were seen in animals treated at 50 mg/kg/day of OS2200 in both sexes, when compared with controls. In the recovery, all lesions of kidney and spleen had resolved.

Conclusions:
The NOAEL for OS-2200 after 13 weeks of oral exposure to rats was determined to be 15 mg/kg bw/day based on the effects on the red blood cell rate of turnover occurring mainly among animals receiving 50 mg/kg bw/day and resulting in associated changes in the spleen.
Executive summary:

A 13 week repeated dose toxicity test followed by a 4 week recovery period was performed to assess the systemic toxicity of OS 2200 under GLP conditions. The test was performed in rats by oral gavage, three groups, each comprising then male and ten female rats received OS2200 at dosages of 5, 15 or 50 mg/kg bodyweight/day. A similarly constituted group received the vehicle, corn oil, at the same volume-dosage. A further five male and five female rats were assigned to each of the groups, these animals were treated for 13 weeks, followed by a 4 week period without treatment to assess recovery from any treatment related effect. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, ophthalmic examination, haematology, blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken. It was concluded that the principal action of OS2200 was to affect the red blood cell rate of turnover occurring mainly among animals receiving 50 mg/kg/day, resulting in associated changes in the spleen. A dosage level of 15 mg/kg/day, however, was considered the NOAEL for OS 2200 on this study.

Endpoint:
sub-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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See attached the rationale for read-across.
See attached the reporting format and data matrix.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Dose descriptor:
NOAEL
Effect level:
13.22 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: red blood cell rate of turnover resulting in associated changes in the spleen.
Remarks on result:
other: Based on a read-across from an analogue substance for which the NOAEL was 15 mg/kg-bw/day.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
44.06 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
erythrocyte development
spleen
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
Based on the read-across approach from the analogue substance OS2200, the sub-chronic NOAEL for Wasox-VMAC2 was determined to be 13.22 mg/kg bw/day for general systemic toxicity.
Executive summary:

A 13 week repeated dose toxicity test followed by a 4 week recovery period was performed to assess the systemic toxicity of the analogue substance OS 2200 by the oral route in rats. It was concluded that the principal action of OS2200 was to affect the red blood cell rate of turnover occurring mainly among animals receiving 50 mg/kg/day, resulting in associated changes in the spleen. A dosage level of 15 mg/kg/day was considered the NOAEL for OS 2200. Based on the read-across approach from the analogue substance OS2200, the sub-chronic NOAEL for Wasox-VMAC2 was determined to be 13.22 mg/kg bw/day for general systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
13.22 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two studies are available: one of 28-days and the other of 90-days duration. Both of them are GLP compliant (Klimisich score = 1). The overall quality of the database was determined to be appropriate for assessment.
The study with the longest duration (90-days) was chosen.
System:
haematopoietic

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Very limited extent of examinations (see: observations and examinations performed)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, N.Y.
- Age at study initiation: 50 days
- Weight at study initiation: mean males 247-256 g; meanfemales 158-163 g
- Fasting period before study: no
- Housing: no data
- Diet (e.g. ad libitum): Purina Laboratory Chow 5001 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 15 days

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
not specified
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4 m³ exposure chambers constructed of glass and stainless steel
- Method of holding animals in test chamber: individual in rack-mounted wire-mesh cages, positions of individual animals rotated on the rack during the study
- Source and rate of air: house supply
- Method of conditioning air: no data
- System of generating particulates/aerosols: Ambient flash evaporation by a Spraying Systems Atomizer (quarter-inch J nozzle with 1A spray set-up), mounted in the inlet port of the exposure chamber; the aerosol droplets evaporated in the inlet air stream to yield a vapor in the exposure chamber
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: 2000 L/min
- Air change rate: 20/h
- Method of particle size determination: not applicable
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: Wilkes MIRAN 1A-CVF infrared analyzer
- Samples taken from breathing zone: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Based on infrared analysis of chamber atmospheres, animals received eposure to mean concentrations of 520, 1980 and 5010 ppm, respectively. Mean nominal concentrations were 550, 2330 and 5330 ppm. Occasional discrepancies between nominal and analytical values were within the limits of expected experimental error (± 10 %).
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 h/d, 5 d/wk
Dose / conc.:
0 ppm
Dose / conc.:
520 ppm (analytical)
Remarks:
(0.69 mg/L)
Dose / conc.:
1 980 ppm (analytical)
Remarks:
(2.63 mg/L)
Dose / conc.:
5 010 ppm (analytical)
Remarks:
(6.67 mg/L)
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice each day
- Cage side observations included: mortality, clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once each week

BODY WEIGHT: Yes
- Time schedule for examinations: prior to study initiation, first day of exposure and weekly thereafter.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: Yes
- Time schedule for examinations: pretest and at termination
- Dose groups that were examined: all animals: lids, lacrimal apparatus, and conjunctiva were examined grossly, the cornea, anterior chamber, lens, vitreous humor, retina, and optic disc were examined by indirect ophthalmoscopy. Mydriasis was induced by atropine.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. The external body surfaces, all orifices, body cavities and their visceral organs, cervical tissues and organs, and carcass were examined. Tissues and organs were examined in situ and after removal. Organ weights were taken for the adrenal glands (paired), heart, kidneys (paired), liver, lungs, spleen, testes (with epididymides paired), and ovaries (paired).
HISTOPATHOLOGY: Yes. Approximately 35 different tissues per animal were preserved for microscopic examination: eyes, testes, epididymides, lungs, bone marrow smears. Slides from the nasal turbinates, trachea, lungs, kidneys, esophagus, liver, and eye (with optic nerve) from all animals from control and high-exposure groups were prepared and examined microscopically. Only one eye (randomly selected) per animal was examined microscopically.
Statistics:
Body weights, organ weights, and organ/body weight ratios were compared by a statistical evaluation of equality of means. This was done using the appropriate one-way analysis of variance technique, followed by a multiple-comparison procedure if needed. First, Bartlett's test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one-way ANOVA used to determine which means were significantly different from the control. If a nonparametric procedure for testing equality of means was needed, the Kruskal-Wallis test was used, and if differences were indicated, a summed-rank test was used to determine which treatments differed from the control. A statistical test for trend in the dose levels was also performed. In the parametric case (i.e., equal variance), standard regression techniques with a test for trend and lack of fit were used. In the nonparametric case, Jonckheere's test for monotonic trend was used. The test for equal variance (Bartlett's) was conduced at the 1%, two-sided risk level. All other statistical tests were conducted at the 5% and 1% two-sided risk levels. Statistical evaluations were not performed when the standard error for the control group or more than one group was 0.0 due to lack of variance. If a standard error for one treated group was 0.0 or when N (number of animals) was less than or equal to 2 animals for any treated group, it was not included in the statistical evaluation.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased frequencies of nasal and eye discharge (lacrimation, mucoid nasal discharge, red nasal discharge, and dried red nasal discharge) were noted in the treated groups. Only mucoid nasal discharge appeared to be dose-related.
Mortality:
no mortality observed
Description (incidence):
All animals survived.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on body weight.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular abnormalities were noted at the terminal examination of the high-dose group.
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Relative spleen weights were significantly (p ≤ 0.05) increased in female rats exposed at 1980 ppm (not at 5010 ppm). However, these differences by themselves are not of any apparent biological significance. No other organ weight effects were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related effects were noted.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related histopathological effects were noted.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
5 010 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects at the highest concentration tested
Key result
Critical effects observed:
no
Conclusions:
In a repeated dose toxicity study with methanol, rats exposed to up to 5010 ppm (6 hr/d, 5d/wk for 4 weeks) showed no treatment-related histopathological effects. Inhalation exposure only resulted in some slight treatment-related signs.
Executive summary:

A short-term repeated dose toxicity study (inhalation route) was performed on methanol according to a similar method to OECD guideline 412. Groups of 5 male and 5 female rats were exposed to the test item by whole body inhalation at concentrations of 0, 520, 1980 and 5010 ppm, 6 hours per day, 5 days per week for 4 weeks. During the study, clinical observations, bodyweight, organ weight, ophthalmoscopic examination, macropathology and histopathology were undertaken. The only treatment and dose-related effect noted was that of mucoid nasal discharge in rats, which was considered reflective of upper respiratory tract irritation. No consistent treatment-related effects were found for organ or body weights or for histopathologic or ophthalmoscopic examinations. These results suggest that a NOAEL of 5010 ppm equivalent to 6.67 mg/L can be established for the test substance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
6 677 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
One study available with Klimisch score=2

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key study: Test substance Wasox-VMAC2 (RDT 28 days, rats):

The test material was administered blended with corn oil, given orally by gavage to 3 groups of 5 male and 5 female rats, once a day for 28 consecutive days. The dose used were 20, 63, and 200 mg/kg/day. A negative control group (treated with the vehicle) was included.In addition, 2 groups of 5 males and 5 females each (i.e. one high dose satellite group and one control satellite group) were treated in the same way as their corresponding groups, but were kept for further 14 days without test substance administration in an attempt to observe the reversibility or persistence of test substance induced lesions.

The effects noted at a dose of 200 mg/kg and below were mainly adaptations to damage to peripheral erythrocytes without an impairment of the blood cell production in the bone marrow. All other effects noted were only borderline ones.

Key study: Read-across from experimental data on the analogue OS2200 (RDT 90 days, rats)

A 13 week repeated dose toxicity test followed by a 4 week recovery period was performed to assess the systemic toxicity of the analogue substance OS 2200 by the oral route in rats. It was concluded that the principal action of OS2200 was to affect the red blood cell rate of turnover occurring mainly among animals receiving 50 mg/kg/day, resulting in associated changes in the spleen. A dosage level of 15 mg/kg/day was considered the NOAEL for OS 2200. Based on the read-across approach from the analogue substance OS2200, the sub-chronic NOAEL for the test substance was determined to be 13.22 mg/kg bw/day for general systemic toxicity. Nevertheless, it is important to note the low severity of the observed effect (reductions of haemoglobin less than 10 % without marked organ disfunction) and the absence of persistence of the observed effects (reversibility within 4 weeks recovery-period).

Key study: Degradation product methanol (RDT, inhalation route, 4 weeks, rats)

A short-term repeated dose toxicity study (inhalation route) was performed on methanol according to a similar method to OECD guideline 412. Groups of 5 male and 5 female rats were exposed to the test item by whole body inhalation at concentrations of 0, 520, 1980 and 5010 ppm, 6 hours per day, 5 days per week for 4 weeks. During the study, clinical observations, bodyweight, organ weight, ophthalmoscopic examination, macropathology and histopathology were undertaken. The only treatment and dose-related effect noted was that of mucoid nasal discharge in rats, which was considered reflective of upper respiratory tract irritation. No consistent treatment-related effects were found for organ or body weights or for histopathologic or ophthalmoscopic examinations. These results suggest that a NOAEL of 5010 ppm equivalent to 6.67 mg/L can be established for the test substance.

Justification for classification or non-classification

Based on the available data on a 28-day study, the substance is not classified for repeated dose toxicity since the effects noted at a dose of 200 mg/kg and below were mainly adaptations to damage to peripheral erythrocytes without an impairment of the blood cell production in the bone marrow. All other effects noted were only borderline ones. Effects seen in mature erythrocytes in the peripheral blood did not result in any damage to the bone marrow production/functionality. An adaptative response was shown by haematopoiesis in spleen. The decrease in haemoblobin was compensated without organ damage. Furthermore the satellite group had shown complete recovery of haematological parameters. Therefore according to the Guidance on the Application of Regulation (EC) nº 1272/2008, annex I: 3.9.2.8.1 the adaptative responses seen in the study are not considered toxicologically relevant to classify the substance.

The 13 weeks-NOAEL (oral, rat) was determined to be 13.22 mg/kg bw/day based on haematologic changes resulting in associated changes in the spleen observed at an estimated dose of 44.06 mg/kg bw/day. Nevertheless, given the low severity of this effect (reductions of haemoglobin less than 10 % without marked organ disfunction) and the absence of persistence of the observed effects (reversibility within 4 weeks recovery-period), the substance is not classified for STOT RE according to CLP Regulation (EC) No. 1272/2008.

Furthermore, methanol was determined not to have treatment related effects in a subacute inhalation toxicity test performed in rats up to concentrations of 5010 ppm (equivalent to 6.67 mg/L). Thus, the test substance also does not need to be classified STOT RE according to CLP Regulation (EC) No. 1272/2008 because of its degradation product methanol.