Registration Dossier

Administrative data

Description of key information

From a valid subacute oral toxicity study with the rat a LOAEL of 50 mg/kg bw/d, and a NOAEL of 15 mg/kg bw/d resulted.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-04-24 to 2002-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: - OECD guideline compliant - GLP compliant
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
as at 1995-July-27
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
as described in Commission Directive 96/54/EC
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050 (28-Day Oral Toxicity in Rodents) as at 2000-July
Deviations:
no
Qualifier:
according to
Guideline:
other: Ministry of Health and Welfare (MHW) Guidelines 1986, Japanese Chemical Substances Control Law 1973 of the Ministry of International Trade and Industry (MITI) amended 1986
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague-Dawley Crl:CD ® (SD) IGS BR
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: five to six wks
- Weight at study initiation: males: 129 to 171 g, females: 122 to 159 g,
- Fasting period before study: no
- Housing: group housing of 5 animals per sex and per cage in polypropylene grid-floor cages suspended over trays lined with absorbent paper, environmental enrichment: wooden chew blocks (B & K Universal Ltd., Hull, UK)
- Diet (e.g. ad libitum): ad libitum, pelleted diet (5LF2 (Certified) Diet, PMT Nutrition International, Nottingham, UK)
- Water (e.g. ad libitum): ad libitum, mains drinking water
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
On several occasions, the relative humidity and temperature were outside these ranges, however, this was not thought to have affected the purpose or integrity of the study.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- prepared at the appropriate concentrations as a suspension
- stability and homogeneity of the test material formulations determined by Safepharm Analytical Laborator
- prepared weekly and stored at approximately +4°C in the dark

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported, but arachis oil is a standard vehicle in rodent oral studies
- Concentration in vehicle: 2.5, 7.5 and 25 mg/mL
- Amount of vehicle (if gavage): 2 ml/kg bw
- Lot/batch no. (if required): not reported
- Purity: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Technique:
- gas chromatography (GC) using an external standard technique.

Sample preparation:
- test material formulations extracted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1 mg/mL

Standards:
- standard solutions of test material prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.

Procedure:
The standard and sample solutions analysed by GC using the following conditions:
- GC system: Agilent Technologies 5890 or 6890, incorporating autosampler and workstation
- Column: DB-5 (15 m x 0.53 mm id x 1.5 pm film),
- Oven temperature program: initial 200°C for 3 min, rate 15°C/min, final 300°C for 5 min
- Injection temperature: 300°C
- Flame ionisation detector temperature: 300°C
- Injection volume: 1 µL
- Retention time: 8 min

Homogeneity Determinations
- thorough mixing of test material formulations and sampling in triplicate from the top, middle and bottom of the container

Stability Determinations
- test material formulations sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days

Verification of Test Material Formulation Concentrations
- test material formulations sampled and analysed within three days of preparation
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
5, 15 and 50 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): animals randomly allocated to treatment groups using random letter tables and determination of group mean bodyweights to ensure similarity between the treatment groups
- Rationale for selecting satellite groups: high dose group and vehicle control group
- Post-exposure recovery period in satellite groups: 14 d
- Section schedule rationale (if not random): after the last treatment
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing and one and five hours after dosing during the working week, immediately before dosing and one hour after dosing at weekends, during the treatment-free period twice daily, morning and afternoon (once daily at weekends)
- Cage side observations: overt signs of toxicity, ill-health or behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 7, 14, 21 and 25 all animals, observed for signs of functional/behavioural toxicity
- Detailed clinical observations:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

BODY WEIGHT: Yes
- Time schedule for examinations: d 0 (the day before the start of treatment), d 7, 14, 21 and 28 and, in the case of recovery group animals, d 35 and 42 and at terminal kill.

FOOD CONSUMPTION: Yes
- weekly per cage


FOOD EFFICIENCY:
- No

WATER CONSUMPTION : Yes
- Time schedule for examinations: daily, visually per cage


OPHTHALMOSCOPIC EXAMINATION: No, except for checks for Exophthalmia in the detailed clinical observations

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the respective study end (28 d or 42 d (recovery group)), where necessary repeat samples obtained by cardiac puncture prior to necropsy on Day 29 and Day 43 respectively
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the respective study end (28 d or 42 d (recovery group)), where necessary repeat samples obtained by cardiac puncture prior to necropsy on Day 29 and Day 43 respectively
- Animals fasted: No
- How many animals: all per group
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: during wk 4 (non-recovery animals) or during wk 6 (recovery animals)
- Metabolism cages used for collection of urine: Yes, collected overnight, maintained under conditions of normal hydration during collection but without access to food
- Animals fasted: No
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during wk 4
- Dose groups that were examined: all
- Battery of functions tested:
• sensory activity: animals individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli: grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex (ST1058 Startle Test Meter (Benwick Electronics)), tail pinch, blink reflex
• grip strength: forelimb and hindlimb, automated grip strength meter
• motor activity / other: 44 infra-red beam automated activity monitors, animals randomly allocated to the activity monitors, performed at approximately the same time each day, under similar laboratory condition, evaluation period: sixteen hours per animal
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2), animals killed by intravenous overdose of sodium pentobarbitone followed by exsanguination at d 29 or d 43 (recovery animals)
HISTOPATHOLOGY: Yes (see table 2)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, not fully reversible
Mortality:
mortality observed, treatment-related
Description (incidence):
at 50 mg/kg bw, not fully reversible
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw , fully reversible
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw , fully reversible
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
at 50 mg/kg bw, fully reversible
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
at 15 and 50 mg/kg bw, not fully reversible
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- no fatalities during the study period
- clinical signs: At 50 mg/kg/d (males and females) increased salivation prior to and/or up to ten minutes after dosing from Day 6 onwards
starting during the third week of treatment: hunched posture, pilo-erection, tiptoe gait, waddling gait, increased inhumation, respiratory pattern changes and staining of the external body fur. Observations began to regress following cessation of treatment with only hunched posture reported in recovery 50 mg/kg/d animals by the end of the 14-day treatment-free period.

BODY WEIGHT AND WEIGHT GAIN
- 50 mg/kg, males: reduction in bodyweight throughout the study period, no recovery to normal during the recovery period
- 50 mg/kg, females: reduced bodyweight during week 4 of the treatment period, reversion to normal during the recovery period

FOOD CONSUMPTION
- 50 mg/kg, males: reduction in dietary intake throughout the study period, most pronounced during weak 3 and 4 and only reverted to normal during the last weak of the recovery period
- 50 mg/kg, females: slightly reduced dietary intake during weeks 3 and 4 of the treatment period, reversion to normal during the recovery period

FOOD EFFICIENCY
- 50 mg/kg, males: reduction in food efficiency throughout the study period, most pronounced during weak 3 and 4 and decreased still in week 5 of the recovery period
- 50 mg/kg, females: slightly reduced food efficiency during weeks 3 and 4 of the treatment period, reversion to normal during the recovery period

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
- no effects in any treatment group

OPHTHALMOSCOPIC EXAMINATION
- not conducted

HAEMATOLOGY
- 50 mg/kg, males: increased platelet count and erythrocyte count; all values reverted to normal during the recovery period
- 50 mg/kg, females: increased platelet count; all values reverted to normal during the recovery period

CLINICAL CHEMISTRY
- 50 mg/kg, males: increased AST, ALT, cholesterol, reduced plasma glucose, albumin and total protein concentration; all values except ALT reverted to normal during the recovery period
- 50 mg/kg, females: increased AST, ALT, cholesterol; all values except ALT reverted to normal during the recovery period

URINALYSIS
- 50 mg/kg, males: increased urine volume along with reduced specific gravity, increased hemoglobin concentration in urine; all values reverted to normal during the recovery period
- 50 mg/kg, females: increased hemoglobin concentration in urine; all values reverted to normal during the recovery period

NEUROBEHAVIOUR
- behavioural assessment: 50 mg/kg, males and females: hunched position and pilo-erection
- sensory activity: no treatment related effects in any dose group
- motor activity/grip strength: 50 mg/kg, males and females: reduction in overall motor activity

ORGAN WEIGHTS
- 50 mg/kg/d, males: increased relative liver, kidney and adrenal weights; all values reverted to normal during the recovery period
- 50 mg/kg, females: increased relative liver and kidney; only liver weights reverted to normal during the recovery period

GROSS PATHOLOGY
- no treatment related effects in any dose group

HISTOPATHOLOGY: NON-NEOPLASTIC
- Liver: At 50 mg/kg/ d(males and females) foamy vacuolation of hepatocytes, generalised hepatocyte enlargement, and vacuolar distension of scattered cells, single cell hepatocyte necrosis (females only), lower incidence of glycogen-type vacuolation(males only); all effects reverted to normal during the recovery period
- Spleen: At 50 mg/kg/d (males and females) lymphoid hyperplasia and vacuolar distension of scattered cells with associated apoptosis; all effects reverted to normal during the recovery period
- Adrenals: At 50 mg/kg/d (males and females) higher grades of severity of vacuolation of cortical cells; all effects reverted significantly during the recovery period and reverted to normal in females at the and of this period
- Heart: At 50 mg/kg/d (and quote from study report also "possibly at 15 mg/kg/d") in females greater incidence of myocarditis was seen in relation to treatment; all effects reverted to normal during the recovery period
- Urinary bladder: At 50 mg/kg/d (females) treatment-related hyperplasia of the transitional epithelium; all effects reverted to normal during the recovery period
- Small intestine: At 50 mg/kg/d (males and females) vacuolation of lamina propria cells in the duodenum, jejunum, and ileum related to treatment; all effects reverted significantly during the recovery period, just a few animals showing residual changes in the ileum. At 15 mg/kg bw/d (males and females vacuolation of lamina propria cells in the ileum in a few animals
- Lungs: At 50 mg/kg/d (males and females) foamy alveolar macrophages prevalent throughout the lung parenchyma; none of these effects reverted during the recovery period
- Mesenteric lymph node: At 50 mg/kg/d (males and females) foamy histiocytes related to treatment with the test material in the mesenteric lymph nodes; residual lesions characterised by accumulations of macrophages with amorphous eosinophilic cytoplasm following completion of the treatment-free period
- Ovaries: At 50 mg/kg foamy vacuolation of corpora luteal cells, the effects reverted only partially during the recovery period
- Skelettal muscle: At 50 mg/kg/d (males and females) muscle fibre degeneration and necrosis, and proliferation of sarcolemmal nuclei. At 15 mg/kg bw/d (males and females) slight muscle fibre degeneration and necrosis, and slight proliferation of sarcolemmal nuclei. Effects reverted significantly during the recovery period.

HISTOPATHOLOGY: NEOPLASTIC
- no effects in any dose group

HISTORICAL CONTROL DATA
- all above effects compared to both control groups and histroricl control data
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at this level
Dose descriptor:
LOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased incidence of myocarditis in females, vacuolation of lamina propria in the ileum in both sexes and muscle fibre degeneration/necrosis in both sexes
Critical effects observed:
not specified

- Table 1: Group Mean Weekly Bodyweights and Standard Deviations (SD)

Dose Level (mg/kg/day)

Number of Animals

 

Bodyweight (g) at Day

0

7

14

21

28

35#

42#

Males

0 (Control)

10

mean

155

223

286

348

367

427

450

 

 

sd

9

13

16

18

23

19

19

5

5

mean

149

216

279

334

336

-

-

 

 

sd

9

14

19

23

27

-

-

15

5

mean

149

213

273

327

334

-

-

 

 

sd

11

16

23

31

30

-

-

50

10

mean

150

209

260

288

281

310

336

 

 

sd

10

15

22

25

21

11

12

Females

0 (Control)

10

mean

137

169

195

214

223

249

246

 

 

sd

9

12

15

15

17

23

20

5

5

mean

139

175

197

219

227

-

-

 

 

sd

7

5

7

8

9

-

-

15

5

mean

133

166

194

214

222

-

-

 

 

sd

6

6

8

5

5

-

-

50

10

mean

135

167

185

196

197

217

223

 

 

sd

11

12

17

18

21

9

8

# = five animals per dose group during treatment-free period

- = not applicable

- Table 2: Group Mean Weekly Bodyweight Gains and Standard Deviations (SD)

Dose Level (mg/kg/day)

Number of Animals

 

Increase in Bodyweight (g) during Week

 

 

 

1

2

3

4

5#

6#

Males

0 (Control)

10

mean

68

64

61

19

44

23

sd

5

5

7

26

2

3

5

5

mean

67

63

56

2

-

-

sd

9

8

6

5

-

-

15

5

mean

64

60

54

7

-

-

sd

6

7

11

9

-

-

50

10

mean

3359

**51

***28

-7

***17

25

sd

7

9

11

25

7

3

Females

0 (Control)

10

mean

33

25

19

9

20

-2

sd

5

8

5

8

4

4

5

5

mean

37

21

22

8

-

-

sd

3

3

7

6

-

-

15

5

mean

33

28

20

8

-

-

sd

4

4

5

5

-

-

50

10

mean

31

18

12

*0

17

*5

sd

4

7

10

7

6

4

* = significantly different from corresponding control group p <0.05

*** = significantly different from corresponding control group p <0.001

# = five animals per dose group during treatment-free period

- = not applicable

Results in support of a LOAEL of 15 mg/kg bw/d:

- Table 3: Summary Incidence of Histopathological Findings

Histopathological Finding

Dose Level (mg/kg/day)

 

0 (Control)

5

15

50

0 (Control) Recovery group

50 Recovery group

Number of animals examined at terminal kill

5

5

5

5

5

5

MALES

Heart

Focal myocarditis

 

 

 

 

 

 

absent

1

1

1

0

2

2

(minimal)

3

4

4

3

3

3

(slight)

0

0

0

2

0

0

(moderate)

1

0

0

0

0

0

Duodenum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

5

4

5

5

(minimal)

0

0

0

1

0

0

Jejunum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

5

0

5

5

(minimal)

0

0

0

1

0

0

(slight)

0

0

0

2

0

0

(moderate)

0

0

0

2

0

0

Ileum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

3

0

5

3

(minimal)

0

0

2

2

0

2

(slight)

0

0

0

3

0

0

Skelettal muscle

Fibre degeneration/necrosis

 

 

 

 

 

 

absent

4

5

5

0

4

3

(minimal)

1

0

0

0

1

2

(slight)

0

0

0

4

0

0

(moderate)

0

0

0

1

0

0

Proliferation sarcolemmal nuclei

 

 

 

 

 

 

absent

4

5

3

0

3

0

(minimal)

1

0

2

0

2

3

(slight)

0

0

0

1

0

1

(moderate)

0

0

0

3

0

1

(marked)

0

0

0

1

0

0

FEMALES

Heart

Focal myocarditis

 

 

 

 

 

 

absent

5

4

2

0

2

0

(minimal)

0

1

3

1

3

5

(slight)

0

0

0

4

0

0

Pericardial inflammation and abscess

 

 

 

 

 

 

absent

5

5

5

5

4

5

present

0

0

0

0

1

0

Duodenum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

5

4

5

5

(minimal)

0

0

0

1

0

0

Jejunum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

5

0

5

4

(minimal)

0

0

0

1

0

1

(slight)

0

0

0

3

0

0

(moderate

0

0

0

1

0

0

Ileum

Vacuolation oflamina propria cells

 

 

 

 

 

 

absent

5

5

2

2

5

2

(minimal)

0

0

3

2

0

0

(slight)

0

0

0

1

0

3

Skelettal muscle

Fibre degeneration/necrosis

 

 

 

 

 

 

absent

5

5

5

0

5

4

(minimal)

0

0

0

3

0

0

(slight)

0

0

0

2

0

1

Proliferation sarcolemmal nuclei

 

 

 

 

 

 

absent

5

5

3

0

4

2

(minimal)

0

0

2

1

1

2

(slight)

0

0

0

1

0

1

(moderate)

0

0

0

2

0

0

(marked)

0

0

0

1

0

0

Conclusions:
Oral administration of the test material MXDA/SM Adduct, to rats for a period of twenty-eight consecutive days (according to OECD 407 and GLP) at dose levels of up to 50 mg/kg/day resulted in toxicologically significant effects at 50 and 15 mg/kg/day. However, the majority of effects in 50 mg/kg/day recovery group were reversible, whereas the effects occured in 15 mg/kg/day dose group were marginal and offten equivalent to findings in the control group.Therefore resulting LOAEL is proposed at 50 mg/kg bw/d, and the NOAEL at 15 mg/kg bw/d.
Executive summary:

Methodology

The study was designed to investigate the systemic toxicity of the test material and complies with the following regulatory guidelines:

OECD Guideline for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in the Rodent".

The test material was administered by gavage to three groups, each of five male and five rats, for twenty-eight consecutive days, at dose levels of 5, 15 and 50 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (50 mg/kg/day) or vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, functional observations, bodyweight development, food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

There were no deaths during the study. No effects observed at all dose levels in endpoints water consumption and gross pathology.

For endpoints body weight / weight gain, food comsumtion, food efficiency, haematology, clinical chemistry, urinalysis, neurobehaviour, andorgan weights, effects were reported at the high dose level 50 mg/kg bw/d, however all effects fully reversible.

Concerning clinical signs, increased salivation prior to and/or up to ten minutes after dosing was detected at the 50 mg/kg/day dose level from Day 6 onwards. The physical condition of the animals began to deteriorate during the third week of treatment with the development of clinical signs including hunched posture, pilo-erection, tiptoe gait, waddling gait, increased inhumation, respiratory pattern changes and staining of the external body fur. Observations began to regress following cessation of treatment with only hunched posture reported in recovery 50 mg/kg/day animals by the end of the fourteen day treatment-free period. No such effects were detected at 15 or 5 mg/kg/day.

In histopathologic analysis several-related changes were observed at 50 mg/kg bw/d which all were fully reversible (foamy vacuolation of hepatocytes, generalised hepatocyte enlargement, and vacuolar distension of scattered cells, lymphoid hyperplasia and vacuolar distension of scattered cells with associated apoptosis, highher grades of severity of vacuolation of cortical cells,

hyperplasia of the transitional epithelium, foamy histiocytes in the mesenteric lymph nodes).

A greater incidence of myocarditis was seen in relation to treatment for females dosed at 50 mg/kg/day and possibly at 15 mg/kg/day. There was no toxicologically significant difference in the incidence or severity of myocarditis between recovery control and 50 mg/kg/day animals following completion of the recovery period.

Vacuolation of lamina propria cells in the duodenum, jejunum, and ileum was related to treatment for rats of either sex dosed at 50 mg/kg/day and for the ileum only in a few animals of either sex dosed at 15 mg/kg/day. Appreciable regression of the condition was observed for animals from the recovery 50 mg/kg/day treatment group, with just a few animals showing residual changes in the ileum.

Foamy alveolar macrophages were prevalent throughout the lung parenchyma in animals of either sex dosed at 50 mg/kg/day. There was no evidence of regression of the condition among recovery 50 mg/kg/day group animals following an additional fourteen days without treatment.

Foamy vacuolation of corpora luteal cells was seen in relation to treatment for females dosed at 50 mg/kg/day. Partial regression of the condition was observed for recovery 50 mg/kg/day females.

Muscle fibre degeneration and necrosis, and proliferation of sarcolernrnal nuclei were observed in relation to treatment for animals of either sex dosed at 50 mg/kg/day and to a much lesser extent for animals dosed at 15 mg/kg/day. Significant regression of both conditions was apparent for recovery 50 mg/kg/day animals following an additional fourteen days without treatment.

Conclusions

Finally it is concluded that oral administration of the test material MXDA/SM Adduct, to rats for a period of twenty-eight consecutive days at dose levels of up to 50 mg/kg/day resulted in toxicologically significant effects at 50 and 15 mg/kg/day. However, the majority of effects in 50 mg/kg/day recovery group were reversible, whereas the effects occured in 15 mg/kg/day dose group were marginal and offten equivalent to findings in the control group. No such effects were detected for animals of either sex at 5 mg/kg/day. Therefore resulting LOAEL is proposed at 50 mg/kg bw/d, and the NOAEL at 15 mg/kg bw/d.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study, Klimisch 1
Organ:
ileum
heart
other: Skeletal muscle

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 8 weeks (range finding study) or 8-9 weeks (main study)
- Weight at study initiation: In the range-finding study were 240 and 163 grams for male and female animals, respectively. Mean body weights at the start of treatment in the main study were 261 and 178 grams for male and female animals, respectively.
- Housing: In groups of five of the same sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly.
- Diet: Cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Whitham, England), ad libitum
- Water: Domestic mains tap-water, ad libitum
- Acclimation period: 13 days (range-finding study) or 15 (males) / 16 (females) days (main study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
In the main study, particle size measurement by means of a cascade impactor was not feasible for the low and mid concentration because these concentrations were too low to obtain weighable amounts of material on the filters. Therefore, particle size distribution measurements were carried out using an Aerodynamic Particle Sizer (APS) once weekly and at least once during preliminary generation of the test atmosphere for each exposure condition. At the mid- and high concentration, the results of the APS were compared to particle size measurements obtained using a 10-stage cascade impactor. For the mid-concentration MMAD results were 1.11 and 1.11 μm by APS and 0.86 μm by cascade. Values for the high concentration were as follows (APS / cascade): 1.20 and 1.20 / 0.93 μm on 08 February 2016, 1.22 and 1.23 / 0.89 μm on 23 February 2016, and 1.18 / 0.86 μm on 24 March 2016. Compared to the MMAD by cascade, the mean MMAD by APS was 25% higher (range 23- 27%).
Details on inhalation exposure:
Exposure equipment:
The animals were exposed to the test atmosphere in nose-only inhalation units consisting of a cylindrical polypropylene column (Group 1 main study only; P. Groenendijk Kunststoffen BV) or a stainless steel column (a modification of the design of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom), surrounded by a transparent cylinder. In the range finding study, the columns had a volume of 57 liters (Groups 1 and 4) or 46 liters (Groups 2 and 3) and consisted of a top assembly with the entrance of the unit, one mixing chamber, two (Groups 2 and 3) or three (Groups 1 and 4) rodent tube sections, and at the bottom the base assembly with the exhaust port. In the main study, the columns had volumes of about 48 liters (Group 1), 46 liters (Groups 2 and 3), or 57 liters (Group 4) and consisted of a top assembly with the entrance of the unit, one mixing chamber, two (Groups 1, 2 and 3) or three (Group 4) rodent tube sections, and at the bottom the base assembly with the exhaust port. An additional exposure chamber (stainless steel column with a volume of 45 liters; top and base assembly, one mixing chamber and one rodent tube section) was used in the main study for generation of the starting mixture from which the test atmospheres were prepared. Each rodent tube section had 20 ports for animal exposure. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column. Male and female rats of each group were placed in alternating order (exception: from 29 March 2016 until the end of the exposure period, male and female animals were placed in separate rodent tube sections due to the use of larger sized animal holders for the males). Animals were rotated weekly with respect to their position in the column. Habituation to the restraint in the animal holders was not performed because in our experience habituation does not help to reduce possible stress (Staal et al., 2012). Several empty ports were used for test atmosphere sampling, and measurement of temperature, relative humidity, oxygen and carbon dioxide. The remaining ports were closed. In our experience, the animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animal’s thorax to the nose was avoided. The units were illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the unit was at least 1 liter/min per animal. The air entering the unit was maintained between 22 ± 3˚C and the relative humidity between 30 and 70%.

Generation of the test atmosphere:
- Range finding study: Test atmospheres were obtained by nebulizing the test material using an air-driven atomizer (Schlick type 970/S, Coburg, Germany) placed at the top inlet of the exposure chamber. The amount of test material delivered to the atomizer was controlled using a motor-driven syringe pump (WPI Type SP220i, World Precision Instruments, Sarasota FL, USA). The atomizer was supplied with humidified compressed air, the flow of which was measured using a mass-view meter (Bronkhorst Hi Tec, Ruurlo, the Netherlands) and controlled by a reducing valve. The resulting test atmosphere was directed downward and led to the noses of the animals. In the high-concentration exposure unit (Group 4) extra air was added with a bypass stream of humidified compressed air (using a mass flow controller; Bronkhorst Hi Tec, Ruurlo, the Netherlands) and then led to the noses of the animals. The extra air was added to ensure that the air flow in the exposure unit was sufficiently high. At the bottom of the unit, the test atmosphere was exhausted. The exposure chamber for the control animals (Group 1) was supplied with humidified compressed air only, the flow of which was measured using a massview meter and controlled by a reducing valve.
- Main study: All three test atmospheres (target concentrations 0.15, 0.6 and 3 mg/m3) were obtained by diluting a starting mixture containing approximately 20 mg/m3 of the test material in humidified compressed air. The starting mixture was generated in a similar exposure chamber as used for the test atmospheres (but without animals) by nebulizing a controlled amount of the test material (controlled by a motor-driven syringe pump; WPI Type SP220i, World Precision Instruments, Sarasota FL, USA) using an air-driven atomizer (Schlick type 970/S, Coburg, Germany) placed at the top inlet of the exposure chamber. The test atmospheres were prepared by diluting the starting mixture with humidified compressed air by means of eductors (AirVac Eductor from Air-Vac Engineering Company, Seymour, CT, USA), followed by dilution with a bypass stream of humidified compressed air. For Group 2, the flow of the bypass was controlled by a mass flow controller (Bronkhorst Hi Tec, Ruurlo, the Netherlands). The bypass flow for Groups 3 and 4 was measured using a mass-view meter (Bronkhorst Hi Tec, Ruurlo, the Netherlands) and controlled by a reducing valve. The test atmospheres entered the exposure chamber at the top and from the top they were led to the noses of the animals. The test atmosphere was exhausted at the bottom of the unit. The eductors were calibrated by measuring the total air flow (coming out of the eductor) at a range of driving air pressures encompassing the driving pressures used during the study. To calculate the dilution factor (used for calculation of the nominal concentration), the eductors were calibrated with and without the aspiration air flow. The exposure chamber for the control animals (Group 1) was supplied with humidified compressed air only. This air flow was measured using a mass-view meter and controlled by a reducing valve. The animals were placed in the exposure unit after stabilization of the test atmosphere. Test atmosphere generation and animal exposure were performed in an illuminated laboratory at room temperature.

Analysis of exposure conditions:
- Time to attain chamber equilibration (T95): The time to reach 95% of the steady state concentration (T95) was calculated as: 3V/F. This follows from the formula C = C∞ (1 – e-(FT/V)), describing the increase in concentration C in a perfectly stirred chamber with volume V [L] and flow F [L/min], where T [min] is the time and C∞ is the steady state concentration.
- Nominal test material use and generation efficiency:
-- Range finding study: The daily amount of test material used could not be determined accurately by weighing the syringe containing the test material at the start and end of the exposure period, especially at the low- and mid-concentration. This was due to the low amount of test material required for test atmosphere generation. Instead, the nominal concentration was calculated from the daily consumption of the test material calculated from the pump rate and the daily mean air flow. The test material consumption was calculated from the time weighted average pump rate during the generation period and a correction factor. The correction factor was the ratio of the test material flow at the pump setting used (set pump rate) and the calibrated test material flow (measured pump rate). The calibrated test material flow was calculated from the amount of test material pumped (determined by weighing the syringe containing the test material) and the time between start and end of pumping. Pump rate was calibrated for each test concentration, in duplicate, at the pump rate settings used for test atmosphere generation. Generation efficiency was calculated from the actual concentration (determined gravimetrically) and the nominal concentration (efficiency = actual concentration as percentage of the nominal concentration).
-- Main study: The nominal concentration of the starting mixture was determined by dividing the daily amount of test material used (by weight, expressed in mg/min) by the mean daily air flow (L/min) passed through the inhalation unit in which the starting mixture was generated. The low-, mid- and high-concentration test atmospheres were prepared by diluting the starting mixture. Consequently, nominal concentration of each test atmosphere could not be directly calculated from the volume of air passed through the exposure unit and the daily weighed amount of test material used. Instead, the nominal concentration for the low-, mid- and highconcentration groups was calculated from the actual concentration of the starting mixture and the dilution factor used at test atmosphere generation, i.e. the ratio between the aspirated flow from the starting mixture and the total flow of the test atmosphere through the exposure unit of the group. For each test concentration, the aspirated flow was determined indirectly at calibration of the eductor used to extract test material from the starting mixture. It should be remarked that this indirect measurement of the aspirated flow was conducted at very low pressures, which decreased the accuracy of the measurement. Therefore, the nominal concentration and generation efficiency values calculated for the test concentrations should be considered as rough estimates of these exposure characteristics. Their main use is to assess whether generation efficiency was in line with what can be expected for generation of a liquid aerosol and whether the efficiency, as indicator of the generation process, was stable during the study. Generation efficiency was calculated from the actual concentration (determined gravimetrically) and the nominal concentration (efficiency = actual concentration as percentage of the nominal concentration).
- Particle size:
-- Range finding study: Particle size distribution measurements were carried out using a 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA), once weekly and at least once during preliminary generation of test atmospheres for each concentration (exception: particle size for the high concentration was not measured in the second exposure week because all animals of this test group were sacrificed early on study Day 6). The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated (Lee, 1972). Additionally, particle size distribution during exposure was measured using an Aerodynamic Particle Sizer (APS, model 3321, TSI Incorporated, Shoreview, MN, USA). The APS measurements were conducted on the same dates as those made by the cascade impactor. They were made to establish the relationship between APS and cascade results for possible use in the main study. They were not used due to the different generation procedures used in the range-finding and main study (the dilution step introduced in the main study is known to influence particle size distribution).
-- Main study: See "Remarks on MMAD"
- Total air flow, temperature, relative humidity, oxygen and carbon dioxide concentration: In the range finding study, the total air flow through the exposure units of Groups 1-3 was recorded hourly by recording the readings of the mass view-meters. For Group 4, total air flow was calculated from the hourly readings of the mass-view meter and those of the mass flow controller used for the bypass flow. In the main study, the total air flow through the exposure units of Groups 2-4 consisted of the air flow from the eductor (measured at calibration of the eductors) and the bypass flow (recorded hourly by recording the readings of the mass-view meter (Groups 3 and 4) or mass flow controller (Group 2)). The total air flow of Group 1 (control) and the starting mixture was recorded hourly by recording the readings of the mass-view meter. The temperature and the relative humidity of the test atmospheres were measured continuously at the animals’ breathing zone and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The concentrations of oxygen (Oxygen analyzer type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70 probe with MI70 read-out unit, Vaisala, Helsinki, Finland) in the test atmosphere were measured twice for each group, i.e. during the first and last week of the exposure period (when males and females were present in the exposure unit).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of Gaskamine 240 in the test atmospheres (and in the starting mixture used in the main study) was measured by means of gravimetric analysis. Representative samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) amounts of test atmosphere (or starting mixture) at 4.6 Ln/min through fiber glass filters (Sartorius, 13400-47). In the range finding study, samples of 1542, 308 and 62 Ln1 of test atmosphere were obtained for Groups 2, 3 and 4 respectively. Sample sizes in the main study were 2 x 1518 Ln for Group 2, 1518 Ln for Group 3, 460 Ln for Group 4 and 185 Ln for the starting mixture. Measurements of test atmospheres were conducted at least three times per day during exposure, except for Group 2 of the range finding study and Groups 2 and 3 of the main study. For these groups only one measurement per day could be made. Due to the low test material concentration in these test atmospheres, long sampling times (about 5.5 hours) were necessary to enable collection of an amount of test material on the filter that could be accurately weighed. The concentration in the starting mixture was generally measured twice during exposure. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. For each measurement a new filter was used (exception: for Groups 2 and 3 of the main study samples of up to five exposure days were collected on the same filter because of the low test material concentrations; a new filter was used at the start of each exposure week). During preliminary measurements, it was established that filter weights needed to be corrected for hygroscopy of the test material. Known amounts of test material were applied to glass fiber filters (duplicate measurement: 53.59 and 54.44 mg per filter) and the filters were kept in ambient air until a stable filter weight was reached (filter weight was monitored up to 13 days). The mean percentage of captured (stable) weight on the filters was 113.2% of the applied weight, indicating hygroscopy of the test material. This percentage was used to correct the filter weights recorded after test atmosphere sampling for gravimetric analysis. The actual concentration was calculated by dividing the corrected amount of test material captured on the filter by the total volume (in liters) of the sample taken.
Duration of treatment / exposure:
- Range finding study: 14 days
- Main study: 90 days
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
0.15 mg/m³ air (analytical)
Remarks:
Main study
Dose / conc.:
0.6 mg/m³ air (analytical)
Remarks:
Main study
Dose / conc.:
2.99 mg/m³ air (analytical)
Remarks:
Main study
Dose / conc.:
3.65 mg/m³ air (analytical)
Remarks:
Range finding study
Dose / conc.:
15.6 mg/m³ air (analytical)
Remarks:
Range finding study
Dose / conc.:
76.7 mg/m³ air (analytical)
Remarks:
Range finding study
No. of animals per sex per dose:
- Range finding study: 5
- Main study: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Target concentrations: Range finding study: 0, 3, 15, 75 mg/m3; Main study: 0.15, 0.6, 3 mg/m3
- Dose selection rationale: For the selection of target concentrations for the range finding study the known corrosive properties of the test material and the results of a 28-day oral (gavage) toxicity study in rats with the test material were taken into account (information available on the ECHA website). In the oral 28-day study significant systemic toxicity (in the absence of local adverse effects on the stomach) was observed at 50 mg/kg bw/day while 15 mg/kg bw/day was considered to be a No Observed Adverse Effect Level (NOAEL). A systemic dose level which is non-toxic in a 28-day study might induce systemic toxicity in a 90-day study because of the longer exposure period. Therefore, the oral NOAEL of 15 mg/kg bw/day was taken as starting point to derive a high-concentration for the inhalation study. An oral dose of 15 mg/kg bw/day would be equivalent to a concentration in air of 52 mg/m3 (based on 6 hours inhalation exposure per day, a respiratory volume of 0.8 L/kg bw/min for rats, and similar absorption of the test material by the inhalation and the oral route). The high-concentration for the range finding was set somewhat higher than 52 mg/m3 (namely at 75 mg/m3). Although this exposure level (on a mg/kg bw/day basis) is not much higher than the NOAEL in the oral 28-day study (and might turn out to be a systemic no-effect concentration in the inhalation study), a higher level might cause local adverse effects in the respiratory tract due to the corrosivity of the test material. It should be remarked that the threshold of respiratory tract irritation is not known. Target concentrations for the main study were selected on the basis of the results of the range finding study with the test material.
- Post-exposure recovery period in satellite groups: In the control and high-concentration groups, ten additional males and ten additional females were included (recovery groups), which were exposed similarly and kept for a recovery period of four weeks after the last exposure.
Observations and examinations performed and frequency:
- Clinical signs: On exposure days, each animal was observed daily in the morning, prior to exposure, by cageside observations and, if necessary, handled to detect signs of toxicity. All animals were thoroughly checked again after exposure. During exposure, the animals were also observed about halfway the 6-hour exposure period. On Saturdays, Sundays and public holidays during the main study, all animals were observed for clinical signs in the morning and checked for signs of morbidity and mortality at the end of the day. On weekend days in the range finding study, only one check per day was carried out. During exposure, when observation was hindered by the restraining tubes, attention was directed to breathing abnormalities and restlessness. All abnormalities, signs of ill health, and reactions to treatment were recorded.
- Ophthalmoscopic examination: Ophthalmoscopic examination was not performed in animals of the range finding study. In the main study, ophthalmoscopic observations were made prior to the start of exposure in all animals (on Day -13) and towards the end of the exposure period in the animals of the control group and the high-concentration group (males on Day 86, females on Day 85). Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Since no exposure-related ocular changes were observed, eye examinations were not extended to the animals of the intermediate concentration groups at the end of the exposure period, or to animals of the recovery groups.
- Body weights: Range finding study: The body weight of each animal was recorded once before the start of exposure (Day -5; these pre-test weights served as a basis for animal allocation), once prior to exposure on the first exposure day (Day 0), twice weekly thereafter (Mondays and Fridays), and on the day of scheduled sacrifice (Day 14). The high-concentration animals, sacrificed early on Day 6, were weighed for the last time on the morning of sacrifice. Main study: The body weight of each animal was recorded twice before the start of the exposure period: on Days -11 and -1 (males) or Days -12 and -2 (females). The weights recorded on Days -11 and -12 were used for animal allocation. During the exposure period, the animals were weighed just before exposure on the first day (Day 0), twice a week (Mondays and Fridays) during the first four weeks, and once a week (Fridays) thereafter (body weights were recorded less frequently after Week 4 because no exposure-related changes were seen during the first four weeks). Finally, the animals were weighed on the day before overnight fasting prior to necropsy, and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.
- Food consumption: Food consumption of the animals was measured per cage by weighing the feeders. The results were expressed in g per animal per day. In the range finding study, food consumption of the control, low- and mid-concentration animals was measured over two 7-day periods, starting on Day 0. Food consumption of the high-concentration animals, sacrificed early on Day 6, was measured over the period Day 0-6. In the main study, food consumption was measured from Day 0 over a 3-day (males) or 2-day (females) period in the first week, and subsequently over successive periods of 7 days, until the last week in which food consumption was measured over a 3-day (males) or 4-day period (females).
- Haematology: Haematology was not performed in animals of the range finding study. In the main study, haematology was conducted at the end of the treatment period on all animals of the main groups. Additionally, due to (possible) treatment-related changes at the end of treatment, haematology was conducted at the end of the recovery period in all animals of the recovery groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anaesthesia at sacrifice. Citrate (for prothrombin time only) or EDTA was used as anticoagulant. The samples were discarded after analysis. In each sample the following determinations were carried out haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell, count (lymphocytes, neutrophils, eosinophils, basophils, monocytes), prothrombin time (PT), thrombocyte count (platelet count). The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).
- Clinical chemistry: Clinical chemistry was not performed in animals of the range finding study. In the main study, clinical chemistry was conducted at the end of the treatment period on all animals of the main groups. Additionally, due to (possible) treatment-related changes at the end of treatment, clinical chemistry was conducted at the end of the recovery period in all animals of the recovery groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anaesthesia at sacrifice. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. After analysis, remaining plasma was stored frozen (<-18 °C) to enable reanalysis if necessary and then discarded. The following measurements were made in the plasma: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate.
Sacrifice and pathology:
Range finding study:
- Sacrifice and macroscopic examination: At the end of the exposure period, the surviving animals were sacrificed in such a sequence that the average time of killing was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes. A thorough necropsy was also performed on all high-concentration animals which were sacrificed early after exposure on Day 6 due to conditional decline.
- Organ weights: At scheduled sacrifice, the following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. No organ weights were recorded for the high-concentration animals sacrificed early on Day 6. Relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, heart, kidneys, liver, lung with trachea and larynx (After weighing, the lung was infused with the fixative), spleen, testes
- Tissue preservation: The complete respiratory tract of all animals and all gross lesions were preserved in a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent). The lungs (after weighing) were infused with the fixative uncer ca. 15 cm water pressure to ensure fixation. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded.
- Slide preparation: Tissue for microscopy was embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. The tissues of groups 1, 2, 3 and 4 were processed concurrently.
- Histopathological examination: All preserved tissues of all animals of the control group and the high-concentration group were examined histopathologically (by light microscopy). Additionally, all gross lesions observed in rats of the intermediate concentration groups were examined microscopically. Further, microscopic examination of the nasopharyngeal tissues, larynx, trachea and lungs was extend to all animals of the low- and mid-concentration groups due to treatment-related changes observed in the nose and larynx of high-concentration animals. Although no treatment-related alterations were noted in the trachea and lungs of high concentration animals, microscopy of these organs was extended to the low- and mid concentration animals for scientific reasons. The absence of visible treatment-related alterations in the trachea and lungs of high- concentration animals could have been due to their early termination (on Day 6 instead of on Day 14). The nasopharyngeal tissues were examined at six levels (Woutersen et al., 1994) with one level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT), the larynx at three levels (one level to include the base of the epiglottis), the trachea at three levels (including a longitudinal section through the carina of the bifurcation), and each lung lobe at one level.
Main study:
- Sacrifice and macroscopic examination: At the end of the exposure period (Day 98; main group males and females on 24 and 25 May 2016, respectively) or the recovery period (Day 126; recovery group males and females on 21 and 22 June 2016, respectively), the animals were sacrificed in such a sequence that the average time of killing was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes.
- Organ weights: At scheduled sacrifice, the following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, brain, epididymides, heart, kidneys, liver, lung with trachea and larynx (After weighing, the lung was infused with the fixative), ovaries, spleen, testes, thymus, thyroid, uterus
- Tissue preservation Samples of the following tissues and organs of all animals (main and recovery groups) were preserved in a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded. Adrenals, aorta, axillary lymph nodes, brain (three levels were examined microscopically: brain stem, cerebrum, cerebellum), caecum, colon, epididymides, eyes (with optic nerve), exorbital lachrymal glands, femur with joint, Harderian glands, heart, kidneys, liver, lungs (Each lung lobe was examined microscopically at one level)/trachea (Three levels were examined microscopically: including a longitudinal section through the carina of the bifurcation)/larynx (Three levels; one including the base of the epiglottis; were examined microscopically), mammary glands (females), cervical lymph nodes, nasopharyngeal tissue (with teeth) (Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [nose associated lymphoid tissue, NALT]), nerve peripheral (sciatic nerve), oesophagus, olfactory bulb, ovaries, pancreas, parathyroids, pharynx, parotid salivary glands, pituitary, prostate, rectum, seminal vesicles with coagulating glands, skeletal muscle (thigh), skin (flank), small intestines (duodenum, ileum, jejunum), spinal cord (Retained in vertebral column, at least three levels were examined microscopically: cervical, mid-thoracic and lumbar), spleen, sternum with bone marrow, stomach (Non-glandular and glandular parts were examined microscopically), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, tongue, tracheobronchial (mediastinal) lymph nodes, ureter, urethra, urinary bladder, uterus (with cervix), all gross lesions.
- Slide preparation: Tissues to be examined were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Unless required for histopathological examination, the tissues of the animals of the low- and mid-concentration groups (main Groups 2 and 3) and the recovery groups (recovery Groups 1 and 4) were not processed. The noses of the animals of main Groups 2 and 3 were decalcified and embedded in paraffin concurrently with the noses of the animals of main Groups 1 (control) and 4 (high-concentration).
- Histopathological examination: All preserved tissues of all animals of the control and high-concentration main groups were examined histopathologically (by light microscopy). In addition, all gross lesions observed in rats of the low- and mid-concentration groups were examined microscopically. Further, microscopic examination of the nasopharyngeal tissues, larynx and trachea was extended to all animals of the low- and mid-concentration groups and the recovery groups. These tissues were examined at the same levels as those examined in the animals of the control and highconcentration main groups.
Statistics:
See "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were observed up to highest concentration (3 mg/m3). The only finding of note was the presence of soiled fur towards the end of the treatment period (from Day 90) in a few treated females: one at 0.15 mg/m3, five at 0.6 mg/m3 and six at 3 mg/m3 (including four of the recovery group). From Day 121-122 (fourth week of the recovery period), soiled fur was no longer observed. In the absence of corroborative signs of toxicity, this finding was considered not to be toxicologically relevant. A few other clinical signs were noted incidentally (observations of the skin/fur or tail) represented background findings which were unrelated to treatment with the test item. No abnormalities were seen at the group-wise observations made about halfway each 6-hour exposure period.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in body weight up to the highest concentration (3 mg/m3). Occasional, statistically significant differences noted at the low- or midconcentration reflected normal biological variation and were unrelated to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by exposure to the test material up to the highest concentration (3 mg/m3). An occasional, statistically significant difference noted at 3 mg/m3 in females (Day 93-100) reflected normal biological variation and was unrelated to treatment.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination did not reveal any exposure-related abnormalities.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, statistically significant differences between animals exposed to the test material and controls were limited to lower mean values for the absolute number and percentage of monocytes at the high-concentration in males. Monocyte values at the end of the recovery period showed no differences between high-concentration animals and controls. As the differences at the end of the treatment period were small (absolute numbers of monocytes in high-concentration males were generally in the range of concurrent control values), they were considered not to be toxicologically relevant and probably unrelated to treatment. At the end of the recovery period, haematology parameters in the high-concentration group showed the following statistically significant differences compared to recovery controls: higher MCV (males); higher total white blood cell count (females); higher absolute number of lymphocytes (females); and lower percentage of eosinophils (females). The differences from controls were small and within normal limits (values in high-concentration recovery animals were in the range of the concurrent control values measured at the end of the treatment period). Moreover, these parameters were neither affected at the end of the treatment period nor in the opposite sex. Therefore, the slight differences noted at the end of the recovery period were considered to be unrelated to treatment and not toxicologically relevant.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, clinical chemistry parameters showed the following statistically significant differences between animals exposed to the test material and controls: lower mean plasma level of urea at the high-concentration in males, and lower fasting glucose at the mid- and high-concentration in females. Urea and glucose levels at the end of the recovery period showed no differences between high-concentration animals and controls. The differences at the end of the treatment period were small (values in high-dose animals were within or close to the concurrent control range) and not corroborated by changes in other parameters examined in this study. The fasting glucose levels showed no clear dose-related response (values at the mid- and high-concentration were similar despite the five-fold difference between the mid- and high-concentration). Moreover, toxicity is generally indicated by increases rather than decreases in urea and fasting glucose. Therefore, the slight differences in urea and fasting glucose at the end of the treatment period were considered not to be toxicologically relevant and unrelated to treatment. At the end of the recovery period, clinical chemistry parameters in the high-concentration group showed the following statistically significant differences compared to recovery controls: higher plasma levels of total protein and calcium (both in males). These isolated, minor differences were considered to be chance findings reflecting normal biological variation.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, organ weight data showed the following statistically significant differences between animals exposed to the test material and controls: higher relative brain weight in the low- and high-concentration group (females), and higher absolute and relative adrenal weight in the high-concentration group (males). The differences in brain weight were considered to be unrelated to treatment because there was no concentrationrelated response. The increase in the weight of the adrenals was fully reversible and considered not to be toxicologically significant because there were no histopathological changes in the adrenals or relevant changes in other parameters. At the end of the recovery period, statistically significant differences were noted in the absolute weights of adrenals (lower in the high-concentration group of males) and lung (higher in the high-concentration group of females). In the absence of significant changes in the relative weights of these organs, these slight differences in absolute organ weights were considered to be chance findings reflecting normal biological variation.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy, no treatment related gross changes were observed in the animals of the main and recovery groups. The few gross changes observed represented background pathology in rats of this strain and age and/or occurred only incidentally.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Main Groups: Microscopic examination revealed treatment-related histopathological changes in the upper airways (nose, larynx and trachea). The histopathological changes in the upper airways were observed in all three treatment groups. In general, the incidence and/or severity of the histopathological changes clearly increased with increasing concentration of the test material, indicative of a concentration-effect relationship. The histopathological changes in the nose were characterised by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation. The inflammation was called mixed because of the presence of a variety of inflammatory cells: polymorphonuclear cells, lymphocytes, macrophages, plasma cells, granulated intra-epithelial cells and globular leukocytes. These mixed inflammations were considered related to the treatment with the test material. In some animals focal mononuclear inflammations were observed, but that type of inflammation is a common finding in the nose. It is considered part of the background pathology and not related to treatment. Although the treatment-related histopathological changes were observed in all levels of the nose, it was clear that the rostral levels were most affected and the caudal levels were somewhat less affected. The changes were predominantly seen in the rostroventral parts of the nose, specifically the ventral meatus and nasal septum. Consequently, because of the typical distribution of the different epithelial cell types covering the nasal cavity, most histopathological changes were observed in areas covered by squamous or respiratory epithelium, whereas the olfactory epithelium (predominantly covering the dorsocaudal parts of the nasal cavity) was hardly affected. Epithelial hyperplasia was occasionally seen and considered a reactive process related to the inflammation. The amount of hyaline droplets (visible as highly eosinophilic globular structures scattered through the epithelial layer and occasionally seen in normal epithelium of the respiratory tract) was considerably increased in treated animals. In most cases these droplets were seen in close relation to the epithelium affected by the inflammatory process, but in the hind levels 5 and 6 of the nose the hyaline droplet accumulation was also seen in olfactory epithelium not affected by inflammation. The histopathological changes in the larynx were characterised by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation, generally comparable to the changes in the nose. In addition, several high-concentration animals showed epithelial ulceration. In some animals focal mononuclear inflammations were observed, but that type of inflammation is a common finding in the larynx. It is considered part of background pathology and not related to treatment. In all cases the treatment related histopathological changes were located at the epiglottis and with increasing severity, extended to the ventral pouch and other parts of the epiglottis and even the proximal part of the trachea. The caudal part of the trachea and the bronchi were generally not affected. However, in several high-concentration animals and in one mid-concentration male focal accumulation of macrophages was seen in the carina. Although the incidence of this finding was not statistically significantly different from that in controls, this change is not a common background finding and is likely to be related to the treatment. The histopathological changes observed in the lungs and other organs and tissues were considered part of background pathology and not related to treatment. These findings occurred in only one or a few animals and/or at random incidences in the different groups.
Microscopic examination Recovery Groups: Because of the histopathological changes observed in the main groups at the end of the treatment period, microscopic examination was extended to the nose, larynx and trachea of the recovery groups. In general, the incidence and severity of the histopathological changes in the high-concentration recovery animals had largely subsided in the nose and the larynx and were absent in the trachea. However, recovery was not complete after the 4-week treatment-free period.
Details on results:
Analysis of exposure conditions
- Actual concentration: The overall mean actual concentration (± standard deviation) of Gaskamine 240 in the test atmospheres as determined gravimetrically was 0.15 (± 0.02), 0.60 (± 0.06) and 2.99 (± 0.37) mg/m3 for the low-, mid- and high-concentration, respectively. These mean actual concentrations were very close to the respective target concentrations of 0.15, 0.60 and 3.0 mg/m3. The overall mean actual concentration (± standard deviation) of the starting mixture was 20.0 (± 3.8) mg/m3.
- Time to attain chamber equilibration (T95): The time to reach 95% of the steady state concentration (T95), based on chamber volume and the total air flow range was calculated to be about 6 minutes for Group 2 and about 4 minutes four Groups 3 and 4. Since the test atmospheres for these groups were obtained by diluting the starting mixture (for which T95 was about 5 minutes), actual T95 of the test atmospheres was slightly longer than these calculated values. The animals were placed in the exposure units at least 12 minutes after the start of atmosphere generation.
- Nominal test material use and generation efficiency: The mean nominal concentration of the starting mixture (± standard deviation) was 53.9 ± 8.2 mg/m3, indicating a generation efficiency of 37%. This generation efficiency is in the range to be expected for test atmosphere generation from a liquid test material. As explained before, the nominal concentrations calculated for the test atmospheres were rough estimates rather than accurate determinations. The overall mean nominal concentration (± standard deviation) was 0.38 (± 0.07), 0.94 (± 0.18) and 6.54 (± 1.41) mg/m3 for the low-, mid- and high-concentration, respectively. The corresponding mean generation efficiencies were 41%, 66% and 47%, respectively, which is in line with what can be expected for generation of liquid aerosols.
- Particle size: The overall mean (± standard deviation) mass median aerodynamic diameter (MMAD) of the test aerosol as measured by APS was 1.09 (± 0.05), 1.16 (± 0.04) and 1.13 ± (0.05) μm for the low-, mid- and high-concentration group, respectively. The corresponding mean (± standard deviation) geometric standard deviations (gsd) were 1.58 (± 0.04), 1.64 (± 0.04) and 1.61 (± 0.02) for the low-, mid- and high-concentration group, respectively.
- Total air flow, temperature, relative humidity, oxygen and carbon dioxide concentration: The overall mean total air flows (± standard deviation) were 50.5 (± 3.0), 26.3 (± 3.1), 54.7 (± 4.5) and 51.5 (± 5.6) L/min for the control, low-, mid- and high concentration group, respectively. The mean air flow in the chamber of the starting mixture was 34.3 (± 2.8) L/min. Measured temperatures during exposure were generally within the range of 19-25˚C. Occasionally, the temperature slightly exceeded these target limits (lowest value: 18.5˚C, highest value: 25.7˚C). The overall mean temperature (± standard deviation) was 23.2 (± 0.8), 21.5 (± 0.3), 21.5 (± 0.4) and 22.6 (± 0.4) ˚C for the control, low-, mid- and high-concentration group, respectively. The relative humidity during exposure generally remained within the range 30-70%. The humidity in the chamber of the control group occasionally exceeded this range (lowest value: 24.5% v/v; highest value 80.5%). The overall mean relative humidity during exposure was 45.1 (± 3.6), 48.6 (± 3.8), 39.4 (± 1.0) and 39.5 (± 1.7) % for the control, low-, mid- and high-concentration group, respectively. The oxygen concentrations measured in the exposure chambers was in the range 20.0 – 20.5% v/v. These concentrations meet the requirement described in OECD guideline 413 (i.e. >19% oxygen). The carbon dioxide concentrations measured in the exposure chambers were in the range 0.282 – 0.684% v/v. These concentrations meet the requirement described in OECD guideline 413 (i.e. <1% carbon dioxide).
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
2.99 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed up to and including the highest dose tested
Key result
Dose descriptor:
LOAEC
Remarks:
Local
Effect level:
0.15 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.15 mg/m³ air (analytical)
System:
respiratory system: upper respiratory tract
Organ:
larynx
nasal cavity
trachea
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Short summary range finding study: Exposure at 75 mg/m3 exceeded the maximum tolerable concentration, leading to early termination of this test group at the end of the fifth exposure day. Exposure to 15 mg/m3 was associated with respiratory difficulties, body weight loss, decreased food consumption, higher spleen and heart weights, and toxicologically relevant histopathological changes in the larynx and nose. The main treatment-related finding at 3 mg/m3 consisted of histopathological changes in the larynx and, to a lesser extent, the nose. Additional findings at 3 mg/m3 consisted of a slightly lower body weight gain in males and higher spleen weight in females. The histopathological changes noted in the nose and larynx at 3 and 15 mg/m3 showed a dose-related response, except for mixed cell inflammation and ulceration in the larynx.

Conclusions:
Under the conditions of this study, inhalation exposure to Gaskamine 240 up to 2.99 mg/m3 did not result in systemic toxicity. Based on this result, the NOAEC for systemic toxicity was at least 2.99 mg/m3. As toxicologically relevant local effects were noted at all concentrations tested, a NOAEC for local toxicity could not be established. The lowest concentration tested in this study, 0.15 mg/m3 (actual concentration), was a LOAEC for local toxicity.
Executive summary:

In a inhalation sub-chronic (90-day) toxicity performed in accordance with OECD Guideline 413 and GLP, Wistar Hannover rats were exposed to the test substance. Four main groups of 10 male and 10 female rats each were exposed (nose-only) to target concentrations of 0 (control), 0.15, 0.6 or 3 mg/m3 for 6 hours/day, 5 days/week over a 14-week period (65 exposure days). Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of 10 male and 10 female animals each, were simultaneously exposed with the main group animals to the control or 3 mg/m3 test atmospheres, and were sacrificed after a 4-week treatment-free period following the last exposure. Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were macroscopically examined at sacrifice, and a large number of organs and tissues were examined microscopically. The concentrations to be tested in the sub-chronic study were selected on the basis of a 14- day range finding study in which groups of five male and five female Wistar Hannover rats were exposed to target concentrations of 3, 15 and 75 mg/m3 for 6 hours/day, 5 days/week. The target concentrations were accurately achieved as demonstrated by the results of the gravimetric analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation) were 0.15 (± 0.02), 0.60 (± 0.06) and 2.99 (± 0.37) mg/m3 for the low-, mid- and high-concentration groups, respectively. All animals survived until scheduled necropsy. Clinical observations revealed no treatment-related signs of toxicity. Soiled fur was noted in some treated females but considered not to be toxicologically relevant. No abnormalities were noted at the observations made about halfway through the 6-hour exposure period. Ophthalmoscopic examination did not reveal any treatment-related ocular abnormalities. There were no treatment-related changes in body weight or food consumption. Haematology and clinical chemistry parameters were not adversely affected by the exposure to the test material. Organ weight data showed an increase in adrenal weight (absolute and relative to body weight) at the high-concentration in males. This change was fully reversible, and in the absence of histopathological correlates or relevant changes in other parameters, considered not to be toxicologically relevant. Macroscopic examination at scheduled termination revealed no exposure-related gross changes. Microscopic examination revealed histopathological changes in the upper airways (nose, larynx and trachea) of male and female rats of the low-, mid- and high-concentration groups. In general, the incidence and/or severity of the changes increased with increasing concentration. The changes in the nose were characterized by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation. The mixed inflammation was characterized by the presence of a variety of inflammatory cells: polymorphonuclear cells, lymphocytes, macrophages, plasma cells, granulated intra-epithelial cells and globular leukocytes. Although the nasal changes were seen at all six nose levels examined, the rostral levels were most affected and the caudal levels were somewhat less affected. The changes were predominantly seen in the rostroventral parts of the nose, specifically the ventral meatus and nasal septum. Consequently, because of the typical distribution of the different epithelial cell types covering the nasal cavity, most histopathological changes were observed in areas covered by squamous or respiratory epithelium, whereas the olfactory epithelium (predominantly covering the dorsocaudal parts of the nasal cavity) was hardly affected. Epithelial hyperplasia was occasionally seen and considered a reactive process related to the inflammation. The amount of hyaline droplets was considerably increased in treated animals. In most cases these droplets occurred in close relation to the epithelium affected by the inflammatory process, but in the hind levels 5 and 6 of the nose the hyaline droplet accumulation was also seen in olfactory epithelium unaffected by inflammation. The histopathological changes in the larynx were characterised by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation, generally comparable to the changes in the nose. In addition, several high-concentration animals showed epithelial ulceration. The changes were located at the epiglottis and with increasing severity, extended to the ventral pouch and other parts of the epiglottis and even the proximal part of the trachea. The caudal part of the trachea and the bronchi were generally not affected. However, in several high-concentration animals and in one mid-concentration male focal accumulation of macrophages was seen in the carina. As this change is not a common background finding, it was likely to be treatment-related. The above local effects in the upper airways were considered to be related to the corrosive properties of the test material. Under the conditions of this study, inhalation exposure to 2.99 mg/m3 (actual concentration) did not result in systemic toxicity. Based on this result, the NOAEC for systemic toxicity was at least 2.99 mg/m3 (actual concentration). Adverse local effects, consisting of histopathological changes in the upper airways (nose, larynx, trachea), occurred at all three concentrations tested. The incidence and/or severity of the histopathological changes generally showed a dose-related response. The changes in the trachea were fully reversible after the 4-week treatment-free period. The incidence and severity of the changes in the nose and larynx decreased considerably after cessation of exposure but recovery was not complete at the end of the 4-week treatment-free period. As toxicologically relevant local effects were noted at all concentrations tested, a NOAEC for local toxicity could not be established. The lowest concentration tested in this study, 0.15 mg/m3 (actual concentration), was a LOAEC for local toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2.99 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP study, Klimisch 1

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 8 weeks (range finding study) or 8-9 weeks (main study)
- Weight at study initiation: In the range-finding study were 240 and 163 grams for male and female animals, respectively. Mean body weights at the start of treatment in the main study were 261 and 178 grams for male and female animals, respectively.
- Housing: In groups of five of the same sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly.
- Diet: Cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Whitham, England), ad libitum
- Water: Domestic mains tap-water, ad libitum
- Acclimation period: 13 days (range-finding study) or 15 (males) / 16 (females) days (main study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
In the main study, particle size measurement by means of a cascade impactor was not feasible for the low and mid concentration because these concentrations were too low to obtain weighable amounts of material on the filters. Therefore, particle size distribution measurements were carried out using an Aerodynamic Particle Sizer (APS) once weekly and at least once during preliminary generation of the test atmosphere for each exposure condition. At the mid- and high concentration, the results of the APS were compared to particle size measurements obtained using a 10-stage cascade impactor. For the mid-concentration MMAD results were 1.11 and 1.11 μm by APS and 0.86 μm by cascade. Values for the high concentration were as follows (APS / cascade): 1.20 and 1.20 / 0.93 μm on 08 February 2016, 1.22 and 1.23 / 0.89 μm on 23 February 2016, and 1.18 / 0.86 μm on 24 March 2016. Compared to the MMAD by cascade, the mean MMAD by APS was 25% higher (range 23- 27%).
Details on inhalation exposure:
Exposure equipment:
The animals were exposed to the test atmosphere in nose-only inhalation units consisting of a cylindrical polypropylene column (Group 1 main study only; P. Groenendijk Kunststoffen BV) or a stainless steel column (a modification of the design of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom), surrounded by a transparent cylinder. In the range finding study, the columns had a volume of 57 liters (Groups 1 and 4) or 46 liters (Groups 2 and 3) and consisted of a top assembly with the entrance of the unit, one mixing chamber, two (Groups 2 and 3) or three (Groups 1 and 4) rodent tube sections, and at the bottom the base assembly with the exhaust port. In the main study, the columns had volumes of about 48 liters (Group 1), 46 liters (Groups 2 and 3), or 57 liters (Group 4) and consisted of a top assembly with the entrance of the unit, one mixing chamber, two (Groups 1, 2 and 3) or three (Group 4) rodent tube sections, and at the bottom the base assembly with the exhaust port. An additional exposure chamber (stainless steel column with a volume of 45 liters; top and base assembly, one mixing chamber and one rodent tube section) was used in the main study for generation of the starting mixture from which the test atmospheres were prepared. Each rodent tube section had 20 ports for animal exposure. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column. Male and female rats of each group were placed in alternating order (exception: from 29 March 2016 until the end of the exposure period, male and female animals were placed in separate rodent tube sections due to the use of larger sized animal holders for the males). Animals were rotated weekly with respect to their position in the column. Habituation to the restraint in the animal holders was not performed because in our experience habituation does not help to reduce possible stress (Staal et al., 2012). Several empty ports were used for test atmosphere sampling, and measurement of temperature, relative humidity, oxygen and carbon dioxide. The remaining ports were closed. In our experience, the animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animal’s thorax to the nose was avoided. The units were illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the unit was at least 1 liter/min per animal. The air entering the unit was maintained between 22 ± 3˚C and the relative humidity between 30 and 70%.

Generation of the test atmosphere:
- Range finding study: Test atmospheres were obtained by nebulizing the test material using an air-driven atomizer (Schlick type 970/S, Coburg, Germany) placed at the top inlet of the exposure chamber. The amount of test material delivered to the atomizer was controlled using a motor-driven syringe pump (WPI Type SP220i, World Precision Instruments, Sarasota FL, USA). The atomizer was supplied with humidified compressed air, the flow of which was measured using a mass-view meter (Bronkhorst Hi Tec, Ruurlo, the Netherlands) and controlled by a reducing valve. The resulting test atmosphere was directed downward and led to the noses of the animals. In the high-concentration exposure unit (Group 4) extra air was added with a bypass stream of humidified compressed air (using a mass flow controller; Bronkhorst Hi Tec, Ruurlo, the Netherlands) and then led to the noses of the animals. The extra air was added to ensure that the air flow in the exposure unit was sufficiently high. At the bottom of the unit, the test atmosphere was exhausted. The exposure chamber for the control animals (Group 1) was supplied with humidified compressed air only, the flow of which was measured using a massview meter and controlled by a reducing valve.
- Main study: All three test atmospheres (target concentrations 0.15, 0.6 and 3 mg/m3) were obtained by diluting a starting mixture containing approximately 20 mg/m3 of the test material in humidified compressed air. The starting mixture was generated in a similar exposure chamber as used for the test atmospheres (but without animals) by nebulizing a controlled amount of the test material (controlled by a motor-driven syringe pump; WPI Type SP220i, World Precision Instruments, Sarasota FL, USA) using an air-driven atomizer (Schlick type 970/S, Coburg, Germany) placed at the top inlet of the exposure chamber. The test atmospheres were prepared by diluting the starting mixture with humidified compressed air by means of eductors (AirVac Eductor from Air-Vac Engineering Company, Seymour, CT, USA), followed by dilution with a bypass stream of humidified compressed air. For Group 2, the flow of the bypass was controlled by a mass flow controller (Bronkhorst Hi Tec, Ruurlo, the Netherlands). The bypass flow for Groups 3 and 4 was measured using a mass-view meter (Bronkhorst Hi Tec, Ruurlo, the Netherlands) and controlled by a reducing valve. The test atmospheres entered the exposure chamber at the top and from the top they were led to the noses of the animals. The test atmosphere was exhausted at the bottom of the unit. The eductors were calibrated by measuring the total air flow (coming out of the eductor) at a range of driving air pressures encompassing the driving pressures used during the study. To calculate the dilution factor (used for calculation of the nominal concentration), the eductors were calibrated with and without the aspiration air flow. The exposure chamber for the control animals (Group 1) was supplied with humidified compressed air only. This air flow was measured using a mass-view meter and controlled by a reducing valve. The animals were placed in the exposure unit after stabilization of the test atmosphere. Test atmosphere generation and animal exposure were performed in an illuminated laboratory at room temperature.

Analysis of exposure conditions:
- Time to attain chamber equilibration (T95): The time to reach 95% of the steady state concentration (T95) was calculated as: 3V/F. This follows from the formula C = C∞ (1 – e-(FT/V)), describing the increase in concentration C in a perfectly stirred chamber with volume V [L] and flow F [L/min], where T [min] is the time and C∞ is the steady state concentration.
- Nominal test material use and generation efficiency:
-- Range finding study: The daily amount of test material used could not be determined accurately by weighing the syringe containing the test material at the start and end of the exposure period, especially at the low- and mid-concentration. This was due to the low amount of test material required for test atmosphere generation. Instead, the nominal concentration was calculated from the daily consumption of the test material calculated from the pump rate and the daily mean air flow. The test material consumption was calculated from the time weighted average pump rate during the generation period and a correction factor. The correction factor was the ratio of the test material flow at the pump setting used (set pump rate) and the calibrated test material flow (measured pump rate). The calibrated test material flow was calculated from the amount of test material pumped (determined by weighing the syringe containing the test material) and the time between start and end of pumping. Pump rate was calibrated for each test concentration, in duplicate, at the pump rate settings used for test atmosphere generation. Generation efficiency was calculated from the actual concentration (determined gravimetrically) and the nominal concentration (efficiency = actual concentration as percentage of the nominal concentration).
-- Main study: The nominal concentration of the starting mixture was determined by dividing the daily amount of test material used (by weight, expressed in mg/min) by the mean daily air flow (L/min) passed through the inhalation unit in which the starting mixture was generated. The low-, mid- and high-concentration test atmospheres were prepared by diluting the starting mixture. Consequently, nominal concentration of each test atmosphere could not be directly calculated from the volume of air passed through the exposure unit and the daily weighed amount of test material used. Instead, the nominal concentration for the low-, mid- and highconcentration groups was calculated from the actual concentration of the starting mixture and the dilution factor used at test atmosphere generation, i.e. the ratio between the aspirated flow from the starting mixture and the total flow of the test atmosphere through the exposure unit of the group. For each test concentration, the aspirated flow was determined indirectly at calibration of the eductor used to extract test material from the starting mixture. It should be remarked that this indirect measurement of the aspirated flow was conducted at very low pressures, which decreased the accuracy of the measurement. Therefore, the nominal concentration and generation efficiency values calculated for the test concentrations should be considered as rough estimates of these exposure characteristics. Their main use is to assess whether generation efficiency was in line with what can be expected for generation of a liquid aerosol and whether the efficiency, as indicator of the generation process, was stable during the study. Generation efficiency was calculated from the actual concentration (determined gravimetrically) and the nominal concentration (efficiency = actual concentration as percentage of the nominal concentration).
- Particle size:
-- Range finding study: Particle size distribution measurements were carried out using a 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA), once weekly and at least once during preliminary generation of test atmospheres for each concentration (exception: particle size for the high concentration was not measured in the second exposure week because all animals of this test group were sacrificed early on study Day 6). The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated (Lee, 1972). Additionally, particle size distribution during exposure was measured using an Aerodynamic Particle Sizer (APS, model 3321, TSI Incorporated, Shoreview, MN, USA). The APS measurements were conducted on the same dates as those made by the cascade impactor. They were made to establish the relationship between APS and cascade results for possible use in the main study. They were not used due to the different generation procedures used in the range-finding and main study (the dilution step introduced in the main study is known to influence particle size distribution).
-- Main study: See "Remarks on MMAD"
- Total air flow, temperature, relative humidity, oxygen and carbon dioxide concentration: In the range finding study, the total air flow through the exposure units of Groups 1-3 was recorded hourly by recording the readings of the mass view-meters. For Group 4, total air flow was calculated from the hourly readings of the mass-view meter and those of the mass flow controller used for the bypass flow. In the main study, the total air flow through the exposure units of Groups 2-4 consisted of the air flow from the eductor (measured at calibration of the eductors) and the bypass flow (recorded hourly by recording the readings of the mass-view meter (Groups 3 and 4) or mass flow controller (Group 2)). The total air flow of Group 1 (control) and the starting mixture was recorded hourly by recording the readings of the mass-view meter. The temperature and the relative humidity of the test atmospheres were measured continuously at the animals’ breathing zone and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The concentrations of oxygen (Oxygen analyzer type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70 probe with MI70 read-out unit, Vaisala, Helsinki, Finland) in the test atmosphere were measured twice for each group, i.e. during the first and last week of the exposure period (when males and females were present in the exposure unit).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of Gaskamine 240 in the test atmospheres (and in the starting mixture used in the main study) was measured by means of gravimetric analysis. Representative samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) amounts of test atmosphere (or starting mixture) at 4.6 Ln/min through fiber glass filters (Sartorius, 13400-47). In the range finding study, samples of 1542, 308 and 62 Ln1 of test atmosphere were obtained for Groups 2, 3 and 4 respectively. Sample sizes in the main study were 2 x 1518 Ln for Group 2, 1518 Ln for Group 3, 460 Ln for Group 4 and 185 Ln for the starting mixture. Measurements of test atmospheres were conducted at least three times per day during exposure, except for Group 2 of the range finding study and Groups 2 and 3 of the main study. For these groups only one measurement per day could be made. Due to the low test material concentration in these test atmospheres, long sampling times (about 5.5 hours) were necessary to enable collection of an amount of test material on the filter that could be accurately weighed. The concentration in the starting mixture was generally measured twice during exposure. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. For each measurement a new filter was used (exception: for Groups 2 and 3 of the main study samples of up to five exposure days were collected on the same filter because of the low test material concentrations; a new filter was used at the start of each exposure week). During preliminary measurements, it was established that filter weights needed to be corrected for hygroscopy of the test material. Known amounts of test material were applied to glass fiber filters (duplicate measurement: 53.59 and 54.44 mg per filter) and the filters were kept in ambient air until a stable filter weight was reached (filter weight was monitored up to 13 days). The mean percentage of captured (stable) weight on the filters was 113.2% of the applied weight, indicating hygroscopy of the test material. This percentage was used to correct the filter weights recorded after test atmosphere sampling for gravimetric analysis. The actual concentration was calculated by dividing the corrected amount of test material captured on the filter by the total volume (in liters) of the sample taken.
Duration of treatment / exposure:
- Range finding study: 14 days
- Main study: 90 days
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
0.15 mg/m³ air (analytical)
Remarks:
Main study
Dose / conc.:
0.6 mg/m³ air (analytical)
Remarks:
Main study
Dose / conc.:
2.99 mg/m³ air (analytical)
Remarks:
Main study
Dose / conc.:
3.65 mg/m³ air (analytical)
Remarks:
Range finding study
Dose / conc.:
15.6 mg/m³ air (analytical)
Remarks:
Range finding study
Dose / conc.:
76.7 mg/m³ air (analytical)
Remarks:
Range finding study
No. of animals per sex per dose:
- Range finding study: 5
- Main study: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Target concentrations: Range finding study: 0, 3, 15, 75 mg/m3; Main study: 0.15, 0.6, 3 mg/m3
- Dose selection rationale: For the selection of target concentrations for the range finding study the known corrosive properties of the test material and the results of a 28-day oral (gavage) toxicity study in rats with the test material were taken into account (information available on the ECHA website). In the oral 28-day study significant systemic toxicity (in the absence of local adverse effects on the stomach) was observed at 50 mg/kg bw/day while 15 mg/kg bw/day was considered to be a No Observed Adverse Effect Level (NOAEL). A systemic dose level which is non-toxic in a 28-day study might induce systemic toxicity in a 90-day study because of the longer exposure period. Therefore, the oral NOAEL of 15 mg/kg bw/day was taken as starting point to derive a high-concentration for the inhalation study. An oral dose of 15 mg/kg bw/day would be equivalent to a concentration in air of 52 mg/m3 (based on 6 hours inhalation exposure per day, a respiratory volume of 0.8 L/kg bw/min for rats, and similar absorption of the test material by the inhalation and the oral route). The high-concentration for the range finding was set somewhat higher than 52 mg/m3 (namely at 75 mg/m3). Although this exposure level (on a mg/kg bw/day basis) is not much higher than the NOAEL in the oral 28-day study (and might turn out to be a systemic no-effect concentration in the inhalation study), a higher level might cause local adverse effects in the respiratory tract due to the corrosivity of the test material. It should be remarked that the threshold of respiratory tract irritation is not known. Target concentrations for the main study were selected on the basis of the results of the range finding study with the test material.
- Post-exposure recovery period in satellite groups: In the control and high-concentration groups, ten additional males and ten additional females were included (recovery groups), which were exposed similarly and kept for a recovery period of four weeks after the last exposure.
Observations and examinations performed and frequency:
- Clinical signs: On exposure days, each animal was observed daily in the morning, prior to exposure, by cageside observations and, if necessary, handled to detect signs of toxicity. All animals were thoroughly checked again after exposure. During exposure, the animals were also observed about halfway the 6-hour exposure period. On Saturdays, Sundays and public holidays during the main study, all animals were observed for clinical signs in the morning and checked for signs of morbidity and mortality at the end of the day. On weekend days in the range finding study, only one check per day was carried out. During exposure, when observation was hindered by the restraining tubes, attention was directed to breathing abnormalities and restlessness. All abnormalities, signs of ill health, and reactions to treatment were recorded.
- Ophthalmoscopic examination: Ophthalmoscopic examination was not performed in animals of the range finding study. In the main study, ophthalmoscopic observations were made prior to the start of exposure in all animals (on Day -13) and towards the end of the exposure period in the animals of the control group and the high-concentration group (males on Day 86, females on Day 85). Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Since no exposure-related ocular changes were observed, eye examinations were not extended to the animals of the intermediate concentration groups at the end of the exposure period, or to animals of the recovery groups.
- Body weights: Range finding study: The body weight of each animal was recorded once before the start of exposure (Day -5; these pre-test weights served as a basis for animal allocation), once prior to exposure on the first exposure day (Day 0), twice weekly thereafter (Mondays and Fridays), and on the day of scheduled sacrifice (Day 14). The high-concentration animals, sacrificed early on Day 6, were weighed for the last time on the morning of sacrifice. Main study: The body weight of each animal was recorded twice before the start of the exposure period: on Days -11 and -1 (males) or Days -12 and -2 (females). The weights recorded on Days -11 and -12 were used for animal allocation. During the exposure period, the animals were weighed just before exposure on the first day (Day 0), twice a week (Mondays and Fridays) during the first four weeks, and once a week (Fridays) thereafter (body weights were recorded less frequently after Week 4 because no exposure-related changes were seen during the first four weeks). Finally, the animals were weighed on the day before overnight fasting prior to necropsy, and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.
- Food consumption: Food consumption of the animals was measured per cage by weighing the feeders. The results were expressed in g per animal per day. In the range finding study, food consumption of the control, low- and mid-concentration animals was measured over two 7-day periods, starting on Day 0. Food consumption of the high-concentration animals, sacrificed early on Day 6, was measured over the period Day 0-6. In the main study, food consumption was measured from Day 0 over a 3-day (males) or 2-day (females) period in the first week, and subsequently over successive periods of 7 days, until the last week in which food consumption was measured over a 3-day (males) or 4-day period (females).
- Haematology: Haematology was not performed in animals of the range finding study. In the main study, haematology was conducted at the end of the treatment period on all animals of the main groups. Additionally, due to (possible) treatment-related changes at the end of treatment, haematology was conducted at the end of the recovery period in all animals of the recovery groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anaesthesia at sacrifice. Citrate (for prothrombin time only) or EDTA was used as anticoagulant. The samples were discarded after analysis. In each sample the following determinations were carried out haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell, count (lymphocytes, neutrophils, eosinophils, basophils, monocytes), prothrombin time (PT), thrombocyte count (platelet count). The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).
- Clinical chemistry: Clinical chemistry was not performed in animals of the range finding study. In the main study, clinical chemistry was conducted at the end of the treatment period on all animals of the main groups. Additionally, due to (possible) treatment-related changes at the end of treatment, clinical chemistry was conducted at the end of the recovery period in all animals of the recovery groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anaesthesia at sacrifice. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. After analysis, remaining plasma was stored frozen (<-18 °C) to enable reanalysis if necessary and then discarded. The following measurements were made in the plasma: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate.
Sacrifice and pathology:
Range finding study:
- Sacrifice and macroscopic examination: At the end of the exposure period, the surviving animals were sacrificed in such a sequence that the average time of killing was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes. A thorough necropsy was also performed on all high-concentration animals which were sacrificed early after exposure on Day 6 due to conditional decline.
- Organ weights: At scheduled sacrifice, the following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. No organ weights were recorded for the high-concentration animals sacrificed early on Day 6. Relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, heart, kidneys, liver, lung with trachea and larynx (After weighing, the lung was infused with the fixative), spleen, testes
- Tissue preservation: The complete respiratory tract of all animals and all gross lesions were preserved in a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent). The lungs (after weighing) were infused with the fixative uncer ca. 15 cm water pressure to ensure fixation. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded.
- Slide preparation: Tissue for microscopy was embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. The tissues of groups 1, 2, 3 and 4 were processed concurrently.
- Histopathological examination: All preserved tissues of all animals of the control group and the high-concentration group were examined histopathologically (by light microscopy). Additionally, all gross lesions observed in rats of the intermediate concentration groups were examined microscopically. Further, microscopic examination of the nasopharyngeal tissues, larynx, trachea and lungs was extend to all animals of the low- and mid-concentration groups due to treatment-related changes observed in the nose and larynx of high-concentration animals. Although no treatment-related alterations were noted in the trachea and lungs of high concentration animals, microscopy of these organs was extended to the low- and mid concentration animals for scientific reasons. The absence of visible treatment-related alterations in the trachea and lungs of high- concentration animals could have been due to their early termination (on Day 6 instead of on Day 14). The nasopharyngeal tissues were examined at six levels (Woutersen et al., 1994) with one level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT), the larynx at three levels (one level to include the base of the epiglottis), the trachea at three levels (including a longitudinal section through the carina of the bifurcation), and each lung lobe at one level.
Main study:
- Sacrifice and macroscopic examination: At the end of the exposure period (Day 98; main group males and females on 24 and 25 May 2016, respectively) or the recovery period (Day 126; recovery group males and females on 21 and 22 June 2016, respectively), the animals were sacrificed in such a sequence that the average time of killing was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes.
- Organ weights: At scheduled sacrifice, the following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, brain, epididymides, heart, kidneys, liver, lung with trachea and larynx (After weighing, the lung was infused with the fixative), ovaries, spleen, testes, thymus, thyroid, uterus
- Tissue preservation Samples of the following tissues and organs of all animals (main and recovery groups) were preserved in a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded. Adrenals, aorta, axillary lymph nodes, brain (three levels were examined microscopically: brain stem, cerebrum, cerebellum), caecum, colon, epididymides, eyes (with optic nerve), exorbital lachrymal glands, femur with joint, Harderian glands, heart, kidneys, liver, lungs (Each lung lobe was examined microscopically at one level)/trachea (Three levels were examined microscopically: including a longitudinal section through the carina of the bifurcation)/larynx (Three levels; one including the base of the epiglottis; were examined microscopically), mammary glands (females), cervical lymph nodes, nasopharyngeal tissue (with teeth) (Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [nose associated lymphoid tissue, NALT]), nerve peripheral (sciatic nerve), oesophagus, olfactory bulb, ovaries, pancreas, parathyroids, pharynx, parotid salivary glands, pituitary, prostate, rectum, seminal vesicles with coagulating glands, skeletal muscle (thigh), skin (flank), small intestines (duodenum, ileum, jejunum), spinal cord (Retained in vertebral column, at least three levels were examined microscopically: cervical, mid-thoracic and lumbar), spleen, sternum with bone marrow, stomach (Non-glandular and glandular parts were examined microscopically), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, tongue, tracheobronchial (mediastinal) lymph nodes, ureter, urethra, urinary bladder, uterus (with cervix), all gross lesions.
- Slide preparation: Tissues to be examined were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Unless required for histopathological examination, the tissues of the animals of the low- and mid-concentration groups (main Groups 2 and 3) and the recovery groups (recovery Groups 1 and 4) were not processed. The noses of the animals of main Groups 2 and 3 were decalcified and embedded in paraffin concurrently with the noses of the animals of main Groups 1 (control) and 4 (high-concentration).
- Histopathological examination: All preserved tissues of all animals of the control and high-concentration main groups were examined histopathologically (by light microscopy). In addition, all gross lesions observed in rats of the low- and mid-concentration groups were examined microscopically. Further, microscopic examination of the nasopharyngeal tissues, larynx and trachea was extended to all animals of the low- and mid-concentration groups and the recovery groups. These tissues were examined at the same levels as those examined in the animals of the control and highconcentration main groups.
Statistics:
See "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were observed up to highest concentration (3 mg/m3). The only finding of note was the presence of soiled fur towards the end of the treatment period (from Day 90) in a few treated females: one at 0.15 mg/m3, five at 0.6 mg/m3 and six at 3 mg/m3 (including four of the recovery group). From Day 121-122 (fourth week of the recovery period), soiled fur was no longer observed. In the absence of corroborative signs of toxicity, this finding was considered not to be toxicologically relevant. A few other clinical signs were noted incidentally (observations of the skin/fur or tail) represented background findings which were unrelated to treatment with the test item. No abnormalities were seen at the group-wise observations made about halfway each 6-hour exposure period.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in body weight up to the highest concentration (3 mg/m3). Occasional, statistically significant differences noted at the low- or midconcentration reflected normal biological variation and were unrelated to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by exposure to the test material up to the highest concentration (3 mg/m3). An occasional, statistically significant difference noted at 3 mg/m3 in females (Day 93-100) reflected normal biological variation and was unrelated to treatment.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination did not reveal any exposure-related abnormalities.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, statistically significant differences between animals exposed to the test material and controls were limited to lower mean values for the absolute number and percentage of monocytes at the high-concentration in males. Monocyte values at the end of the recovery period showed no differences between high-concentration animals and controls. As the differences at the end of the treatment period were small (absolute numbers of monocytes in high-concentration males were generally in the range of concurrent control values), they were considered not to be toxicologically relevant and probably unrelated to treatment. At the end of the recovery period, haematology parameters in the high-concentration group showed the following statistically significant differences compared to recovery controls: higher MCV (males); higher total white blood cell count (females); higher absolute number of lymphocytes (females); and lower percentage of eosinophils (females). The differences from controls were small and within normal limits (values in high-concentration recovery animals were in the range of the concurrent control values measured at the end of the treatment period). Moreover, these parameters were neither affected at the end of the treatment period nor in the opposite sex. Therefore, the slight differences noted at the end of the recovery period were considered to be unrelated to treatment and not toxicologically relevant.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, clinical chemistry parameters showed the following statistically significant differences between animals exposed to the test material and controls: lower mean plasma level of urea at the high-concentration in males, and lower fasting glucose at the mid- and high-concentration in females. Urea and glucose levels at the end of the recovery period showed no differences between high-concentration animals and controls. The differences at the end of the treatment period were small (values in high-dose animals were within or close to the concurrent control range) and not corroborated by changes in other parameters examined in this study. The fasting glucose levels showed no clear dose-related response (values at the mid- and high-concentration were similar despite the five-fold difference between the mid- and high-concentration). Moreover, toxicity is generally indicated by increases rather than decreases in urea and fasting glucose. Therefore, the slight differences in urea and fasting glucose at the end of the treatment period were considered not to be toxicologically relevant and unrelated to treatment. At the end of the recovery period, clinical chemistry parameters in the high-concentration group showed the following statistically significant differences compared to recovery controls: higher plasma levels of total protein and calcium (both in males). These isolated, minor differences were considered to be chance findings reflecting normal biological variation.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, organ weight data showed the following statistically significant differences between animals exposed to the test material and controls: higher relative brain weight in the low- and high-concentration group (females), and higher absolute and relative adrenal weight in the high-concentration group (males). The differences in brain weight were considered to be unrelated to treatment because there was no concentrationrelated response. The increase in the weight of the adrenals was fully reversible and considered not to be toxicologically significant because there were no histopathological changes in the adrenals or relevant changes in other parameters. At the end of the recovery period, statistically significant differences were noted in the absolute weights of adrenals (lower in the high-concentration group of males) and lung (higher in the high-concentration group of females). In the absence of significant changes in the relative weights of these organs, these slight differences in absolute organ weights were considered to be chance findings reflecting normal biological variation.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy, no treatment related gross changes were observed in the animals of the main and recovery groups. The few gross changes observed represented background pathology in rats of this strain and age and/or occurred only incidentally.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Main Groups: Microscopic examination revealed treatment-related histopathological changes in the upper airways (nose, larynx and trachea). The histopathological changes in the upper airways were observed in all three treatment groups. In general, the incidence and/or severity of the histopathological changes clearly increased with increasing concentration of the test material, indicative of a concentration-effect relationship. The histopathological changes in the nose were characterised by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation. The inflammation was called mixed because of the presence of a variety of inflammatory cells: polymorphonuclear cells, lymphocytes, macrophages, plasma cells, granulated intra-epithelial cells and globular leukocytes. These mixed inflammations were considered related to the treatment with the test material. In some animals focal mononuclear inflammations were observed, but that type of inflammation is a common finding in the nose. It is considered part of the background pathology and not related to treatment. Although the treatment-related histopathological changes were observed in all levels of the nose, it was clear that the rostral levels were most affected and the caudal levels were somewhat less affected. The changes were predominantly seen in the rostroventral parts of the nose, specifically the ventral meatus and nasal septum. Consequently, because of the typical distribution of the different epithelial cell types covering the nasal cavity, most histopathological changes were observed in areas covered by squamous or respiratory epithelium, whereas the olfactory epithelium (predominantly covering the dorsocaudal parts of the nasal cavity) was hardly affected. Epithelial hyperplasia was occasionally seen and considered a reactive process related to the inflammation. The amount of hyaline droplets (visible as highly eosinophilic globular structures scattered through the epithelial layer and occasionally seen in normal epithelium of the respiratory tract) was considerably increased in treated animals. In most cases these droplets were seen in close relation to the epithelium affected by the inflammatory process, but in the hind levels 5 and 6 of the nose the hyaline droplet accumulation was also seen in olfactory epithelium not affected by inflammation. The histopathological changes in the larynx were characterised by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation, generally comparable to the changes in the nose. In addition, several high-concentration animals showed epithelial ulceration. In some animals focal mononuclear inflammations were observed, but that type of inflammation is a common finding in the larynx. It is considered part of background pathology and not related to treatment. In all cases the treatment related histopathological changes were located at the epiglottis and with increasing severity, extended to the ventral pouch and other parts of the epiglottis and even the proximal part of the trachea. The caudal part of the trachea and the bronchi were generally not affected. However, in several high-concentration animals and in one mid-concentration male focal accumulation of macrophages was seen in the carina. Although the incidence of this finding was not statistically significantly different from that in controls, this change is not a common background finding and is likely to be related to the treatment. The histopathological changes observed in the lungs and other organs and tissues were considered part of background pathology and not related to treatment. These findings occurred in only one or a few animals and/or at random incidences in the different groups.
Microscopic examination Recovery Groups: Because of the histopathological changes observed in the main groups at the end of the treatment period, microscopic examination was extended to the nose, larynx and trachea of the recovery groups. In general, the incidence and severity of the histopathological changes in the high-concentration recovery animals had largely subsided in the nose and the larynx and were absent in the trachea. However, recovery was not complete after the 4-week treatment-free period.
Details on results:
Analysis of exposure conditions
- Actual concentration: The overall mean actual concentration (± standard deviation) of Gaskamine 240 in the test atmospheres as determined gravimetrically was 0.15 (± 0.02), 0.60 (± 0.06) and 2.99 (± 0.37) mg/m3 for the low-, mid- and high-concentration, respectively. These mean actual concentrations were very close to the respective target concentrations of 0.15, 0.60 and 3.0 mg/m3. The overall mean actual concentration (± standard deviation) of the starting mixture was 20.0 (± 3.8) mg/m3.
- Time to attain chamber equilibration (T95): The time to reach 95% of the steady state concentration (T95), based on chamber volume and the total air flow range was calculated to be about 6 minutes for Group 2 and about 4 minutes four Groups 3 and 4. Since the test atmospheres for these groups were obtained by diluting the starting mixture (for which T95 was about 5 minutes), actual T95 of the test atmospheres was slightly longer than these calculated values. The animals were placed in the exposure units at least 12 minutes after the start of atmosphere generation.
- Nominal test material use and generation efficiency: The mean nominal concentration of the starting mixture (± standard deviation) was 53.9 ± 8.2 mg/m3, indicating a generation efficiency of 37%. This generation efficiency is in the range to be expected for test atmosphere generation from a liquid test material. As explained before, the nominal concentrations calculated for the test atmospheres were rough estimates rather than accurate determinations. The overall mean nominal concentration (± standard deviation) was 0.38 (± 0.07), 0.94 (± 0.18) and 6.54 (± 1.41) mg/m3 for the low-, mid- and high-concentration, respectively. The corresponding mean generation efficiencies were 41%, 66% and 47%, respectively, which is in line with what can be expected for generation of liquid aerosols.
- Particle size: The overall mean (± standard deviation) mass median aerodynamic diameter (MMAD) of the test aerosol as measured by APS was 1.09 (± 0.05), 1.16 (± 0.04) and 1.13 ± (0.05) μm for the low-, mid- and high-concentration group, respectively. The corresponding mean (± standard deviation) geometric standard deviations (gsd) were 1.58 (± 0.04), 1.64 (± 0.04) and 1.61 (± 0.02) for the low-, mid- and high-concentration group, respectively.
- Total air flow, temperature, relative humidity, oxygen and carbon dioxide concentration: The overall mean total air flows (± standard deviation) were 50.5 (± 3.0), 26.3 (± 3.1), 54.7 (± 4.5) and 51.5 (± 5.6) L/min for the control, low-, mid- and high concentration group, respectively. The mean air flow in the chamber of the starting mixture was 34.3 (± 2.8) L/min. Measured temperatures during exposure were generally within the range of 19-25˚C. Occasionally, the temperature slightly exceeded these target limits (lowest value: 18.5˚C, highest value: 25.7˚C). The overall mean temperature (± standard deviation) was 23.2 (± 0.8), 21.5 (± 0.3), 21.5 (± 0.4) and 22.6 (± 0.4) ˚C for the control, low-, mid- and high-concentration group, respectively. The relative humidity during exposure generally remained within the range 30-70%. The humidity in the chamber of the control group occasionally exceeded this range (lowest value: 24.5% v/v; highest value 80.5%). The overall mean relative humidity during exposure was 45.1 (± 3.6), 48.6 (± 3.8), 39.4 (± 1.0) and 39.5 (± 1.7) % for the control, low-, mid- and high-concentration group, respectively. The oxygen concentrations measured in the exposure chambers was in the range 20.0 – 20.5% v/v. These concentrations meet the requirement described in OECD guideline 413 (i.e. >19% oxygen). The carbon dioxide concentrations measured in the exposure chambers were in the range 0.282 – 0.684% v/v. These concentrations meet the requirement described in OECD guideline 413 (i.e. <1% carbon dioxide).
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
2.99 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed up to and including the highest dose tested
Key result
Dose descriptor:
LOAEC
Remarks:
Local
Effect level:
0.15 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.15 mg/m³ air (analytical)
System:
respiratory system: upper respiratory tract
Organ:
larynx
nasal cavity
trachea
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Short summary range finding study: Exposure at 75 mg/m3 exceeded the maximum tolerable concentration, leading to early termination of this test group at the end of the fifth exposure day. Exposure to 15 mg/m3 was associated with respiratory difficulties, body weight loss, decreased food consumption, higher spleen and heart weights, and toxicologically relevant histopathological changes in the larynx and nose. The main treatment-related finding at 3 mg/m3 consisted of histopathological changes in the larynx and, to a lesser extent, the nose. Additional findings at 3 mg/m3 consisted of a slightly lower body weight gain in males and higher spleen weight in females. The histopathological changes noted in the nose and larynx at 3 and 15 mg/m3 showed a dose-related response, except for mixed cell inflammation and ulceration in the larynx.

Conclusions:
Under the conditions of this study, inhalation exposure to Gaskamine 240 up to 2.99 mg/m3 did not result in systemic toxicity. Based on this result, the NOAEC for systemic toxicity was at least 2.99 mg/m3. As toxicologically relevant local effects were noted at all concentrations tested, a NOAEC for local toxicity could not be established. The lowest concentration tested in this study, 0.15 mg/m3 (actual concentration), was a LOAEC for local toxicity.
Executive summary:

In a inhalation sub-chronic (90-day) toxicity performed in accordance with OECD Guideline 413 and GLP, Wistar Hannover rats were exposed to the test substance. Four main groups of 10 male and 10 female rats each were exposed (nose-only) to target concentrations of 0 (control), 0.15, 0.6 or 3 mg/m3 for 6 hours/day, 5 days/week over a 14-week period (65 exposure days). Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of 10 male and 10 female animals each, were simultaneously exposed with the main group animals to the control or 3 mg/m3 test atmospheres, and were sacrificed after a 4-week treatment-free period following the last exposure. Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were macroscopically examined at sacrifice, and a large number of organs and tissues were examined microscopically. The concentrations to be tested in the sub-chronic study were selected on the basis of a 14- day range finding study in which groups of five male and five female Wistar Hannover rats were exposed to target concentrations of 3, 15 and 75 mg/m3 for 6 hours/day, 5 days/week. The target concentrations were accurately achieved as demonstrated by the results of the gravimetric analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation) were 0.15 (± 0.02), 0.60 (± 0.06) and 2.99 (± 0.37) mg/m3 for the low-, mid- and high-concentration groups, respectively. All animals survived until scheduled necropsy. Clinical observations revealed no treatment-related signs of toxicity. Soiled fur was noted in some treated females but considered not to be toxicologically relevant. No abnormalities were noted at the observations made about halfway through the 6-hour exposure period. Ophthalmoscopic examination did not reveal any treatment-related ocular abnormalities. There were no treatment-related changes in body weight or food consumption. Haematology and clinical chemistry parameters were not adversely affected by the exposure to the test material. Organ weight data showed an increase in adrenal weight (absolute and relative to body weight) at the high-concentration in males. This change was fully reversible, and in the absence of histopathological correlates or relevant changes in other parameters, considered not to be toxicologically relevant. Macroscopic examination at scheduled termination revealed no exposure-related gross changes. Microscopic examination revealed histopathological changes in the upper airways (nose, larynx and trachea) of male and female rats of the low-, mid- and high-concentration groups. In general, the incidence and/or severity of the changes increased with increasing concentration. The changes in the nose were characterized by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation. The mixed inflammation was characterized by the presence of a variety of inflammatory cells: polymorphonuclear cells, lymphocytes, macrophages, plasma cells, granulated intra-epithelial cells and globular leukocytes. Although the nasal changes were seen at all six nose levels examined, the rostral levels were most affected and the caudal levels were somewhat less affected. The changes were predominantly seen in the rostroventral parts of the nose, specifically the ventral meatus and nasal septum. Consequently, because of the typical distribution of the different epithelial cell types covering the nasal cavity, most histopathological changes were observed in areas covered by squamous or respiratory epithelium, whereas the olfactory epithelium (predominantly covering the dorsocaudal parts of the nasal cavity) was hardly affected. Epithelial hyperplasia was occasionally seen and considered a reactive process related to the inflammation. The amount of hyaline droplets was considerably increased in treated animals. In most cases these droplets occurred in close relation to the epithelium affected by the inflammatory process, but in the hind levels 5 and 6 of the nose the hyaline droplet accumulation was also seen in olfactory epithelium unaffected by inflammation. The histopathological changes in the larynx were characterised by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation, generally comparable to the changes in the nose. In addition, several high-concentration animals showed epithelial ulceration. The changes were located at the epiglottis and with increasing severity, extended to the ventral pouch and other parts of the epiglottis and even the proximal part of the trachea. The caudal part of the trachea and the bronchi were generally not affected. However, in several high-concentration animals and in one mid-concentration male focal accumulation of macrophages was seen in the carina. As this change is not a common background finding, it was likely to be treatment-related. The above local effects in the upper airways were considered to be related to the corrosive properties of the test material. Under the conditions of this study, inhalation exposure to 2.99 mg/m3 (actual concentration) did not result in systemic toxicity. Based on this result, the NOAEC for systemic toxicity was at least 2.99 mg/m3 (actual concentration). Adverse local effects, consisting of histopathological changes in the upper airways (nose, larynx, trachea), occurred at all three concentrations tested. The incidence and/or severity of the histopathological changes generally showed a dose-related response. The changes in the trachea were fully reversible after the 4-week treatment-free period. The incidence and severity of the changes in the nose and larynx decreased considerably after cessation of exposure but recovery was not complete at the end of the 4-week treatment-free period. As toxicologically relevant local effects were noted at all concentrations tested, a NOAEC for local toxicity could not be established. The lowest concentration tested in this study, 0.15 mg/m3 (actual concentration), was a LOAEC for local toxicity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
0.15 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP study, Klimisch 1

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

Repeated dose toxicity, oral:

One fully reliable study is available (Ford 2002/KS K1/repeated dose toxicity: oral - 28 d rats) conducted according to OECD TG 407 and GLP (28 d, oral gavage, doses: 5, 15 and 50 mg/kg/d, Sprague Dawley rats).

The NOEL was identified by the author to be 5 mg/kg bw/d and the LOAEL correspondingly 15 mg/kg bw/day based on an increased incidence of myocarditis in females, vacuolation of lamina propria in the small intestines in both sexes and muscle fibre degeneration/necrosis in both sexes at 15 mg/kg bw/d. At the high dose of 50 mg/kg bw/d treatment related effects were seen in multiple endpoints including clinical signs, body weight effects, haematology, clinical chemistry, urinalysis, organ weights and histopathology (various organs and tissues) where nearly all were completely reverted in the recovery period. Due to the high reversibility of the effects in the recovery group, they might be regarded as non-adverse effects. However for human safety, the conservative LOAEL is set by the applicant at 50 mg/kg bw/d and NOAEL at 15 mg/kg bw/d.

Repeated dose toxicity, inhalation:

In a inhalation sub-chronic (90-day) toxicity performed in accordance with OECD Guideline 413 and GLP, Wistar Hannover rats were exposed to the test substance. Four main groups of 10 male and 10 female rats each were exposed (nose-only) to target concentrations of 0 (control), 0.15, 0.6 or 3 mg/m3 for 6 hours/day, 5 days/week over a 14-week period (65 exposure days). Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of 10 male and 10 female animals each, were simultaneously exposed with the main group animals to the control or 3 mg/m3 test atmospheres, and were sacrificed after a 4-week treatment-free period following the last exposure. Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were macroscopically examined at sacrifice, and a large number of organs and tissues were examined microscopically. The concentrations to be tested in the sub-chronic study were selected on the basis of a 14- day range finding study in which groups of five male and five female Wistar Hannover rats were exposed to target concentrations of 3, 15 and 75 mg/m3 for 6 hours/day, 5 days/week. The target concentrations were accurately achieved as demonstrated by the results of the gravimetric analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation) were 0.15 (± 0.02), 0.60 (± 0.06) and 2.99 (± 0.37) mg/m3 for the low-, mid- and high-concentration groups, respectively. All animals survived until scheduled necropsy. Clinical observations revealed no treatment-related signs of toxicity. Soiled fur was noted in some treated females but considered not to be toxicologically relevant. No abnormalities were noted at the observations made about halfway through the 6-hour exposure period. Ophthalmoscopic examination did not reveal any treatment-related ocular abnormalities. There were no treatment-related changes in body weight or food consumption. Haematology and clinical chemistry parameters were not adversely affected by the exposure to the test material. Organ weight data showed an increase in adrenal weight (absolute and relative to body weight) at the high-concentration in males. This change was fully reversible, and in the absence of histopathological correlates or relevant changes in other parameters, considered not to be toxicologically relevant. Macroscopic examination at scheduled termination revealed no exposure-related gross changes. Microscopic examination revealed histopathological changes in the upper airways (nose, larynx and trachea) of male and female rats of the low-, mid- and high-concentration groups. In general, the incidence and/or severity of the changes increased with increasing concentration. The changes in the nose were characterized by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation. The mixed inflammation was characterized by the presence of a variety of inflammatory cells: polymorphonuclear cells, lymphocytes, macrophages, plasma cells, granulated intra-epithelial cells and globular leukocytes. Although the nasal changes were seen at all six nose levels examined, the rostral levels were most affected and the caudal levels were somewhat less affected. The changes were predominantly seen in the rostroventral parts of the nose, specifically the ventral meatus and nasal septum. Consequently, because of the typical distribution of the different epithelial cell types covering the nasal cavity, most histopathological changes were observed in areas covered by squamous or respiratory epithelium, whereas the olfactory epithelium (predominantly covering the dorsocaudal parts of the nasal cavity) was hardly affected. Epithelial hyperplasia was occasionally seen and considered a reactive process related to the inflammation. The amount of hyaline droplets was considerably increased in treated animals. In most cases these droplets occurred in close relation to the epithelium affected by the inflammatory process, but in the hind levels 5 and 6 of the nose the hyaline droplet accumulation was also seen in olfactory epithelium unaffected by inflammation. The histopathological changes in the larynx were characterised by mixed inflammation, epithelial hyperplasia and increased hyaline droplet accumulation, generally comparable to the changes in the nose. In addition, several high-concentration animals showed epithelial ulceration. The changes were located at the epiglottis and with increasing severity, extended to the ventral pouch and other parts of the epiglottis and even the proximal part of the trachea. The caudal part of the trachea and the bronchi were generally not affected. However, in several high-concentration animals and in one mid-concentration male focal accumulation of macrophages was seen in the carina. As this change is not a common background finding, it was likely to be treatment-related. The above local effects in the upper airways were considered to be related to the corrosive properties of the test material. Under the conditions of this study, inhalation exposure to 2.99 mg/m3 (actual concentration) did not result in systemic toxicity. Based on this result, the NOAEC for systemic toxicity was at least 2.99 mg/m3 (actual concentration). Adverse local effects, consisting of histopathological changes in the upper airways (nose, larynx, trachea), occurred at all three concentrations tested. The incidence and/or severity of the histopathological changes generally showed a dose-related response. The changes in the trachea were fully reversible after the 4-week treatment-free period. The incidence and severity of the changes in the nose and larynx decreased considerably after cessation of exposure but recovery was not complete at the end of the 4-week treatment-free period. As toxicologically relevant local effects were noted at all concentrations tested, a NOAEC for local toxicity could not be established. The lowest concentration tested in this study, 0.15 mg/m3 (actual concentration), was a LOAEC for local toxicity.

Justification for classification or non-classification

Even though the study director in the oral subacute toxicity study of Ford 2002 set the LOAEL for Gaskamine 240 to 15 mg/kg bw/d, the effects leading to that LOAEL are not considered as classification relevant. The effect level for classification relevant effects which occurred in the study to a significant extent was 50 mg/kg bw/d. The NOAEL is proposed by applicant at 15 mg/kg bw/d.

Discussion of the classification relevant effects observed during the subacute toxicity study:

- Clinical observations: Effects were reported at 50 mg/kg bw/d, all effects showed reversibility within the 14 days recovery period.

- Heart: Increased myocarditis in females observed at 50 mg/kg bw/d, and according to the study report also "possibly at 15 mg/kg bw/d"; all effects fully reversible.

- Small intestines: Effects reported at 50 mg/kg bw/d, and in a few animals at 15 mg/kg bw/d. The recovery group (50 mg/kg bw/d) eventually showed appreciable regression of the condition, with just a few animals showing residual changes in the ileum.

- Skeletal muscle: Muscle fibre degradation and necrosis and proliferation of sacrolemnal nuclei was observed at 50 mg/kg bw/d and to a much lesser extent at 15 mg/kg bw/d. Significant regression of both conditions apparent in the 50 mg/kg bw/d recovery group.

The study of Braun 2012 performed according to OECD TG 421 Reproduction/Developmental screening propose for the systemic toxicity a NOAEL of 15 mg/kg bw/d. Respectively the LOAEL for systemic toxicity could be assumed at 40 mg/kg bw/d, the next ascending dose. These data support the approach to consider the LOAEL in Ford 2002 study at 50 mg/kg bw/d.

In addition a 90-day inhalation study is available. In that study only effects on the upper respiratory tract were observed which were due to the corrosive characteristics of the test substance. As the substance has already been classified as corrosive, additional classification is not warrented. However, based on 28-day oral toxicity study (Ford 2002) and above mentioned grounds, for protective measures, following classification is being proposed:

Regulation (EC) No 1272/2008 (CLP): CLP: As the relevant effect level is 50 mg/kg bw/d, and therefore falls within the bracket 30 - 300 mg/kg bw/d, the applicable classification is STOT RE 2; H373 "May cause damage to organs through prolonged or repeated exposure".