Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 September 1985 to 01 October 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study was designed to assess the toxic effects of the test material when administered by inhalation as an vapour/aerosol to CD® (Sprague-Dawley derived) rats (35/sex/group) for six hours per day, five days per week, for four weeks at target concentrations of 1, 10 and 30 milligrams per cubic meter (mg/m^3).
GLP compliance:
yes (incl. certificate)
Remarks:
Statement of compliance with US EPA's GLP Standards and applicable Standard Operating Procedures with some exceptions noted.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: CD (Sprague-Dawley derived)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at initiation of exposure: approx. 9 weeks old (males and females)
- Initial body weights: 278-337 g (males, mean = 308) and 160-204 g (females, mean = 182).
- Acclimation: Animals were acclimated for 4 weeks and were examined by the staff veterinarian. Animals considered unsuitable for the study were excluded prior to random selection for group assignment.
- Housing: During non-exposure periods animals were doubly-housed in suspended stainless steel wire mesh cages during the first week of acclimation and then individually housed during the rest of the acclimation period and all non-exposure periods. During exposure periods: Animals were doubly-housed in similar cages when the number of animals was even and any remaining odd animal was housed individually. Chamber temperature ranged between 21-30 °C and chamber RH between 44-100 %.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 67-76 °F
- Humidity: 30-70 %
- Photoperiod: 12 hour light/dark cycle (7 am to 7 pm).

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
other: administered in breathing zone of animal
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: The MMAD were as follows:
Group I (control): N/A
Group II: 0.98 microns
Group III: 1.7 microns
Group IV: 1.5 microns
Details on inhalation exposure:
- The test material was administered as a vapour/aerosol to male and female CD (Sprague-Dawley derived) rats, via inhalation (administered in the breathing zone of the animals).   
- Target exposure levels: 0 (control), 1, 10, and 30 mg/m^3. Exposure levels were selected on the basis of multiples of the current TLV at the time.
- The stainless steel and glass chambers in which the animals were exposed had a total volume of 1 m^3 with an effective volume of 760 L. The airflow rates in groups I-IV were 205-209 L/min, air change was 4.8-4.9 min, and 99 % equilibrium time was 22.0-22.5 min.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Air samples from all exposure chambers were collected (via impinger) at least 4 times over the test period. Additionally, distribution samples (i.e., one at the top and one at the bottom of the chamber) were collected from the test material exposure chambers over the course of the exposure period.
- Samples were sent to the sponsor for analysis by atomic absorption spectroscopy (AAS). Particle size distribution measurements were made weekly over the exposure period.
- Mean measured (target) exposure concentrations were 1.33 (1.0), 7.17 (10), and 9.54 (30) mg Sn/m^3 (ca. 2.4, 23.8 and 71.3 mg test material/m^3 target conc). The median particle diameter in the exposure chambers ranged from 0.98-1.7 µm. The particulate level in the low concentration exposure chamber (1 mg/m^3) was comparable to that found in room air, indicating the test material was respirable to the rat.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily, 6 hours/day, 5 days/week
Total 20 exposures/sex/group
Doses / concentrationsopen allclose all
Dose / conc.:
1 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
10 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
30 mg/m³ air
Remarks:
Target concentration
No. of animals per sex per dose:
- Groups of animals (35/sex/dose) were exposed daily to the test material at concentrations of 1, 10, and 30 mg Sn/m^3 (ca. 2.4, 23.8 and 71.3 mg test material/m^3 or 1.33, 7.17 and 9.54 mg/m^3 of organic tin), respectively, for 6 hours/day, 5 days/week for 4 weeks.
- Control animals (35/sex) were exposed to house-supply air for the same  length of time.
Control animals:
yes, concurrent no treatment
Details on study design:
- Post-exposure recovery period: 0 weeks (15 animals/sex/dose), 2 weeks (10 animals/sex/dose), or 4 weeks (10 animals/sex/dose).
- The test material concentrations were calculated from the target tin (Sn) concentrations using the conversion factor [282.17/118.69] consisting of the Mol weights of the test material and Sn, respectively. All measured Sn was attributed to the test material.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
In-life observations:
- For mortality and gross signs of toxic effects - twice daily, once in the morning and once in the afternoon.
- Animals underwent physical examination pre-test and weekly thereafter.  
- Ophthalmoscopic examinations were performed pre-test and on weeks 5, 7, and 8.
- Body weights were recorded prior to testing, weekly during exposure, and prior to sacrifice (after fasting).  
Sacrifice and pathology:
-After 4 weeks exposure, 10 animals/sex/group were sacrificed and subjected to clinical study, postmortem examination, organ weight determination, and histopathological evaluation. An overnight urine sample was collected from an additional 5 animals/sex/group. These animals were then sacrificed and selected tissues (blood, lungs, liver, kidneys and brain) were removed and sent to the sponsor for assay. No postmortem examinations of these animals were conducted at the test laboratory.
-The remaining animals were retained for a 2- or 4-week recovery period. After the 2- and 4-week recovery periods, 10 animals/sex/group were sacrificed. Five animals/sex/group underwent clinical study, postmortem  examination, organ weight determination, and histopathological evaluation (when visibly abnormal organs were noted) at the test laboratory. The remaining 5 animals/sex/group were processed as described previously and sent to the sponsor for assay.
- Sacrifice method: Exsanguination under ether anaesthesia.
Other examinations:
- Blood samples were obtained from fasted animals, and separated into two aliquots for haematology and clinical chemistry.
- Urinalysis was also performed.
Statistics:
Body weights, haematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analysed. Mean values of all treated groups were compared to control at each time interval.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Physical observations, including mucoid nasal discharge, rales, lacrimation, salivation, rough coat, abdominal distension (males), ano-genital staining, and fur discoloration, were observed primarily in the high-exposure group (30 mg/m^3). These symptoms reduced during the recovery period.
Mortality:
mortality observed, treatment-related
Description (incidence):
- Three male rats and one female rat in the high exposure group (30 mg/m^3) died during exposure. The deaths occurred after 13-15 days of exposure and were considered test material-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of male and female rats in the high-exposure group (30 mg/m^3) were significantly lower than controls during all (male) or part (females) of the 4-week exposure period. The differences in animal weights (i.e., treated vs. control) decreased during the recovery period.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Post-exposure (pre-recovery) haematology data indicated slight to sometimes-significant differences from the control group. In the high-exposure group (30 mg/m^3) these included increased mean haemoglobin values (males and females), increased mean erythrocyte counts (males and females), and increased mean haematocrit values (females). Female rats in all exposure groups exhibited increased mean erythrocyte counts. Differences from controls were slight, though sometimes significant.
- Haematology results in female rats in the recovery groups were comparable to controls. Male rats in the recovery groups exhibited increased mean erythrocyte counts (high dose group - 2 weeks recovery) and increased haematocrit, haemoglobin, and erythrocyte counts (mid and high dose group - 4 weeks recovery).
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy results reported primarily involved lung tissue. Observations related to exposed males and females included increased discolouration, amorphous material in lungs (potential test material or hydrolysis product), alveolar oedema (dose-related), peribronchial lymphoid cell accumulation, perivascular lymphoid cell infiltrate, extravasated alveolar erythrocytes (males only, dose-related), and accumulation of alveolar macrophages and neutrophilic infiltrates.
- The lesions and their severity scores represent the expected response of pulmonary tissue to introduction of a foreign and known corrosive material. Low-, mid- and high-dose males and females had increased lesion mean severity scores for amorphous material in their lungs compared to controls. The no-effect-level would be expected to be <1 mg Sn/m^3 (<2.4 mg test material/m^3).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Skin sections from high-dose males and females showed epidermal acanthosis and hyperkeratosis.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
< 3 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Dose descriptor:
LOAEC
Effect level:
3 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
other: local effects (microscopic findings in lung). 3 mg/m^3 (test material) = 1.33 mg Sn/m^3
Key result
Dose descriptor:
NOAEC
Effect level:
16.18 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
other: Systemic effects. Mortality and several clinical signs were observed at 21.53 mg/m^3 (test material). 16.18 mg/m^3 (test material) = 7.17 mg Sn/m^3

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1 : Mean Body Weight Values

Sex

Dose (mg/m3)

Exposure period

Recovery period

W0

W1

W2

W3

W4

W5

W6

W7

W8

M

0

308

+/-13

342

+/-18

367

+/-20

388

+/-21

405

+/-27

422

+/-30

438

+/-34

463

+/-32

480

+/-35

M

1

308

+/-13

344

+/-18

353*

+/-20

378

+/-23

398

+/-26

417

+/-30

437

+/-37

441

+/-38

455

+/-36

M

10

308

+/-14

339

+/-17

356

+/-20

376

+/-23

398

+/-27

419

+/-29

444

+/-30

470

+/-41

483

+/-43

M

30

307

+/-13

331*

+/-15

346**

+/-17

354**

+/-19

376**

+/-23

394*

+/-25

425

+/-29

451

+/-41

467

+/-41

F

0

182

+/-11

240

+/-12

222

+/-14

237

+/-14

248

+/-15

261

+/-19

261

+/-19

271

+/-21

277

+/-23

F

1

182

+/-10

204

+/-11

218

+/-12

236

+/-13

248

+/-14

254

+/-16

263

+/-17

266

+/-16

276

+/-18

F

10

183

+/-11

229**

+/-15

218

+/-15

233

+/-16

246

+/-17

248

+/-18

256

+/-19

262

+/-21

268

+/-20

F

30

182

+/-10

198

+/-13

214

+/-14

227*

+/-15

235**

+/-17

243*

+/-18

254

+/-21

259

+/-22

266

+/-19

*, ** results statistically significant (*p<0.05, **p<0.01)

Table 2 : Mean Haematology Values – Week 5

Sex

Dose (mg/m^3)

HGB (g/dl)

HCT (%)

RBC (mil/ul)

PLAT (100 T/ul)

RETIC (% RBC)

WBC (thous/ul)

M

0

17.3

+/-0.5

40

+/-1

7.65

+/-0.21

9.76

+/-1.59

1.9

+/-0.4

13.8

+/-3.4

M

1

17.5

+/-0.9

40

+/-2

7.66

+/-0.41

9.78

+/-0.84

1.7

+/-0.4

13.5

+/-4.2

M

10

18.2

+/-1.6

40

+/-1

7.75

+/-0.36

9.81

+/-1.18

2.4

+/-0.6

13.3

+/-2.7

M

30

18.0

+/-0.7

40

+/-2

7.91

+/-0.44

9.55

+/-1.25

1.9

+/-0.7

13.8

+/-3.4

F

0

16.2

+/-0.5

37

+/-1

6.79

+/-0.20

10.72

+/-2.08

2.4

+/-0.5

9.0

+/-1.9

F

1

16.7

+/-0.5

39*

+/-1

7.13*

+/-0.19

10.21

+/-1.64

2.2

+/-0.6

10.4

+/-2.4

F

10

16.8

+/-0.6

39

+/-2

7.06*

+/-0.33

9.69

+/-1.76

1.9

+/-0.4

11.4

+/-2.5

F

30

17.4**

+/-0.5

40**

+/-1

7.33**

+/-0.21

9.92

+/-0.76

2.6

+/-0.8

11.5

+/-3.5

*, ** results statistically significant (*p<0.05, **p<0.01)

HGB = haemoglobin; HCT = haematocrit; RBC = red blood cells ; PLAT = Platelet count; RETIC = reticulocyte count; WBC = white blood cells

Table 3 : Mean Haematology Values – Week 7

Sex

Dose (mg/m^3)

HGB (g/dl)

HCT (%)

RBC (mil/ul)

PLAT (100 T/ul)

RETIC (% RBC)

WBC (thous/ul)

M

0

16.3

+/-0.3

46

+/-1

7.25

+/-0.23

11.39

+/-1.21

1.1

+/-0.3

13.6

+/-3.6

M

1

16.7

+/-0.5

48

+/-1

7.65

+/-0.27

9.76

+/-1.28

1.2

+/-0.4

15.6

+/-3.6

M

10

16.9

+/-0.6

48

+/-3

7.61

+/-0.22

11.11

+/-1.29

1.0

+/-0.4

13.4

+/-2.8

M

30

16.8

+/-0.5

47

+/-2

7.73*

+/-0.28

10.05

+/-1.34

0.9

+/-0.3

14.0

+/-4.4

F

0

16.4

+/-0.2

46

+/-2

6.85

+/-0.29

10.57

+/-0.62

0.9

+/-0.3

8.7

+/-1.1

F

1

16.5

+/-0.5

48

+/-2

7.11

+/-0.21

9.82

+/-1.28

0.8

+/-0.3

10.8

+/-3.1

F

10

16.6

+/-0.3

48

+/-1

7.18

+/-0.26

10.34

+/-1.34

0.7

+/-0.2

9.1

+/-1.3

F

30

16.9

+/-0.2

49

+/-1

7.24

+/-0.19

11.62

+/-2.58

0.8

+/-0.4

9.9

+/-1.2

*, ** results statistically significant (*p<0.05, **p<0.01)

HGB = haemoglobin; HCT = haematocrit; RBC = red blood cells ; PLAT = Platelet count; RETIC = reticulocyte count; WBC = white blood cells

Table 4 : Mean Haematology Values – Week 9

Sex

Dose (mg/m^3)

HGB (g/dl)

HCT (%)

RBC (mil/ul)

PLAT (100 T/ul)

RETIC (% RBC)

WBC (thous/ul)

M

0

15.9

+/-0.3

46

+/-1

7.43

+/-0.34

9.59

+/-1.77

0.7

+/-0.3

12.8

+/-1.9

M

1

16.1

+/-0.4

47

+/-1

7.58

+/-0.25

9.75

+/-1.38

0.7

+/-0.2

15.3

+/-2.3

M

10

16.9*

+/-0.7

48

+/-3

7.75

+/-0.39

10.97

+/-2.00

0.8

+/-0.4

13.7

+/-1.0

M

30

17.2**

+/-0.7

49*

+/-1

7.60

+/-0.43

9.43

+/-1.19

0.5

+/-0.2

13.3

+/-3.5

F

0

16.5

+/-0.2

48

+/-1

7.19

+/-0.19

9.65

+/-1.43

0.7

+/-0.2

9.7

+/-3.0

F

1

16.5

+/-1.3

48

+/-4

7.32

+/-0.68

9.16

+/-0.86

0.5

+/-0.2

8.9

+/-1.4

F

10

16.0

+/-1.5

46

+/-5

6.96

+/-0.60

9.09

+/-1.22

0.6

+/-0.2

8.6

+/-1.3

F

30

16.4

+/-1.6

48

+/-5

7.22

+/-0.48

9.37

+/-0.95

0.7

+/-0.2

9.9

+/-2.9

*, ** results statistically significant (*p<0.05, **p<0.01)

+/-HGB = haemoglobin; HCT = haematocrit; RBC = red blood cells ; PLAT = Platelet count; RETIC = reticulocyte count; WBC = white blood cells

Applicant's summary and conclusion

Conclusions:
For the local effects (microscopic findings in lungs), the LOAEL is 3.00 mg test material/m^3 (1.33 mg Sn/m^3).
For the systemic effect, the NOAEL is 16.18 mg test material/m^3 (7.17 mg Sn/m^3). Mortality and several clinical signs were observed at 21.53 mg/m^3. As the mode of action of the test material's apparent toxicity seen in the repeat dose inhalation study is not deemed to be as a result of systemic toxic effects as a result of inhalation, but rather the test materials particular mode of action (corrosive effects) it is proposed that the test material is not classified for repeated toxicity according to Regulation EC no.1272/2008 and according to the EU Directive 67/548/EEC.
Executive summary:

This study was designed to assess the toxic effects of the test material when administered by inhalation as a vapour/aerosol to 210 CD (Sprague-Dawley derived) rats (35/sex/group) for six hours per day, five days per week, for four weeks at target concentrations of 1, 10 and 30 mg/m^3. Control animals (35/sex) received house-supply air only while in chamber.

After four weeks of exposure (Week 5), up to 15 animals/sex/group were sacrificed. The first 10 animals/sex/group were pre-designated for clinical laboratory studies, gross post-mortem examinations, organ weight measurements and histopathological evaluations (Groups I and IV animals only). An over-night urine sample was collected on the remaining 5 animals/sex/group (Week 5) prior to sacrifice. No gross post-mortem examination was performed on these animals; however, selected tissues (blood, lungs, liver, kidneys and brain) were removed, weighed and shipped frozen to the sponsor for assay.

The remaining animals remained on test for a two- or four-week recovery period. After two and four complete weeks post-exposure, 10 animals/sex/group were sacrificed (Week 7 and Week 9). Five animals/sex/group were evaluated for clinical laboratory studies, gross post-mortem examinations, organ weight measurements and histopathological evaluations, while the remaining 5 animals/sex/group were processed for chemical assays by the sponsor.

The Group II, III and IV animals were exposed to cumulative mean analytical concentrations of 1.33, 7.17 and 9.54 mg/m^3 of organic tin respectively. Daily weighted mean nominal concentrations for Groups II, III and IV were 4.6, 25.1 and 484 mg/m^3 tin. Particle size distribution measurements indicated the test atmosphere contained particles having a mass median aerodynamic diameter in the range 0.98 to 1.5 microns. The particle size distribution measurement revealed a significant level of aerosol in all chambers containing test material. The level of particulate in the Group I chamber was comparable to the level or particulate found in room air. This result indicated the test material was respirable to the rat.

Three Group IV males and one Group IV female died during the exposure phase of the study. These deaths all occurred after 13 to 15 days of exposure and were considered related to exposure to the test material.

A number of findings were notable in the physical observation data of the treated animals. For the most part, these findings occurred at the high exposure level during the exposure phase of the study. These findings included mucoid nasal discharge, rales, lacrimation, salivation, rough coat, abdominal distension (males only), ano-genital staining and discoloration of the fur. In general these findings were not evident during the recovery phase of the study.

The mean body weights of the males and females in the high-exposure level (Group IV) were significantly reduced compared to their respective controls during part (females) or all (males) of the 4-week exposure phase of the study. The differences from control noted in the mean body weights of the Group IV animals progressively decreased during the recovery period.

The haematology data of the treated males and females exhibited slight, though in some cases, statistically significant differences from control. These included increased mean haemoglobin value and erythrocyte counts in the high-dose males at Week 5 (end of the exposure phase) and, increased mean haemoglobin and haematocrit values and erythrocyte counts in the high-dose females at Week 5 as well as increased erythrocyte counts in the low- and mid-dose female groups. Differences from control noted following 2 weeks and 4 weeks of recovery (Weeks 7 and 9) included increased mean erythrocyte counts in the high-dose males (Week 7) and, increased mean haemoglobin, haematocrit and erythrocyte counts in the mid- and high-dose males (Week 9). The haematology data of the females following 2 and 4 weeks of recovery were considered unremarkable.

Necropsy was performed on animals marked for the terminal, first and second recovery sacrifices as well as unscheduled deaths. Grossly, the incidence of lung discolouration was increased in exposed males and females. Microscopically, amorphous material, (perhaps the test material or hydrolysis products) and alveolar oedema were evident in the lungs of exposed males and females. Other lung changes which occurred with increased incidence and severity in the exposed groups included peribronchial lymphoid cell accumulation and perivascular lymphoid cell infiltrate, extravagated erythrocytes (males only), and accumulation of alveolar macrophages. Dose related responses were shown only by alveolar oedema in both sexes and by alveolar erythrocytes in males only.

For the local effects (microscopic findings in lungs), the LOAEL is 3.00 mg test material/m^3 (1.33 mg Sn/m^3).

For the systemic effect, the NOAEL is 16.18 mg test material/m^3 (7.17 mg Sn/m^3). Mortality and several clinical signs were observed at 21.53 mg test material/m^3.

Although apparently the NOAEC obtained in the 28-day inhalation toxicity study is lower than suggested reference values given under GHS/CLP, the specific mode of action of the test material i.e. corrosiveness, does not support the need for any classification for repeated exposure systemic effects. Animals in the study were exposed whole body to very small particles of the test material (mass mean aerodynamic diameter in the range 0.98 to 1.5 microns) and actually signs of the same irritating action exerted on all the sites-of-contact (i.e. lungs, skin and stomach) were noted. These included clinical signs such as mucoid nasal discharge, rales, lachrymation, salivation, rough coat and discolouration of the fur, all indicative of irritation, and macro- and microscopic findings. Alveolar oedema and extravagated erythrocytes (accompanied by peribronchial/perivascular lymphoid accumulation/ infiltration and accumulation of alveolar macrophages) were observed in the lungs; mucous/purulent exudates were found in the nose turbinate sections; acanthosis and hyperkeratosis were noted microscopically in the skin but also purulent dermatitis involving the more exposed areas of the body (i.e. the external ear, extremities and skin) were observed, especially in animals which died. In the stomach, infarction (haemorrhage in one case) was also present, especially affecting animals which died. These findings are all most feasibly of local, site-of-contact origin, and are addressed by classification for site-of-contact effects.

There is no evidence of other organ toxicity. The haematological changes in the number of erythrocytes and mean haemoglobin concentration were slight, although attaining statistical significance, and were considered secondary to the lesions, often haemorrhagic, occurring in affected organs and tissues. Clinical chemistry did not evidence any change indicative of specific organ toxicity, either on liver function, as was instead noted in the 90-day dietary oral study.

Based on the lack of indications of any specific target organ systemic toxicity and on the specific mode of action of the test material, no classification for repeated exposure on the basis of this study is proposed for the test material.