Registration Dossier

Administrative data

Description of key information

The repeated toxicity profile of benzene, mono-C10-13-alkyl derivatives, distillation residues, sulfonated, sodium salt (registered substance/target substance)  was not determined by actual repeated toxicity studies. Instead, read across substances were used to predict the repeated toxicity of the registered substance.

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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines to to GLP, and therefore meets the requirements for Klimisch code 1. However as this study is used in the context of a read across, Klimisch 2 is assigned.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
A fuctional observation battery for neurotoxicity was not performed since this test was not part of the OECD 407 guideline at the time the study was performed
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 41 days
- Weight at study initiation: males, 179-215g; female 141-170g
- Housing: hanging stainless steel wire-bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23°C
- Humidity (%): 48-66%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Mixed weekly weight/volume in peanut oil


DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis of dosing solutions was conducted to confirm that they were homogeneous and met the desired concentrations.
Duration of treatment / exposure:
29 day treatment duration with a 14 day recovery period.
Frequency of treatment:
7 days/week.
Remarks:
Doses / Concentrations:
0 mg/kg-bw day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg-bw day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg-bw day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg-bw day
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes
Details on study design:
- Dose selection rationale: Data from a pilot two week repeated dose oral study
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily


BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly


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


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined:


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: Yes
- How many animals: All



URINALYSIS: Yes
- Time schedule for collection of urine: Overnight before termination
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Tests applied included; parametric ANOVA with Dunnetts post-hoc test, non parametric Kruskal-Wallis and Mann-Whitney U test, Bartletts test for equal variances, Students t test and Dixons test for rejection of outlying values
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One animal was sacrificed on Day 0 and one animal was found dead on Day 9, a result of probable misdosing.

Stained fur was observed in high dose animals, scabbed skin occurred in one control male and high dose female displayed sneezing and abnormal respiratory sounds.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences were observed in mean bodyweights or body weight gains.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A statistically significant increase in food consumption was observed in low dose males compared with controls.

FOOD EFFICIENCY
No statistically significant differences were observed in food efficiency.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not recorded

OPHTHALMOSCOPIC EXAMINATION
Not recorded

HAEMATOLOGY
Male mean cell haemoglobin concentrations were significantly decreased compared with the controls at all dose levels. However, these were not considered to be biologically significant, as there was no dose response trend. A statistically significant increase in partial thromboplastin time was observed in mid and top dose males compared with controls. Prothrombin time was significantly increased in the mid and high dose females during the treatment period, and was significantly reduced in males in the recovery group. This were within normal limits and therefore not considered to be biologically significant. A statistically significant increase in the reticulocyte count was observed in treated males in the recovery group, however, was not considered to be biologically significant.

CLINICAL CHEMISTRY
A statistically significant decrease in serum cholesterol was observed in high dose males and females and persisted in females into the recovery period. This was considered to be treatment related.

Statistically significant increases were observed in alanine aminotransferase, lactic dehydrogenase, aspartate aminotransferase, sodium, phosphorus and triglycerides, as well as decreases in albumin and chloride.There was no dose related trend with these changes, therefore they are not considered to be treatment related.

URINALYSIS
A statistically significant increase in specific gravity was observed in low dose males. Urine volume was significantly reduced in treated males in the recovery group. This was not considered to be biologically significant.

NEUROBEHAVIOUR
Not recorded

ORGAN WEIGHTS
No statistically significant differences were observed.

GROSS PATHOLOGY
No substance related macroscopic changes were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
No substance related microscopic changes were observed.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not recorded

HISTORICAL CONTROL DATA (if applicable)
Not recorded
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Mean serum cholesterol levels were significantly reduced in the 1000 mg/kg males and females at termination of dosing. This was still significantly reduced in 1000 mg/kg females at the end of the 14 day recovery period.
Critical effects observed:
not specified

Table 1: Average body weights and body weight gains during xx days of treatment

 

Dose rate (ppm)

Body Weights (g)

 

Total Weight Gain

Week 0

Week 1

Week 2

Week 3

Week 4

g               

% of control

Male

 0

195

243

286

323

352

157

181

100

196

245

298

340

374

175

191

500

200

245

289

329

362

162

181

1000

193

240

283

315

346

154

179

Female

  0

155

175

194

213

223

67

144

100

156

174

194

215

228

72

146

500

154

175

190

212

221

67

144

1000

155

174

195

212

224

69

145

 

Conclusions:
A NOAEL of 500 mg/kg bw/day was identified in this study.
Executive summary:
In a subchronic toxicity study calcium sulphonate was administered to 12 rats/sex in the control and top dose and 6 rats/sex in the low and mid dose via gavage at dose levels of 0, 100, 500 or 1000 mg/kg bw/day.

 

A decrease in serum cholesterol levels occurred in the top dose group. The LOAEL is 1000 mg/kg bw/day, based on  a decrease in serum cholesterol at the top dose.  The NOAEL is 500 mg/kg bw/day.

 

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study conducted to OECD guidelines to to GLP, and therefore meets the requirements for Klimisch code 1. However as this study is used in the context of a read across, Klimisch 2 is assigned.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. In addition, as this study is used in the context of a read across, Klimisch 2 is assigned.
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: males, 6 weeks; females, 7 weeks
- Weight at study initiation: males, 205-232g; females 156-186g
- Housing: suspended wire mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26°C
- Humidity (%): 20-76%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD 3.3-3.7 µm
GSD 2.0-2.1 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Glass and stainless steel exposure chambers
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: 21-26°C, 20-76%
- Air flow rate: 210-215 pm
- Air change rate: 4.8/minute
- Method of particle size determination: Delron DCI-6 cascade impactor


TEST ATMOSPHERE
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Concentration measured by gravimetric analysis.
Duration of treatment / exposure:
6 hours per day for 28 days.
Frequency of treatment:
5 days per week.
Remarks:
Doses / Concentrations:
49.5, 156, 260 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed
Positive control:
None.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


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


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals
- Parameters examined: Haemoglobin concentration, haematocrit, erythrocyte count, platelet count, clotting time, total and differential leukocyte count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Animals fasted: No data
- How many animals: All animals



URINALYSIS: Yes
- Time schedule for collection of urine: Prior to terminal sacrifice
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data



NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
gross pathology on all animals
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Statistics:
ANOVA with Dunnets test
Regression analysis
Krusal-Walis and Dunns summed rank test
Jonsheere's test for monotonic trend
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the test.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain was decreased in treated animals, although this was not statistically significant.

FOOD CONSUMPTION
Not measured

FOOD EFFICIENCY
Not measured

WATER CONSUMPTION
Not measured

OPHTHALMOSCOPIC EXAMINATION
Not measured

HAEMATOLOGY
Statistically significant differences were observed in females. Specifically; increased haematocrit in the low dose group.
CLINICAL CHEMISTRY
Statistically significant differences were observed in females. Specifically; increased creatine phospholinase in the mid and top dose groups and sodium in the top dose group.

NEUROBEHAVIOUR
Not measured

ORGAN WEIGHTS
Statistically significant increased lung weights and lung to body weight ratios were observed in a dose dependant manner in the mid and top dose groups.

GROSS PATHOLOGY
Gross lesions were sporadic and were not considered to be caused by the test article.

HISTOPATHOLOGY: NON-NEOPLASTIC
A higher incidence of accumulations of intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium was seen in the lungs of the treated animals. These were dose related at the mid and top dose levels.
Dose descriptor:
NOAEL
Effect level:
49.5 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: Statistically significant dose related increase in lung weight and relative lung weights with corresponding accumulations of intraalveolar macrophages and hyperplasia/hypertrophy of the bronchiole epithelium.
Critical effects observed:
not specified

 

Table 1: Average body weights and body weight gains during 28 days of treatment

 

Analytical concentration (mg/L)

Body Weights (g)

Total Weight Gain

Week 0

Week 1

Week 2

Week 3

Week 4

g

% of control

Male

    0

 217

 249

 295

336

 369

 152

 

LCT

 216

248

294

331

359 

 143

 94

MCT

 215

247 

302 

346

384 

 169

 111

HCT

 215

243 

285 

319

347 

 132

 87

Female

    0

 171

 185

 206

223

 239

 68

 

LCT

 169

 185

 205

220

 232

 63

 93

MCT

 168

 181

 203

219

 229

 61

 90

HCT

 172

 186

 205

223

 238

 66

 97

 

Table 2 Selected haematology, clinical chemistry and pathology findings

 

Doses (unit)

0

50

150

250

0

50

150

250

male

female

Number of animals/group

5

5

5

5

5

5

5

5

Haematology(day x)

 

 

 

 

 

 

 

 

- RBC (TERA/L)

6.62

6.89 

6.66 

6.52 

6.60 

6.90 

6.59 

6.70

- MCV (FL)

 -

- HCT (L/L)

 46

47 

46 

43 

 46

48 

47 

44 

- HGB (MMOL/L)

 16.1

16.7 

16.1 

15.5 

15.5 

16.6 

16.1 

15.5 

- WBC (GIGA/L)

 13.9

13.7 

12.9 

10.5 

9.0 

12.8 

11.5 

13.8 

Blood chemistry(day x)

 

 

 

 

 

 

 

 

- sodium (MMOL/L)

 146

146 

147 

146 

 145

143 

144 

141 

- potassium (MMOL/L)

 4.4

4.4 

4.5 

4.3 

 4.3

4.3 

4.3 

4.3 

- chloride (MMOL/L)

 101

102 

102 

102 

 102

102 

102 

101 

- gobulin (G/L)

 2.2

2.3 

2.4 

2.0 

 2.1

2.1 

2.1 

2.0 

- cholesterol (MMOL/L)

 56

53 

 63

55 

 59

75 

60 

69 

- triglyceride (MMOL/L)

 45

39 

54 

35 

 30

24 

25 

25 

Pathology

 

 

 

 

 

 

 

 

Accumulation of intraalveolar macrophages

 3

5

5

Bronchiolar epithelium: hyperplasia/hypertrophy

 3

Table 3: Absolute and relative organ weights

 

 

Males

Females

DAILY DOSE
(units e.g. mg/kg bw/day)

0

50

150

250

0

50

150

250

NUMBER OF ANIMALS

 5

BODY WEIGHT (g)a

 335

323 

346 

313 

 213

204 

204 

208 

BRAIN

 

 

 

 

 

 

 

 

Absolute Weighta

g

 1.976

1.939 

2.032 

1.943 

 1.900

1.876 

1.856 

1.830 

Per Body Weighta

%

 5.93

6.03 

5.90 

6.21 

 8.96

9.20 

9.12 

8.84 

ADRENALS

 

 

 

 

 

 

 

 

Absolute Weighta

g

 0.051

0.052 

0.054 

0.054 

 0.068

0.069 

0.070 

0.060 

Per Body Weighta

%

 1.52

1.63 

1.56 

1.71 

 3.21

3.37 

3.44 

2.90 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

HEART

 

 

 

 

 

 

 

 

Absolute Weighta

g

 1.220

 1.173

 1.179

 1.122

 0.803

0.801 

0.753 

0.793 

Per Body Weighta

%

 3.64

3.65 

3.41 

3.59 

 3.79

3.92 

3.70 

3.82 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

KIDNEYS

 

 

 

 

 

 

 

 

Absolute Weighta

g

 2.592

2.779 

3.054 

2.704 

 1.745

1.696 

1.694 

1.883 

Per Body Weighta

%

 7.79

8.61 

8.81 

8.61 

 8.16

8.32 

8.29 

9.05 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

 

 

Absolute Weighta

g

 10.775

10.306 

11.470 

9.999 

 7.020

6.464 

6.789 

7.357 

Per Body Weighta

%

 3.22

3.20 

3.31 

3.18 

 3.30

3.17 

3.33 

3.53 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

SPLEEN

 

 

 

 

 

 

 

 

Absolute Weighta

g

 0.670

0.569 

0.652 

0.594 

 0.426

0.441 

0.471 

0.484 

Per Body Weighta

%

 2.00

 1.77

1.88 

1.89 

 2.01

2.17 

2.30 

2.34 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

TESTES

 

 

 

 

n.a.b

n.a.b

n.a.b

n.a.b

Absolute Weighta

g

 3.187

3.072 

2.796 

3.135 

n.a.b

n.a.b

n.a.b

n.a.b

Per Body Weighta

%

 9.54

 9.54

8.16 

10.02 

n.a.b

n.a.b

n.a.b

n.a.b

Per Brain Weighta

%

 

 

 

 

n.a.b

n.a.b

n.a.b

n.a.b

LUNGS

 

 

 

 

 

 

 

 

Absolute Weighta

g

 1.306

1.302 

1.515 

1.537 

 1.051

1.127 

1.138 

1.338 

Per Body Weighta

%

 3.91

 4.05

4.39 

4.91 

 4.93

5.52 

5.59 

6.46 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

OVARIES

n.a.b

n.a.b

n.a.b

n.a.b

 0.0921

0.0768 

0.0943 

0.0742 

Absolute Weighta

g

n.a.b

n.a.b

n.a.b

n.a.b

 4.34

3.77 

4.62 

3.56 

Per Body Weighta

%

n.a.b

n.a.b

n.a.b

n.a.b

 

 

 

 

Per Brain Weighta

%

n.a.b

n.a.b

n.a.b

n.a.b

 

 

 

 

 

Conclusions:
A NOAEL of 49.5 mg/m³ was identified for males and females, based on increased lung weight and microscopic changes of the lung.
Executive summary:

In a subacute inhalation toxicity study, a petroleum derived calcium salt was administered to 5 Sprague-Dawley rats/sex/concentration by whole body exposure at concentrations of 0, 49.5, 156 or 260 mg/m³ for 6 hours per day, 5 days/week for a total of 28 days.

 

Statistically significant, dose related increases in lung weights occurred in the mid and top dose groups, with corresponding increases in intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium.  The LOAEL is 156 mg/m³, based on effects in the lung. The NOAEL is 49.5 mg/m³.

 

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement (OECD 412) for a subchronic inhalation study in the rat.   

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
881.6 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
System:
other: (based on starting point is obtained by conversion of the oral NOAEL of 500 mg/kg bw from the oral one-generation toxicity study in rats with the calcium sulfonate read-across substance)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. In addition, as this study is used in the context of a read across, Klimisch 2 is assigned.
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: males, 6 weeks; females, 7 weeks
- Weight at study initiation: males, 205-232g; females 156-186g
- Housing: suspended wire mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26°C
- Humidity (%): 20-76%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD 3.3-3.7 µm
GSD 2.0-2.1 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:Glass and stainless steel exposure chambers
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: 21-26°C, 20-76%
- Air flow rate: 210-215 pm
- Air change rate: 4.8/minute
- Method of particle size determination: Delron DCI-6 cascade impactor


TEST ATMOSPHERE
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Concentration measured by gravimetric analysis.
Duration of treatment / exposure:
6 hours per day for 28 days.
Frequency of treatment:
5 days per week.
Remarks:
Doses / Concentrations:
49.5, 156, 260 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed
Positive control:
None.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


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


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals
- Parameters examined: Haemoglobin concentration, haematocrit, erythrocyte count, platelet count, clotting time, total and differential leukocyte count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to terminal sacrifice
- Animals fasted: No data
- How many animals: All animals



URINALYSIS: Yes
- Time schedule for collection of urine: Prior to terminal sacrifice
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data



NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
gross pathology on all animals
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Statistics:
ANOVA with Dunnets test
Regression analysis
Krusal-Walis and Dunns summed rank test
Jonsheere's test for monotonic trend
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the test.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain was decreased in treated animals, although this was not statistically significant.

FOOD CONSUMPTION
Not measured

FOOD EFFICIENCY
Not measured

WATER CONSUMPTION
Not measured

OPHTHALMOSCOPIC EXAMINATION
Not measured

HAEMATOLOGY
Statistically significant differences were observed in females. Specifically; increased haematocrit in the low dose group.
CLINICAL CHEMISTRY
Statistically significant differences were observed in females. Specifically; increased creatine phospholinase in the mid and top dose groups and sodium in the top dose group.

NEUROBEHAVIOUR
Not measured

ORGAN WEIGHTS
Statistically significant increased lung weights and lung to body weight ratios were observed in a dose dependant manner in the mid and top dose groups.

GROSS PATHOLOGY
Gross lesions were sporadic and were not considered to be caused by the test article.

HISTOPATHOLOGY: NON-NEOPLASTIC
A higher incidence of accumulations of intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium was seen in the lungs of the treated animals. These were dose related at the mid and top dose levels.
Dose descriptor:
NOAEL
Effect level:
49.5 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: Statistically significant dose related increase in lung weight and relative lung weights with corresponding accumulations of intraalveolar macrophages and hyperplasia/hypertrophy of the bronchiole epithelium.
Critical effects observed:
not specified

 

Table 1: Average body weights and body weight gains during 28 days of treatment

 

Analytical concentration (mg/L)

Body Weights (g)

Total Weight Gain

Week 0

Week 1

Week 2

Week 3

Week 4

g

% of control

Male

    0

 217

 249

 295

336

 369

 152

 

LCT

 216

248

294

331

359 

 143

 94

MCT

 215

247 

302 

346

384 

 169

 111

HCT

 215

243 

285 

319

347 

 132

 87

Female

    0

 171

 185

 206

223

 239

 68

 

LCT

 169

 185

 205

220

 232

 63

 93

MCT

 168

 181

 203

219

 229

 61

 90

HCT

 172

 186

 205

223

 238

 66

 97

 

Table 2 Selected haematology, clinical chemistry and pathology findings

 

Doses (unit)

0

50

150

250

0

50

150

250

male

female

Number of animals/group

5

5

5

5

5

5

5

5

Haematology(day x)

 

 

 

 

 

 

 

 

- RBC (TERA/L)

6.62

6.89 

6.66 

6.52 

6.60 

6.90 

6.59 

6.70

- MCV (FL)

 -

- HCT (L/L)

 46

47 

46 

43 

 46

48 

47 

44 

- HGB (MMOL/L)

 16.1

16.7 

16.1 

15.5 

15.5 

16.6 

16.1 

15.5 

- WBC (GIGA/L)

 13.9

13.7 

12.9 

10.5 

9.0 

12.8 

11.5 

13.8 

Blood chemistry(day x)

 

 

 

 

 

 

 

 

- sodium (MMOL/L)

 146

146 

147 

146 

 145

143 

144 

141 

- potassium (MMOL/L)

 4.4

4.4 

4.5 

4.3 

 4.3

4.3 

4.3 

4.3 

- chloride (MMOL/L)

 101

102 

102 

102 

 102

102 

102 

101 

- gobulin (G/L)

 2.2

2.3 

2.4 

2.0 

 2.1

2.1 

2.1 

2.0 

- cholesterol (MMOL/L)

 56

53 

 63

55 

 59

75 

60 

69 

- triglyceride (MMOL/L)

 45

39 

54 

35 

 30

24 

25 

25 

Pathology

 

 

 

 

 

 

 

 

Accumulation of intraalveolar macrophages

 3

5

5

Bronchiolar epithelium: hyperplasia/hypertrophy

 3

Table 3: Absolute and relative organ weights

 

 

Males

Females

DAILY DOSE
(units e.g. mg/kg bw/day)

0

50

150

250

0

50

150

250

NUMBER OF ANIMALS

 5

BODY WEIGHT (g)a

 335

323 

346 

313 

 213

204 

204 

208 

BRAIN

 

 

 

 

 

 

 

 

Absolute Weighta

g

 1.976

1.939 

2.032 

1.943 

 1.900

1.876 

1.856 

1.830 

Per Body Weighta

%

 5.93

6.03 

5.90 

6.21 

 8.96

9.20 

9.12 

8.84 

ADRENALS

 

 

 

 

 

 

 

 

Absolute Weighta

g

 0.051

0.052 

0.054 

0.054 

 0.068

0.069 

0.070 

0.060 

Per Body Weighta

%

 1.52

1.63 

1.56 

1.71 

 3.21

3.37 

3.44 

2.90 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

HEART

 

 

 

 

 

 

 

 

Absolute Weighta

g

 1.220

 1.173

 1.179

 1.122

 0.803

0.801 

0.753 

0.793 

Per Body Weighta

%

 3.64

3.65 

3.41 

3.59 

 3.79

3.92 

3.70 

3.82 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

KIDNEYS

 

 

 

 

 

 

 

 

Absolute Weighta

g

 2.592

2.779 

3.054 

2.704 

 1.745

1.696 

1.694 

1.883 

Per Body Weighta

%

 7.79

8.61 

8.81 

8.61 

 8.16

8.32 

8.29 

9.05 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

 

 

Absolute Weighta

g

 10.775

10.306 

11.470 

9.999 

 7.020

6.464 

6.789 

7.357 

Per Body Weighta

%

 3.22

3.20 

3.31 

3.18 

 3.30

3.17 

3.33 

3.53 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

SPLEEN

 

 

 

 

 

 

 

 

Absolute Weighta

g

 0.670

0.569 

0.652 

0.594 

 0.426

0.441 

0.471 

0.484 

Per Body Weighta

%

 2.00

 1.77

1.88 

1.89 

 2.01

2.17 

2.30 

2.34 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

TESTES

 

 

 

 

n.a.b

n.a.b

n.a.b

n.a.b

Absolute Weighta

g

 3.187

3.072 

2.796 

3.135 

n.a.b

n.a.b

n.a.b

n.a.b

Per Body Weighta

%

 9.54

 9.54

8.16 

10.02 

n.a.b

n.a.b

n.a.b

n.a.b

Per Brain Weighta

%

 

 

 

 

n.a.b

n.a.b

n.a.b

n.a.b

LUNGS

 

 

 

 

 

 

 

 

Absolute Weighta

g

 1.306

1.302 

1.515 

1.537 

 1.051

1.127 

1.138 

1.338 

Per Body Weighta

%

 3.91

 4.05

4.39 

4.91 

 4.93

5.52 

5.59 

6.46 

Per Brain Weighta

%

 

 

 

 

 

 

 

 

OVARIES

n.a.b

n.a.b

n.a.b

n.a.b

 0.0921

0.0768 

0.0943 

0.0742 

Absolute Weighta

g

n.a.b

n.a.b

n.a.b

n.a.b

 4.34

3.77 

4.62 

3.56 

Per Body Weighta

%

n.a.b

n.a.b

n.a.b

n.a.b

 

 

 

 

Per Brain Weighta

%

n.a.b

n.a.b

n.a.b

n.a.b

 

 

 

 

 

Conclusions:
A NOAEL of 49.5 mg/m³ was identified for males and females, based on increased lung weight and microscopic changes of the lung.
Executive summary:

In a subacute inhalation toxicity study, a petroleum derived calcium salt was administered to 5 Sprague-Dawley rats/sex/concentration by whole body exposure at concentrations of 0, 49.5, 156 or 260 mg/m³ for 6 hours per day, 5 days/week for a total of 28 days.

 

Statistically significant, dose related increases in lung weights occurred in the mid and top dose groups, with corresponding increases in intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium.  The LOAEL is 156 mg/m³, based on effects in the lung. The NOAEL is 49.5 mg/m³.

 

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement (OECD 412) for a subchronic inhalation study in the rat.   

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
49.5 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study meets the criteria for Klimisch code 1, however as used in a read-across approach Klimisch 2 is chosen.
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
Route of administration: Dermal, 6 hour/day, to the clipped, unabraided, dorsal surface.
The test substance was applied undiluted to the clipped dorsal surface for periods of 6h per day of study. The material was held in place by a gauze patch secured with tape. After each exposure period the treated area was wiped. The procedure was repeated daily for 28d.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 hours followed by a 14 day recovery in the high dose satellite recovery group only. (daily)
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
o mg/kg bw
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
100 mg /kg bw
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5 Sprague Dawley CD rats/sex
Control animals:
yes, sham-exposed
Details on study design:
Control and treatment groups: 5 rats/sex in the control group, in each dose level and in the satellite recovery group at the 1000 mg/kg/day dose. The control group received no treatment (sham control). The test material was administered undiluted to the treated animal based on individual animal body weight.
- Dose selection rationale: Dose rangefinding study - Dose levels were selected based on results of a rangefinding study conducted at dose levels up to 1000mg/kg/day. No signs of toxicity were observed.
- Rationale for selecting satellite groups: not given
- Post-exposure recovery period in satellite groups: 14 days (high dose group)
Positive control:
None
Observations and examinations performed and frequency:
clinical observations daily
dermal responses on days 0, 1, 4, 7, 11, 14, 18, 21, 25 and prior to blood collection on day 28
body weight and food consumption during treatment and recovery
Hematology and clinical chemistry at termination of treatment and recovery
Microscopic examination on all animals
Sacrifice and pathology:
Full range of evaluations performed
Statistics:
ANOVA with Dunnets test, Kruskal-Walis, Dunns summed rank test , Jonkheere test for monotonic trend, Student's t-test

Body weight, food consumption, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of all dose groups were compared to control at each time interval. Tests included parametric ANOVA with a Dunnett’s test and regression analysis for linear response, non-parametric Kruskal-Wallis and Dunn’s Summed Rank Test, Jonckheere’s test for monotonic trend. A Student’s t-test was used to compare the satellite group’s main study termination and recovery blood values and organ weights.
Dermal irritation:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
No mortality occurred during the study. Low incidences of very slight erythema, desquamation and / or pinpoint scabbing were observed sporadically in all treated animals. All animals were free of edema during the study. Body weights and food consumption were unremarkable. There were no treatment related differences in heamatology. Differences from control were noted for several heamatology parameters including a statistically significant increase in mean % of neutrophils of 300 and 1000 mg/kg females and a decrease in mean % of lymphocytes in the 1000mg/kg females compared to controls on day 28. In the absence of differences from control in absolute white blood cell counts, these findings were not considered related to treatment. There was a statistically significant decrease in mean corpuscular haemoglobin concentration in the male satellite animals from day 28-42. In the absence of other significant findings these small differences were not considered clinically significant. Serum chemistry values were unremarkable. Gross postmortem findings were considered incidental and unrelated to treatment. There were no alterations in organ weights that were attributed to treatment.There were no test material related microscopic findings noted in any group. Effects on the skin were seen in all groups including control but tended to increase in male treated animals and females in the 300 and 1000 mg/kg groups, indicating a mild irritant effect of the test article.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Under the conditions of this study, dermal application of this test material resulted in no signs of overt systemic toxicity.
Critical effects observed:
not specified

A NOAEL of 1000 mg/kg was established for this study. No mortality occurred during this study. Low incidences of very slight erythemia, desquamation and/or pinpoint scabbing were observed sporadically in the treated animals. All animals were free of edema during the study. Body weights and food consumption data were unremarkable during the treatment and recovery periods. There were no treatment-related differences from control observed in the hematology data of the treated animals following the dosing or recovery periods. Differences from control were noted for several hematology parameters including a statistically significant increase in the mean percentage of neutrophils of the 300 and 1000 mg/kg females and a decrease in mean percentage of lymphocytes in the 1000 mg/kg females compared to control on day 28. There was a statistically significant decrease in mean percentage of basophils in the satellite females from day 28 to 42. However these values were within the normal range. In the absence of differences from control in absolute white blood cell counts, these findings were considered unrelated to treatment. There was a statistically significant decrease in the mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration of the male satellite animals from day 28 to 42. In the absence of other significant findings in mean hemoglobin or red blood cell parameters, these small differences were not considered clinically significant. Serum chemistry values were unremarkable in the treated animals at termination of the treatment and recovery periods. There was a slight increase in the mean aspartate aminotransferase and alanine aminotransferase of the high dose females at day 28. These increases were attributed to two females with high values. Similar changes were not observed in the satellite females or in the males at day 28. These increases were not considered related to treatment. There were several differences from control noted at the end of recovery. These values were within the range of normal and similar differences were not evident at the end of the treatment period indicating that these findings were not clinically significant or treatment related. Gross postmortem findings were limited to one 300 mg/kg male with small testes, one control female with discolored lungs and liver and black material in the stomach; and single occurrences of scabs in the 100 and 1000 mg/kg and recovery males. These findings were considered incidental and unrelated to treatment. Tape irritation was observed in a number of animals. There were no alterations in organ weights that were attributed to treatment with the test material. Slight alterations were noted in several organ weights at termination of dosing or recovery. There was a statistically significant decrease in mean absolute brain weight of the 300 mg/kg females compared to control. This finding lacked a dose response and was not considered biologically significant. There was a statistically significant decrease in mean relative adrenal and testes weights of the male satellite animals at termination of recovery compared to control at end of treatment. Compared to the high dose at study termination there was a statistically significant decrease in mean relative adrenal, brain and testes weight of the male satellite animals and mean relative adrenal and brain weight of the female satellite animals at recovery termination. These alterations in organ weights were attributed to the cessation of the stress associated with wrapping (adrenal) and the animals continued increase in body weight while organ weights remained constant in adult animals. In the absences of significant organ weight findings following treatment or correlating effects with histopathology these findings were not considered clinically significant. There were no test material related microscopic findings noted in any group. Livers from female rats of all groups (including control) sacrificed after 28 days of treatment exhibited focal necrosis. This finding did not exhibit a dose response. This finding has been seen in other dermal studies and has been attributed to trauma and/or ischemia to the liver resulting from the wrapping and manipulation of the animals. Liver necrosis was not evident in any of the satellite

recovery animals. This finding was not considered treatment related. The treated skin of most animals revealed variable amounts of thickening of the epidermis due to acanthosis and hyperkeratosis, sebaceous gland hyperplasia and focal dermal inflammation. These changes occurred in all groups including control. However the severity of these changes tended to be increased in the male treated group rats and in the females of the 300 and 1000 mg/kg groups, suggesting a mild irritating effect of the test material. Following recovery these findings were less severe.

Conclusions:
Under the conditions of this study, dermal application of this test material resulted in no signs of overt systemic toxicity.
Executive summary:

The test substance exhibited no evidence of systemic toxicity via the dermal route under the conditions of this study when 5 Sprague Dawley CD rats/sex were exposed to the calcium sulfonate read across substance (CAS 68783 -96 -0) over a period of 28 days. A NOAEL of 1000 mg/kg was established for this study. Under the conditions of this study, dermal application of this test material resulted in no signs of overt systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study meets the criteria for Klimisch code 1, however as used in a read-across approach Klimisch 2 is chosen.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. However as this study is used in the context of a read across, Klimisch 2 is assigned.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Principles of method if other than guideline:
To evaluate the potential dermal effects of OS#68022C when administered dermally to Sprague-Dawley rats over four weeks to the shaved intact dorsal skin.
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Test Animals- Source: Sixty-six CD male rats were received in good condition from Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts.

- Age at study initiation: Approximately 6 weeks old

- Weight at study initiation: 140 to 172g

- Fasting period before study:Not applicable

- Housing: Individually in stain less - steel wire-mesh cages suspended above cage paper.

- Diet: The animals were allowed free access to food; basal ration, NIH Open Formula 07 Rat and Mouse Diet (certified), (Zeigler Brothers, Inc, Gardners, PA).

- Water - animals were allowed free access to tap water supplied to the test facility and monitored for contaminants at periodic intervals according to FDRL Standard Operating Procedures.

- Acclimation period: 20-day acclimation and pre test period.

ENVIRONMENTAL CONDITIONS

- Temperature Range: Not advised

- Humidity Range : Not advised

- Air changes (per hr): Not advised

- Photoperiod (hr dark / hrs light): 12 hours continuous light and 12 hours darkness

IN-LIFE DATES: The study was initiated on November 23, 1987 (first day of treatment) and the in-life phase completed on December 21, 1987.
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test articles were applied undiluted to the shaved intact dorsal skin of each test animal five days per week for four weeks for a total of 20 applications.

The test articles were applied evenly by gentle inunction over the test site using a syringe and a glass rod.

The test articles were held in place with 2 layers of gauze and non irritating tape. All rats were fitted with collars to prevent ingestion of the test articles during the exposure period.

At the end of the six hour exposure period, the dressings were removed and the test sites wiped with a mineral oil gauze to remove as much unabsorbed test article as possible. These sites were then wiped with a clean, dry gauge t o remove any excess mineral oil.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not Applicable
Duration of treatment / exposure:
Six hour exposure period.
Frequency of treatment:
Once daily for five days per week over a four week treatment period.
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
Five males per test article
Control animals:
other: other: Although not identified as such, two groups were considered to be control groups
Details on study design:
- Dose selection rationale:

The selected route of administration was topical, since dermal contact is a likely route of exposure for the general population.

- Rationale for animal assignment (if not random):

Random

- Rationale for selecting satellite groups:

Not applicable

- Post-exposure recovery period in satellite groups:

Not applicable

- Section schedule rationale (if not random):

Random
Positive control:
No
Observations and examinations performed and frequency:
- Mortality Check:
All animals were observed for mortality and overt signs of toxicity twice daily at least 5 hours apart.

- Physical Examination: see appended Appendix 2.
All animals received a detailed physical examination weekly.

- Body Weights: see appended Appendices 3 and 4.
Individual body weights were measured on the first day of test article administration and weekly thereafter.

- Food Consumption: see appended Appendix 5.
Individual food consumption was measured weekly beginning with the first day of test article administration.

Dermal Irritation: see appended Appendix 5.
Application sites were examined for dermal irritation once daily throughout the study period.

Dermal irritation was evaluated according to the method of Draize, 1965.
Sacrifice and pathology:
See attached Pathology Report (Appendix 7):

A complete necropsy examination was conducted on all animals sacrificed at study termination.

Necropsies were performed under the supervision of a board certified veterinary pathologist.

Animals were euthanized using CO2 gas.

Gross necropsy included examination of the external surfaces and orifices, the cranial cavity, carcass, the external and cut surfaces of the brain, the thoracic, abdominal and pelvic cavities and their viscera and the cervical tissues and organs. Untreated and treated skin and all gross lesions were removed from each animal and fixed in 10% neutral buffered formalin.
Other examinations:
Microscopic examination of paraffin embedded hematoxylin and eosin stained tissue sections was performed on the treated and untreated skin and gross lesions from all animals. All tissues were examined by S.W. Thompson, D.V.M., M.S., Diplomate A.C.V.P.
Statistics:
Continuous data including body weight, body weight gain and food consumption w ere analyzed using analysis of variance (Snedecor and Cochran,
1967).
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
- Mortality and Observations: Individual weekly observations are presented in Appendix 2.

No deaths occurred during the study period.

No test article related signs of toxicity were noted throughout the 4-week treatment period.

Three instances of sores and two observations of hair loss a l l in the neck region were noted. These findings were attributed to the collars worn by the rats during the 6-hour exposure period.

- Body Weights: See attached Tables 1 and 2 and Appendices 3 and 4.

No differences in mean body weight or body weight gain were noted among the eight treated groups throughout the study period.

- Food Consumption: See attached Table 3 and Appendix 5.

Mean food consumption, evaluated as grams per animal per week, was comparable among the eight treated groups throughout the study period.

- Dermal Irritation: See attached Appendix 6 for iIndividual dermal irritation scores.

No dermal irritation was noted when test articles OS#65657B, OS#68022C, OS#82288, OS#82289 and OS#75788 were applied at 1000 mg/kg to the skin for five days per week for four weeks.

Pinpoint scabbed areas were noted on the skin of one animal on study on Day 13 when OS#54090A was dosed at 1000 mg/kg. No other signs of dermal irritation were noted for this test article throughout the study period.

Pinpoint scabbed areas were noted on the skin of one animal on study Days 10 through 18 when OS#82287 was dosed at 1000 mg/kg. No other signs of dermal irritation were noted for this test article throughout the study period.

All animals exhibited dermal irritation when dosed with OS#44321P. Well-defined erythema was noted for one animal on study Day 3 and a second animal on study Day 3. The other 3 animals exhibited very slight erythema on study Day 3.

Other findings noted for this group included pinpoint scabbed areas and dry flaking skin. Two animals exhibited dermal irritation for the remainder of the 26 day study period. For animal numbers 0025, 0021 and 0023, no dermal irritation was noted on study days 20, 23 and 24 through day 28 respectively.

- Pathology See attached Appendix 7 for individual gross and microscopic findings.

Compound-related findings were noted at necropsy for two animals treated with GS#34321Y.

Animals numbered 0022 and 0023 exhibited pinpoint scabbed areas over the site of administration. These were the same rats that
exhibited dermal irritation to the end of the study period when observed during the in-life phase.


The other findings noted at necropsy were instances of sores around the neck. These findings correlate with the in-life findings of sores and hair loss for the same animals. This was attributed to the collars worn during the 6-hour dosing period and not considered treatment related.

No other findings were noted at necropsy.

Compound-related microscopic effects were noted in 3 of the 5 rats treated with OS#44321Y. The lesions were observed on the treated skin sites and consisted of minimal (rat numbers 0023 and 0025) to mild (rat number 0022) multifocal eschars. In rat number 0022, mild multifocal hemorrhages in the underlaying dermis also were noted.

All rats exhibited minimal hyperkeratosis of the epithelium accompanied by minimal acanthosis of the epidermis at the site of test article administration. Since this finding occurred in all groups and with essentially no differences in severity, it was considered a response of the skin to repeated shaving and exposure to a foreign material.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Dermal applications did not elicit a test article related effect when applied at a dose level of 1000 mg/kg to the skin for five days per week over a four week treatment period
Critical effects observed:
not specified
Conclusions:
Dermal applications of OS#68022C did not elicit a test article related effect as determined by weekly observations for physical changes and skin irritation and weekly determinations of body weight and food consumption when applied at a dose level of 1000 mg/kg to the skin for five days per week over a four week treatment period

OS#54090A and OS#82287 both elicited minimal dermal irritation. These effects appeared to be transitory as they occurred in only one animal for one day (OS#54090A) and nine days (OS#82287). No treatment-related effects were noted at necropsy or microscopically.

OS#44321Y produced signs of dermal irritation in all treated animals throughout the majority of the 28 day study period.

These signs included slight to well defined erythema, pinpoint scabbed areas and dry flaking skin.

Microscopic observations of minimal to mild multifocal eschar were noted in two rats and mild multifocal hemorrhages in the underlaying dermis in one rat.

OS#44321Y was irritating when applied at 1000 mg/kg to the skin five days per week for four weeks.
Executive summary:

Dermal applications of OS#68022C did not elicit a test article related effect as determined by daily observations for physical changes and skin irritation and weekly determinations of body weights and food consumption when applied at a dose level of 1000 mg/kg to the skin for five days per week over a four week treatment period. Notreatment-related effects were noted at necropsy or microscopically.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000
Study duration:
subacute
Species:
rat
Quality of whole database:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Additional information

Repeated Dose - Oral Exposure

An OECD guideline 407 study in rats with calcium sulphonate (CASN 70024-69-0) allocated 12 rats/sex in the control and top dose and 6 rats/sex in the low and mid dose via gavage at dose levels of 0, 100, 500, or 1000 mg/kg/day. A decrease in serum cholesterol levels occurred in the top dose group. A NOAEL was established at 500 mg/kg/day.

An OECD guideline 407 study on a calcium sulfonate (CASN 115733-09-0) in rats administered 0, 50, 150, 500, or 1000 mg/kg/day to 5 rats/sex/dose via oral gavage. No dose related effects were observed. A NOAEL was established at the high dose level of 1000 mg/kg-day.

In a one-generation reproduction (OECD 415) study, a calcium sulfonate read across substance, (CAS 115733-09-0), was administered in corn oil via oral gavage to 28 Sprague-Dawley rats/sex at dose levels of 0, 50, 167 and 500 mg/kg bw/day (Bjorn, 2004). All F0 males were dosed for 70 days prior to mating, mating (maximum 3 weeks) and through the completion of parturition. All F0 females were dosed for up to 70 days (14 days prior to mating, during mating and gestation and through day 20 of lactation. No substance related effects occurred in treated animals, except for the observation of post dosing salivation and dark material around the nose in the mid and high dose groups in F0 males and the negative ammonium sulfide staining in two high dose and one mid dose F0-female.

Repeated Dose - Inhalation Exposure

According to Annex VIII testing by the inhalation route is not justified since the test substance has low vapour pressure (0.01 Pa at 25 °C) and the potential for the generation of aerosols, particles or droplets of inhalable size is low. Having said this, an OECD guideline 412, GLP subacute rat inhalation study is available on calcium sulfonate (CAS 61789-86-4) a read across substance. Sprague Dawley rats were administered in 49.5, 156, or 260 mg/m3 for 6 hours per day, 5 days per week for 28 days. The test article did not elicit systemic affect at the highest dose but caused a range of local effects in the lung; accumulation of intra-alveolar macrophages, hyperplasia/hypertrophy of the bronchiole epithelium. While these effects were also seen in control animals, there was a dose response in mid and high dose treated animals which was considered treatment related. A NOAEL of 49.5 mg/m³ was identified.

Effects observed in the repeat dose inhalation toxicity study available demonstrated enlarged lungs in high and intermediate dose animals which are likely to be physical effects due to the inhalation of mineral oil [Test material: Product as manufactured in mineral oil solvent further diluted in mineral oil (65/35)] and not necessarily a direct toxicological effect of the registered substance. The data is therefore considered unsuitable for determining the intrinsic hazard of the substance as effects due to the registered substance may well be masked by the effects due to inhalation of mineral oil mist. For example, Occupation Exposure Limits (OELs) expressed for human exposure in the work place are typically 5 mg/m³. Considering a typical intra-species assessment factor of 10 the results demonstrate good correlation to the anticipated effect level for mineral oil mist.

Repeated Dose - Dermal Exposure

The calcium sulfonate read across substance (CAS 68783-96-0) exhibited no evidence of systemic toxicity via the dermal route under the conditions of the study (Sanitised, K., 1995) when 5 Sprague Dawley CD rats/sex were exposed to the test item over a period of 28 days. A NOAEL of 1000 mg/kg was established for this study. Under the conditions of this study, dermal application of this test material resulted in no signs of overt systemic toxicity.

Another weight of evidence study (Laveglia, 1988) refers to the dermal applications of the calcium sulfonate read across substance (CAS 61789-86-4). The test item was applied to the skin of 5 male Sprague Dawley rats at a dose level of 1000 mg/kg for five days per week over a four week treatment period. The substance did not elicit related effect as determined by daily observations for physical changes and skin irritation and weekly determinations of body weights and food consumption. No treatment-related effects were noted at necropsy or microscopically.

Summary

The repeated dose toxicity of the substance has been assessed by over a period of 28-days by three routes of exposure: oral, dermal and inhalation. Effects observed by oral exposure demonstrate a reduction in serum cholesterol at the highest tested dose. Furthermore, the results obtained by inhalation exposure are considered unsuitable for determination of the intrinsic hazard of the substance by inhalation due to the high proportion of mineral oil in the test sample [Test material: Product as manufactured in mineral oil solvent. No local or systemic adverse dermal effects were observed at 1000 mg/kg bw further diluted in mineral oil (65/35)].


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
In a subchronic toxicity study calcium sulphonate was administered to 12 rats/sex in the control and top dose and 6 rats/sex in the low and mid dose via gavage at dose levels of 0, 100, 500 or 1000 mg/kg bw/day. A decrease in serum cholesterol levels occurred in the top dose group. The LOAEL is 1000 mg/kg bw/day, based on  a decrease in serum cholesterol at the top dose.  The NOAEL is 500 mg/kg bw/day.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The test article elicited a range of effects in the lung; accumulation of intraalveolar macrophages, hyperplasia/hypertrophy of the bronchiole epithelium but no systemic effects were observed at the highest dose. While these effects were also seen in control animals, there was a dose response in mid and high dose treated animals which was considered treatment related.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
The test article elicited a range of effects in the lung; accumulation of intraalveolar macrophages, hyperplasia/hypertrophy of the bronchiole epithelium. While these effects were also seen in control animals, there was a dose response in mid and high dose treated animals which was considered treatment related. A NOAEL of 49.5 mg/m³ was identified.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The test substance exhibited no evidence of systemic toxicity via the dermal route under the conditions of this study when 5 Sprague Dawley CD rats/sex were exposed to the calcium sulfonate read across substance (CAS 68783 -96 -0) over a period of 28 days. A NOAEL of 1000 mg/kg was established for this study. Under the conditions of this study, dermal application of this test material resulted in no signs of overt systemic toxicity.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Dermal applications did not elicit a test article related effect when applied at a dose level of 1000 mg/kg to the skin for five days per week over a four week treatment period

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: other

Justification for classification or non-classification

The repeated dose toxicity profile of benzene, mono-C10-13-alkyl derivatives, distillation residues, sulfonated, sodium salt (registered substance/target substance) was not determined by actual acute toxicity studies. Instead, read across substances were used to predict the repeated dose toxicity of the registered substance. As the calcium sulfonate read across substance (CAS 115733-09-0) did not cause relevant significant toxicological effects after repeated oral exposure, benzene, mono-C10-13-alkyl derivatives, distillation residues, sulfonated, sodium salt is also not expected to cause significant toxicity. Therefore, benzene, mono-C10-13-alkyl derivatives, distillation residues, sulfonated, sodium salt does not meet the criteria for classification and will not require labelling, according to the European regulation (EC) No. 1272/2008.