Registration Dossier

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Administrative data

Description of key information

Oral NOAEL (Rat): ≥ 1056 mg/Kg (1.28 mL/Kg) (similar to OECD TG 408)

Inhalation NOAEC (Rat): 690 ppm (3950 mg/m3) (similar to OECD TG 413)

Dermal NOAEL (Rat): ≥ 495 mg/Kg (similar to OECD TG 410)

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given:comparable to guidelines/standards.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- Concentration in vehicle: constant volume dosage of 5 ml/kg bw
Duration of treatment / exposure:
30 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0.14 ml/kg/day (~116 mg/kg bw)
Basis:
other: nominal
Remarks:
Doses / Concentrations:
0.42 ml/kg/day (~347 mg/kg bw)
Basis:
other: nominal
Remarks:
Doses / Concentrations:
1.28 ml/kg/day (~1056 mg/kg bw)
Basis:
other: nominal
No. of animals per sex per dose:
5 female/5 male
Control animals:
yes, concurrent vehicle
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, organs examined include kidneys and livers.
HISTOPATHOLOGY: Yes, organs examined include kidneys.
Other examinations:
Clinical chemistry- including plasma glucose
hematology - including lymphocyte and platelet counts, cell volume, hemoglobin concentration, and erythrocyte counts;
Urinanalysis - including protein concentrations
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
The majority of animals in the 0.42 and 1.48 ml/kg/day groups showed salivation and/or brown facial staining from day 4 onwards, as did three animals in the 0.14 ml/kg/day group. Salivation was normally for a short period, and the staining resolved within 24 hrs.

HAEMATOLOGY
Males rats in the 1.28 ml/kg/day group showed higher lymphocyte and platelet numbers, and slightly lower packed cell volume, hemoglobin concentration and erythrocyte counts.

CLINICAL CHEMISTRY
Plasma glucose levels of rats in the 1.28 ml/kg/day group were lower than controls.

URINALYSIS
Urinary protein concentrations were higher in all male rats in the two higher dose groups, and in 2 males in the lowest dose group.

ORGAN WEIGHTS
Male rats showed a dosage related increase in liver and kidney weights. Female rats only showed higher liver weight at the highest dose level.

GROSS PATHOLOGY
One male rat in the 1.28 ml/kg/day dose group had occasional cystic spaces in the parenchyma of the left kidney.

HISTOPATHOLOGY: NON-NEOPLASTIC
The changes in the kidneys were a slight degeneration of the cells lining the proximal tubules in all treatment groups. There was tubular cell degeneration, tubular dilation with intratubular protein and regeneration. These changes were only found in three males in the low dose groups, and four males each in the medium and high dose groups.

Key result
Dose descriptor:
NOAEL
Effect level:
1.28 other: ml/kg/day
Sex:
female
Basis for effect level:
other: 1056 mg/kg bw
Key result
Dose descriptor:
LOAEL
Effect level:
0.14 other: ml/kg/day
Sex:
male
Basis for effect level:
other: This type of renal pathology is specific to male rats due to an alpha2u-globulin-mediated process that is not relevant to humans.
Critical effects observed:
not specified
Conclusions:
The LOAEL for male rats was 0.14 ml/kg/day based on renal damage. This type of renal damage is specific to male rats, and is not relevant to humans. The NOAEL for female rats was 1.28 ml/kg/day.
Executive summary:

The LOAEL for male rats was 0.14 ml/kg/day based on renal damage. This type of renal damage is specific to male rats, and is not relevant to humans. The NOAEL for female rats was 1.28 ml/kg/day. 

 

This study examined the oral 30 -day subchronic toxicity of BP 8313 to rats. Groups of 5 rats of each sex were given doses of 0.14 (116 mg/kg), 0.42 (347 mg/kg), or 1.28 (1056 mg/kg) mL/kg of test substance in corn oil for 30 days. Animals were examined for clinical signs, mortality, body weight, food consumption, water consumption, and food conversion. After sacrifice clinical chemistry, hematology, clinical chemistry, urinalysis, organ weights, histopathology, and gross pathology were examined. There was no mortality during the experiment. Renal damage was observed in male rats at all dose levels. This type of renal pathology is specific to male rats due to a alpha2u-globulin-mediated process that is not relevant to humans. Female rats exhibited adaptive liver changes at the highest dosage.

The LOAEL for male rats was 0.14 ml/kg/day based on renal damage. The female NOAEL was 1.28 (1056 mg/kg) mL/kg.

Endpoint:
sub-chronic toxicity: oral
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
The 'Justification for the read across' is provided in the 'Attached justification' section below.
Species:
rat
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 056 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1 key and 1 supporting read across study from structural analogues available for assessment.

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
Study period:
1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Shell Toxicology Laboratory (Tunstall) Breeding Unit
- Age at study initiation: 10-13 weeks
- Weight at study initiation: male mean weight: 396-398, female mean weight: 244-245
- Fasting period before study: food removed during exposure
- Housing: three per sex in hanging aluminum cages with stainless steel mesh bases 14 x 10 x 7 in, with two layers of cages for a total of twelve cages per exposure chamber
- Diet (e.g. ad libitum): LAD 1, Spillers Spratts Ltd., replenished daily after exposure
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-22
- Humidity (%): 32-61
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m³ aluminum exposure chamber
- Method of holding animals in test chamber: cages
- Source and rate of air: laboratory air
- Method of conditioning air: dust filters
- System of generating particulates/aerosols: Solvent was evaporated into the air stream using micrometering pumps and vaporizers. Vaporizers were quartz tubes heated to a surface temperature required for complete evaporation of the solvent.
- Temperature, humidity, pressure in air chamber: 17-22°C, 32-61%
- Air flow rate: 2.0 ± 0.03 m³/min
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air: Air was exhausted into the laboratory exhaust which exited on the roof of the laboratory.

TEST ATMOSPHERE
- Brief description of analytical method used: total hydrocarbon analyser fitted with a flame-ionization detector

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Total hydrocarbon analyser fitted with a flame-ionization detector.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week for 13 weeks
Remarks:
Doses / Concentrations:
1293 ppm (7400 mg/m3)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
690 ppm (3950 mg/m3)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
345 ppm (1975 mg/m3)
Basis:
analytical conc.
No. of animals per sex per dose:
18 per sex
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: general health and behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of experiment
- How many animals: all animals
- Parameters checked: erythrocyte count, mean cell volume, hemoglobin, leucocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hematocrit, red cell fragilities, reticulocyte count, prothrombin time, kaolin-cephalin coagulation time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of experiment
- How many animals: all animals
- Parameters checked: total protein, urea nitrogen, alkaline phosphatase, aspartate amino transferase, alanine animo transferase, sodium, potassium, chloride, albumin, bilirubin
- Other: Estimations of blood glucose were made after 10 weeks of exposure using samples taken from the tail vein.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals were examined and the following organs weighed: brain, liver, heart, spleen, kidneys, and testes.

HISTOPATHOLOGY: Yes, the following tissues of the high and medium exposure animals and the control animals were examined: mammary gland, mesenteric lymph node, pancreas, stomach, intestine at 5 levels, caecum, spleen, liver, adrenals, kidneys, ovaries or testes, uterus or prostate, seminal vesicles, urinary bladder, thyroid, trachea, heart, lungs, nasal cavity, thymus, eye and lachrymal glands, salivary glands, brain, spinal cord, pituitary, tongue, sciatic nerves, muscle, knee joint and femur, and macroscopic lesions. The kidneys of low exposure males were also examined.
Statistics:
Body and organ weights were analysed using covariance analysis, with initial body weight as the covariance. Means were adjusted if a significant covariance was found. Organs weights were also analysed using terminal body weights as the covariance. Clinical chemistry and hematological parameters were analysed using analysis of variance. Differences between treatment groups and controls were analysed using Williams t-test. Dunnett's test was used if a monotonic dose response could not be assumed.
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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no deaths during the experiment. High exposure animals were lethargic when examined 30 minutes after exposure. Animals were fully recovered by the next morning.

BODY WEIGHT AND WEIGHT GAIN
Males in the medium and high exposure groups had body weights significantly than controls. High exposure females also had body weights slower than controls.

FOOD CONSUMPTION
No significant differences in food consumption were observed.

WATER CONSUMPTION
Exposed animals showed significant increase in water consumption, particularly animals in the high exposure group.

HAEMATOLOGY
Male PCV, erythrocyte count, mean cell volume, and mean corpuscular hemoglobin were significantly different from controls at all exposure levels. In females, the number of white cells, and mean cell volume were increased in the high exposure group.

CLINICAL CHEMISTRY
Alkaline phosphatase in both sexes, male aspartate amino transferase, and female albumin levels were significantly higher in the high exposure group. Female total protein was also significantly increased in the medium and high exposure groups.

ORGAN WEIGHTS
Male kidney weights at all exposure levels, and spleen weights in the medium and high exposure levels were significantly increased. Female kidney weights at the medium and high exposure levels, and liver weights at all exposure levels were also increased. However, there were no lesions in these organs found during the histopathology examination. These changes in femals were likely hyperfunctional adaptations of the organs rather than a toxic effect.

GROSS PATHOLOGY
Males in the medium and high exposure groups showed a low incidence of splenic enlargement, renal pallor, and hepatic darkening.

HISTOPATHOLOGY: NON-NEOPLASTIC
Kidneys - Male rats in all exposure group showed multiple, hyaline intracytoplasmic, inclusion droplets in the epithelium of the proximal convoluted tubules of their kidneys. This change did not seem to be dose related. These males also showed increase in the number and size of lysosomes in the cytoplasm of the proximal convoluted tubules. Exposure males also had more frequent focal tubular basophilia. Three males in the high exposure group also showed focal tubular dilatation and inspissated debris in the tubular laminae.

Spleen - Males in the medium and high exposure groups showed accelerated erythropoietic activity and increased hemosiderin deposition. Females in the high exposure group showed increased hemosiderin deposition, and mild extramedullary hematopoiesis.

Lungs - No treatment related effects were noted.
Key result
Dose descriptor:
NOAEC
Effect level:
690 ppm
Sex:
female
Basis for effect level:
other: 3950 mg/m3
Key result
Dose descriptor:
LOAEC
Effect level:
345 ppm
Sex:
male
Basis for effect level:
other: 1975 mg/m3; Increased kidney weights as a result of an alpha2u-globulin-mediated process that is not regarded as relevant to humans
Key result
Dose descriptor:
LOAEC
Effect level:
1 293 ppm
Sex:
female
Basis for effect level:
other: 7400 mg/m3
Critical effects observed:
not specified

Mean Body Weights of Male Rats (g)

Week

Control

345 ppm

690 ppm

1293 ppm

Standard Deviation of a Single Observation

0

397

396

396

398

24.9

1

422

422

396

396

20.9 (cage effect)

2

435

434

400

402

21.7

3

444

441

407

407

26.0

4

450

444

423

413

25.5

5

455

452

432

423

25.4

6

464

461

443

431

25.0

7

471

473

449

437

34.2 (cage effect)

8

480

479

460

445

28.2

9

486

486

466

448

30.9

10

494

490

469

453

35.6

11

495

491

478

458

34.9

12

502

495

481

466

36.7

13

512

503

491

473

38.4

Mean Body Weights of Female Rats (g)

Week

Control

345 ppm

690 ppm

1293 ppm

Standard Deviation of a Single Observation

0

244

245

244

245

14.2

1

249

253

252

245

6.2

2

256

261

257

248

9.0

3

264

264

263

252

10.1

4

264

269

266

254

13.5 (cage effect)

5

266

271

267

261

12.9 (cage effect)

6

269

274

269

261

11.4

7

274

278

273

264

11.5

8

275

277

274

263

12.5

9

275

277

272

266

13.8 (cage effect)

10

274

278

273

264

11.3

11

275

279

276

267

12.0

12

280

284

280

271

12.1

13

286

291

289

273

13.3

Conclusions:
The 90-day LOAEC for male rats was 345 ppm (inhalation). This value is based on increased kidney weights as a result of an alpha2u-globulin-mediated process that is not regarded as relevant to humans. The LOAEC was established at 1293 ppm (7400 mg/m3) due to a significant body weight reduction. No other effects were noted. The NOAEC for female rats was 690 ppm (3950 mg/m3).
Executive summary:

This study evaluated the subchronic toxicity of low aromatic white spirits to rats when exposed via inhalation. Groups of 18 rats per sex were exposed to 345, 690, or 1293 ppm of test substance for 6 hrs/day, 5 days/week, for 13 weeks. The highest concentration, 1293 ppm, was near the saturation point for test substance vapor. Rats were observed for clinical signs, mortality, food consumption, water consumption, and body weight. At the end of the exposure period, the animals were sacrificed, and clinical chemistry, hematology, gross pathology, and histopathology parameters were examined. Male rats at all exposure levels had degenerative effects of the as a result of an alpha2u-globulin-mediated process that is not regarded as relevant to humans. The LOAEC was established at 1293 ppm (7400 mg/m3) due to a significant body weight reduction. No other effects were noted. The NOAEC for female rats was 690 ppm (3950 mg/m3).  

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
3 950 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
One key read across sub-chronic toxicity study availablle from structural analogues. NOAEC based on effects on body weight at highest concentration tested.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 411: GLP.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Groups of 12 male and 12 female, individually housed, Sprague-Dawley rats aged 7-9 weeks were used. The males weighed 198-328 g and the females weighed 156-249 g at the initiation of the study.
Type of coverage:
semiocclusive
Vehicle:
other: unknown
Details on exposure:
Test material was applied at concentrations of 20, 40 or 60% (v/v) at a rate of 1 ml/kg/day to the shorn intrascapular region of the rats. This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age were administered mineral oil at a dose rate of 1 ml/kg/day; these animals served as vehicle controls. An additional 12 rats/sex/group in the vehicle controls and high dose group were maintained for a 4-week recovery period following dosing for 13 weeks. All animals were fitted with collars to prevent ingestion and these were removed six hours after dosing and any residual test or control material was wiped from the skin. Animals were observed for clinical signs prior to dosing and 1, 6 and 24 hours after the first dose. Subsequently, observations were made prior to each dose being applied. Prior to the administration of each dose, the treated skin site was evaluated for dermal irritation using the Draize scoring method. Body weights were recorded prior to the first dose and weekly thereafter. An ophthalmic examination was conducted on each rat prior to application of the first dose and again prior to sacrifice at the end of the study. During the week prior to the first dose, each rat was subjected to a functional observation battery (FOB). The FOB was conducted again 1 and 24 hours after the first dose and at 7 and 14 days. During the study, the FOB, motor activity and startle response testing was conducted on all rats at weeks 4, 8 and 12. [The details of the FOB, the test for startle response test and the test for motor activity are given in detail in the laboratory report but are not included here]. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups).
Duration of treatment / exposure:
This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks.
Frequency of treatment:
This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks.
Remarks:
Doses / Concentrations:
0, 165, 330 or 495 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
Groups of 12 male and 12 female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 12 male and 12 female, individually housed, Sprague-Dawley rats aged 7-9 weeks were used. The males weighed 198-328 g and the females weighed 156-249 g at the initiation of the study.
Test material was applied at concentrations of 20, 40 or 60% (v/v) at a rate of 1 ml/kg/day to the shorn intrascapular region of the rats. This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age were administered mineral oil at a dose rate of 1 ml/kg/day; these animals served as vehicle controls. An additional 12 rats/sex/group in the vehicle controls and high dose group were maintained for a 4-week recovery period following dosing for 13 weeks. All animals were fitted with collars to prevent ingestion and these were removed six hours after dosing and any residual test or control material was wiped from the skin. Animals were observed for clinical signs prior to dosing and 1, 6 and 24 hours after the first dose. Subsequently, observations were made prior to each dose being applied. Prior to the administration of each dose, the treated skin site was evaluated for dermal irritation using the Draize scoring method. Body weights were recorded prior to the first dose and weekly thereafter. An ophthalmic examination was conducted on each rat prior to application of the first dose and again prior to sacrifice at the end of the study. During the week prior to the first dose, each rat was subjected to a functional observation battery (FOB). The FOB was conducted again 1 and 24 hours after the first dose and at 7 and 14 days. During the study, the FOB, motor activity and startle response testing was conducted on all rats at weeks 4, 8 and 12. [The details of the FOB, the test for startle response test and the test for motor activity are given in detail in the laboratory report but are not included here]. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups).

The following hematological and clinical chemical parameters were measured.
HEMATOLOGY:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Platelet count
Reticulocyte count
Total leukocyte count
Differential leukocyte count
Morphological examination of erythrocytes and platelets Coagulation determinations (prothrombin time & activated partial thromboplastin time) were also carried out on six animals from each group at week 14 and from the recovery groups at the week 18 necropsy.

CLINICAL CHEMISTRY
Blood urea nitrogen,
Creatinine,
Serum aspartate aminotransferase,
Serum alanine aminotransferase,
Alkaline phosphatase,
Lactate dehydrogenase,
Sorbitol dehydrogenase,
Gamma glutamyl transferase,
Creatinine kinase,
Serum glucose
Total, direct and indirect bilirubin
Total protein
Albumin
Calcium
Phosphorus
Sodium
Potassium
Chloride
A complete necropsy was performed on six rats/sex/group following 13 weeks dosing, and on 6 rats/sex/group of the recovery animals (high dose and controls) at week 18. A limited necropsy was performed on the remaining six animals and their organs were not weighed (see below). Each full necropsy included an examination of the external surface of the body, all orifices, cranial, thoracic, abdominal and pelvic cavities and their contents. Gross observations were recorded and the following organs were weighed: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, spleen, testes, thymus and uterus.

The following tissues were collected, processed and then examined microscopically.
Adrenal glands,
Nose (nasal cavity & turbinates),
Animal identification,
Ovaries,
Bone marrow (from sternum),
Oviducts,
Brain,
Pancreas,
Epididymides,
Parathyroid glands,
Esophagus,
Pituitary gland,
Exorbital lacrimal glands,
Prostate Eyes with optic nerve,
Salivary glands,
Femur (incl. articular surface),
Seminal vesicles,
Gross lesions Skin (application site),
Harderian gland,
Skin (inguinal),
Heart and aorta,
Spinal cord (3 levels),
Intestine (3 levels),
Spleen,
Kidneys,
Stomach,
Larynx and pharynx,
Testes,
Liver,
Thymus,
Lungs with mainstream bronchi,
Thyroid gland,
Lymph nodes (mandibular/mesenteric),
Urinary bladder,
Mammary glands with adjacent skin,
Uterus Muscle (thigh),
Vagina Nerve (sciatic).

The remaining six rats of each group were anesthetized with an intraperitoneal injection of Pentothal ® and transcardially perfused in-situ using 10% neutral-buffered formalin and given a limited necropsy. For these rats, no organs were weighed and the following tissues were collected: Head/skull, Sural nerve, Brain, Tibial nerve, Spinal cord, Gross lesions, Sciatic nerve.

The following tissues were examined microscopically in these animals: Brain (forebrain, cerebrum, midbrain, cerebellum, pons and medulla obligata), Gasserian ganglia, Dorsal root ganglia, Dorsal and ventral root fibers, Sural nerve, Tibial nerve, Spinal cord (cervical and lumbar areas), and Sciatic nerve.
Observations and examinations performed and frequency:
Groups of 12 male and 12 female, individually housed, Sprague-Dawley rats aged 7-9 weeks were used. The males weighed 198-328 g and the females weighed 156-249 g at the initiation of the study.
Test material was applied at concentrations of 20, 40 or 60% (v/v) at a rate of 1 ml/kg/day to the shorn intrascapular region of the rats. This was equivalent to doses of test material of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age were administered mineral oil at a dose rate of 1 ml/kg/day; these animals served as vehicle controls. An additional 12 rats/sex/group in the vehicle controls and high dose group were maintained for a 4-week recovery period following dosing for 13 weeks. All animals were fitted with collars to prevent ingestion and these were removed six hours after dosing and any residual test or control material was wiped from the skin. Animals were observed for clinical signs prior to dosing and 1, 6 and 24 hours after the first dose. Subsequently, observations were made prior to each dose being applied. Prior to the administration of each dose, the treated skin site was evaluated for dermal irritation using the Draize scoring method. Body weights were recorded prior to the first dose and weekly thereafter. An ophthalmic examination was conducted on each rat prior to application of the first dose and again prior to sacrifice at the end of the study. During the week prior to the first dose, each rat was subjected to a functional observation battery (FOB). The FOB was conducted again 1 and 24 hours after the first dose and at 7 and 14 days. During the study, the FOB, motor activity and startle response testing was conducted on all rats at weeks 4, 8 and 12. [The details of the FOB, the test for startle response test and the test for motor activity are given in detail in the laboratory report but are not included here].
Sacrifice and pathology:
At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups).

The following hematological and clinical chemical parameters were measured.
HEMATOLOGY:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Platelet count
Reticulocyte count
Total leukocyte count
Differential leukocyte count
Morphological examination of erythrocytes and platelets Coagulation determinations (prothrombin time & activated partial thromboplastin time) were also carried out on six animals from each group at week 14 and from the recovery groups at the week 18 necropsy.

CLINICAL CHEMISTRY
Blood urea nitrogen,
Creatinine,
Serum aspartate aminotransferase,
Serum alanine aminotransferase,
Alkaline phosphatase,
Lactate dehydrogenase,
Sorbitol dehydrogenase,
Gamma glutamyl transferase,
Creatinine kinase,
Serum glucose
Total, direct and indirect bilirubin
Total protein
Albumin
Calcium
Phosphorus
Sodium
Potassium
Chloride
A complete necropsy was performed on six rats/sex/group following 13 weeks dosing, and on 6 rats/sex/group of the recovery animals (high dose and controls) at week 18. A limited necropsy was performed on the remaining six animals and their organs were not weighed (see below). Each full necropsy included an examination of the external surface of the body, all orifices, cranial, thoracic, abdominal and pelvic cavities and their contents. Gross observations were recorded and the following organs were weighed: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, spleen, testes, thymus and uterus.

The following tissues were collected, processed and then examined microscopically.
Adrenal glands,
Nose (nasal cavity & turbinates),
Ovaries,
Bone marrow (from sternum),
Oviducts,
Brain,
Pancreas,
Epididymides,
Parathyroid glands,
Esophagus,
Pituitary gland,
Exorbital lacrimal glands,
Prostate Eyes with optic nerve,
Salivary glands,
Femur (incl. articular surface),
Seminal vesicles,
Gross lesions Skin (application site),
Harderian gland,
Skin (inguinal),
Heart and aorta,
Spinal cord (3 levels),
Intestine (3 levels),
Spleen,
Kidneys,
Stomach,
Larynx and pharynx,
Testes,
Liver,
Thymus,
Lungs with mainstream bronchi,
Thyroid gland,
Lymph nodes (mandibular/mesenteric),
Urinary bladder,
Mammary glands with adjacent skin,
Uterus Muscle (thigh),
Vagina Nerve (sciatic).

The remaining six rats of each group were anesthetized with an intraperitoneal injection of Pentothal ® and transcardially perfused in-situ using 10% neutral-buffered formalin and given a limited necropsy. For these rats, no organs were weighed and the following tissues were collected: Head/skull, Sural nerve, Brain, Tibial nerve, Spinal cord, Gross lesions, Sciatic nerve.

The following tissues were examined microscopically in these animals: Brain (forebrain, cerebrum, midbrain, cerebellum, pons and medulla obligata), Gasserian ganglia, Dorsal root ganglia, Dorsal and ventral root fibers, Sural nerve, Tibial nerve, Spinal cord (cervical and lumbar areas), and Sciatic nerve.
Statistics:
Statistics Normally-distributed in-life data (parametic) were analyzed for test substance effects by analysis of variance and pairwise comparisons made between groups using Dunnett's test. Nonparametric data (nonhomogenous as determined by Bartlett's test) were analyzed using a modified t-test. Statistical significance was reported at the P < 0.05 level. Statistical analyses of neurobehavior data (FOB and motor activity) are described in the results section.
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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:
There were no test substance-related effects on survival, clinical observations (apart from skin irritation), neurobehavioral signs or ophthalmological findings. The only clinical observations during the study were related to skin irritation at the application site. There was a generally dose-related increase in the incidence and severity of erythema, edema, epidermal scaling, scab formation, thickening of the skin and ulceration at the treated site. Males seemed to be more sensitive than females. The FOB screen did not demonstrate any substance-related effects. The areas monitored were: behavioral parameters, including autonomic, muscle tone and equilibrium, sensorimotor responses, central nervous system. In addition the test substance had little effect on motor activity or startle response. Growth rates were unaffected by treatment. At necropsy no substance-related observations were made for males in any group. In the females there was a suggestion of a possible treatment- related effect which occurred in 7 rats across all groups and consisted of skin crusts or ulceration at the site of application of test material. Hematological and serum clinical parameters were unaffected by treatment. The only organ weight effects noted were an increase in spleen/body weight and spleen/brain weight ratios in the high dose group females at the 13 week necropsy and an increase in absolute spleen weight in the same dose group females after the 4 weeks recovery period. Since there were no associated microscopic or clinical chemical findings, these differences were not considered to be of biological relevance. There were no treatment-related microscopic changes in the tissues examined with the exception of the findings in the skin. The skin observations were minimal in nature with a severity score less than 1 on a 1 [low] to 4 [severe] scale. The findings included acanthosis, ulceration, parakeratosis, chronic active inflammation and hyperkeratosis. The males were affected at all doses, however, the effects indicated very little irritation. Recovery group animals revealed complete recovery in the females and minimal hyperkeratosis in the high dose group males. No effects were found in the animals subjected to a detailed neuropathological examination.
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
> 495 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No systemic or neurological effects were noted in the study
Critical effects observed:
not specified
Conclusions:
There were no systemic or neurological effects noted at any of the tested doses. The systemic NOAEL was >495 mg/kg/day.
Executive summary:

Test material was applied at concentrations of 165, 330 or 495 mg/kg/day. Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age was administered mineral oil as vehicle controls and an additional high dose group was maintained for a 4-week recovery period following dosing for 13 weeks. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups). There were no systemic or neurological effects noted at any of the tested doses. The systemic NOAEL was >495 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
495 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
One key read across sub-chronic toxicity study from strucural analogues available for assessment.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No oral, inhalation, or dermal repeat dose toxicity data is available for Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, aromatics (2-30%). However, data is available for structural analogues, Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) and Kerosene. Petroleum substances of similar carbon number and aromatic content, principally kerosene and jet fuel, are typically in the range of C9-C16. These substances also contain similar types of molecules in similar proportions to those in C14-C20 aliphatic [2-30% Aromatics] Hydrocarbon solvents. In general, hydrocarbon solvents are more highly refined than petroleum substances. Accordingly, the petroleum substances typically represent a “worse case” with respect to hydrocarbon solvents and can be used for read across on that basis. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Oral

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)

A key repeat dose sub-chronic oral toxicity study (DHC, 1984a), examined the oral 30 -day subchronic toxicity of Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) in rats. Groups of 5 rats of each sex were given doses of 0.14 (116 mg/kg), 0.42 (347 mg/kg), or 1.28 (1056 mg/kg) mL/kg of test substance in corn oil for 30 days. Animals were examined for clinical signs, mortality, body weight, food consumption, water consumption, and food conversion. After sacrifice clinical chemistry, hematology, clinical chemistry, urinalysis, organ weights, histopathology, and gross pathology were examined. There was no mortality during the experiment. Renal damage was observed in male rats at all dose levels. This type of renal pathology is specific to male rats due to a alpha2u-globulin-mediated process that is not relevant to humans. Female rats exhibited adaptive liver changes at the highest dosage.

 

The LOAEL for male rats was 0.14 mL/Kg/day based on renal damage (specific to male rats, and is not relevant to humans). The female NOAEL was 1.28 (1056 mg/Kg) mL/Kg.

Kerosene

In a supporting study (Mattie et al. 1995), male rats were given 0, 750, 1500, or 3,000 mg/Kg of the test material (JP-8) daily by gavage for 90 days. The test material was administered without a vehicle. Rats were given the last dose on day 90 and sacrificed within 3.8 ± 1.8 hours after dosing. Exposure to high concentrations of JP-8 by the oral route resulted in minimal toxicity except for a decrease in body weight. The NOAEL was determined to be 750 mg/kg/day based on body weight reduction.

Additionally, in order to comply with standard information requirements for Annex X substances, OECD Guideline 90-day sub-chronic (OECD 408) toxicity tests are proposed for structural analogues Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%) (EC# 919-006-8) and Hydrocarbons, C11-C15, aromatics, <1% naphthalene (EC# 922-153-0). The testing proposals for the same have been presented in the lead registrant dossiers for these substances already submitted to ECHA. These studies will be conducted subsequent to ECHA's approval and this endpoint will be updated upon completion of the above studies.

Inhalation

Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)

A key sub-chronic repeated dose study (Shell, 1980a) evaluated the subchronic toxicity of low aromatic white spirits to rats when exposed via inhalation. Groups of 18 rats per sex were exposed to 345, 690, or 1293 ppm of test substance for 6 hrs/day, 5 days/week, for 13 weeks. The highest concentration, 1293 ppm, was near the saturation point for test substance vapor. Rats were observed for clinical signs, mortality, food consumption, water consumption, and body weight. At the end of the exposure period, the animals were sacrificed, and clinical chemistry, hematology, gross pathology, and histopathology parameters were examined. Male rats at all exposure levels had degenerative effects of the as a result of an alpha2u-globulin-mediated process that is not regarded as relevant to humans. The LOAEC was established at 1293 ppm (7400 mg/m3) due to a significant body weight reduction. No other effects were noted. The NOAEC for female rats was 690 ppm (3950 mg/m3).  

 

Kerosene

In a supporting study (API, 1986a), groups of 20 male and 20 female Sprague Dawley rats (aged approximately six weeks) were exposed to a nominal concentration of 25mg/m³ of the test material (hydrodesulfurized kerosene) by inhalation. Exposures were for approximately six hours each day, five days each week for four consecutive weeks. Control groups of 20 male and 20 females were exposed to filtered air. Animals were observed twice daily for overt signs of toxicity and they underwent detailed examination once weekly. Body weights were also recorded weekly. At study termination, the animals were killed and blood samples were taken for the following clinical chemical and hematological investigations.

 

There were no treatment-related effects on clinical condition, growth rate organ or organ body weight ratios or on any of the hematological or clinical chemistry determinations. Furthermore, there were no treatment-related microscopic changes observed in any of the organs examined. The NOAEC was determined to be 25mg/m3, which was the highest concentration tested.

Dermal

C14-C20 aliphatics, 2-30% aromatics are expected to have a low order of repeated dose toxicity by the dermal route of exposure. Available read-across data from the structurally analogous test material hydrodesulfurized kerosene was analysed. All tests were performed in a manner similar or equivalent to currently established OECD guidelines. Test material was applied at concentrations of 165, 330 or 495 mg/Kg/day (American Petroleum Institute (1997)). Dosing was continued daily for five consecutive days each week, five days a week for 13 weeks. In addition a group of 12 male and 12 female rats of similar age was administered mineral oil as vehicle controls and an additional high dose group was maintained for a 4-week recovery period following dosing for 13 weeks. At the 14 week necropsy, blood samples were collected from 12 animals/sex/group and at the week 18 necropsy from the recovery rats (vehicle and high dose groups). There were no systemic or neurological effects noted at any of the tested doses. The systemic NOAEL was ≥495 mg/kg/day.

These results do not warrant classification under the new Reguation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Justification for classification or non-classification

Based on available read-across from structurally related substances, Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, 2-30% aromatics does not meet the criteria for classification for repeated dose toxicity (STOT-RE) under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).