Hydrocarbons, C10-C13, aromatics, <1% naphthalene

Endpoint summary

Description of key information

Repeated Dose Oral 90d - NOAEL = 300 mg/kg for rats (similar to OECD TG 408)
Repeated Dose Inhalation 12 month - NOAEL >= 900 mg/m3 for rats (similar to OECD TG 413), read-across material C9 Aromatics

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990/10/30-1991/02/27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: In agreement to OECD guideline 408.

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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Harlan Sprague Dawley Inc.
Sex: Male (50), Female (50)
Age at study initiation: Approximately 8 weeks
Weight at study initiation: Males: 253.0 - 328.4 g; Females: 172.3 - 229.1 g
Housing: Individually
Diet (e.g. ad libitum): Purina Certified Rodent Chow 5002, ad libitum
Water (e.g. ad libitum): Automatic watering system, ad libitum
Acclimation period: 29d

ENVIRONMENTAL CONDITIONS
Temperature (°F): 68-76
Humidity (%): 40-70%
Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test mixture was administered by oral gavage at a dose of 0, 300, 600, or 1000 mg/ kg at a volume of 2.4 ml/kg, 7 days per week for a period of at least 13 weeks. The control animals received a carrier (corn oil) dose 2.4 ml/kg. The satellite group was dosed at 1000 mg/ kg, 7 days/week for ~13 weeks and was then observed for reversibility, persistence or delayed occurrence of toxic effects for 28 days post-treatment. The amount of test material administered to each animal was recalculated weekly, based on the most recent body weight.

Duration of treatment / exposure:

once daily

Frequency of treatment:

7 days/week for ~13 weeks

Remarks:

Doses / Concentrations:
0, 300, 600, or 1000 mg/ kg
Basis:
other: oral gavage

No. of animals per sex per dose:

Males (10). Females (10) per dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

The test mixture was administered by oral gavage at a dose of 300, 600, or 1000 mg/ kg at a volume of 2.4 ml/kg, 7 days per week for a period of at least 13 weeks. The control animals received a carrier (corn oil) dose 2.4 ml/kg. The satellite group was dosed at 1000 mg/ kg, 7 days/week for ~13 weeks and was then observed for reversibility, persistence or delayed occurrence of toxic effects for 28 days post-treatment. The amount of test material administered to each animal was recalculated weekly, based on the most recent body weight.

The animals were checked for viability twice daily on Monday through Friday, and once daily on Saturday and Sunday. Clinical observations were made daily for toxicological signs. Body weights were recorded prior to dosing and then once a week thereafter. Body weights were also recorded at the scheduled sacrifice at the death of animals that succumbed before the end of the study. Clinical laboratory studies (hematology and serum chemistry) were performed on all animals at main study termination or at the termination of the satellite/recovery study. Blood samples were collected under methoxyflurane anesthesia from the orbital sinus.

Positive control:

none

Observations and examinations performed and frequency:

The animals were checked for viability twice daily on Monday through Friday, and once daily on Saturday and Sunday. Clinical observations were made daily for toxicological signs. Body weights were recorded prior to dosing and then once a week thereafter. Body weights were also recorded at the scheduled sacrifice at the death of animals that succumbed before the end of the study. Clinical laboratory studies (hematology and serum chemistry) were performed on all animals at main study termination or at the termination of the satellite/recovery study. Blood samples were collected under methoxyflurane anesthesia from the orbital sinus.

Sacrifice and pathology:

The necropsy was performed on all animals. The necropsy examined the external surfaces of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. The ovaries, kidneys, adrenal, liver, and testes were weighed prior to fixation. Preserved tissue from the control and high dose group as well from all animals that were sacrificed moribund or succumbed during the study were processed, sectioned, stained (hematoxylin and eosin) and examined microscopically. Abnormalities from the low and mid doses were examined as well as the stomach, spleen, urinary bladder, and the thyroid.

Other examinations:

HEMATOLOGY
-erythrocyte count
-hematocrit
-hemoglobin
-leukocyte count (total and differential)
-mean corpuscular volume
-mean corpuscular hemoglobin
-mean corpuscular hemoglobin conc.
-platelets

SERUM CHEMISTRY
-total bilirubin
-albumin
-blood urea nitrogen
-calcium
-cholesterol
-creatinine
-electrolytes (Na, K, Cl)
-glucose
-total protein
-triglycerides
-phosphorous
-gamma glutamyl transferase
-serum aspartate aminotransferase
-serum alanine aminotransferase

Statistics:

The following parameters were statistically analyzed:
-mean hematology parameters
-mean serum chemistry parameters
-mean organ weights
-mean organ to body weights, by weighing period
-mean food consumption

Comparisons were limited to within sex analysis. Variance was assessed through the use of a one way ANOVA. Bartlett’s test (conducted at the 1% level of significance) was performed to determine if the dose groups had equal variance. If the variances were equal, the test was done using parametric methods, otherwise nonparametric techniques were used.

The parametric procedures used a one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett’s test was used to determine which treatment groups differ significantly from control. In addition, a regression analysis for linear response in the dose group was performed.

The nonparametric procedures tested for the equality of means using the Kruskal-Walls test. If significant differences were indicated, Dunn’s Summed Rank test was used to determine which treatment group differed significantly from control. Jonckheere’s test for monotonic trend in the dose response was performed.

Bartlett’s test was conducted at the 1% level of significance. All other tests were conducted at the 5% and 1% level of significance.

The t-test was used to compare the satellite group’s main study termination and recovery termination hematology and clinical chemistry values. The t-test was also used to compare the satellite and the control groups’ organ weights. The t-test was also used to compare the high dose and satellite groups to ensure similar results in order to accurately evaluate the recovery effects.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

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 a total of 10 animal deaths; four were attributed to dosing trauma or aspiration of the test material. Two animals (1 female at 300 mg/kg and a female in the satellite group) died of unknown causes. The four remaining deaths (1 male, 3 female) at the 1000 mg/kg dose appeared to be treatment-related. Animals in the 0, 300, or 600 mg/kg dose displayed little to no clinical signs. Animals in the 1000 mg/kg dose consistently displayed signs of ano-genital staining, alopecia, and emaciation. Note: The animals in the 1000mg/kg dose were initially treated with a dose of 1200 mg/kg, but displayed severe signs of hypothermia and hypoactivity and after day 3, their dose was reduced to 1000mg/kg. Sporadic clinical signs across all groups included oral discharge, red discharge from the eyes, rales, sores, scabs, soft stool, and little sign of stool.

BODY WEIGHT and WEIGHT GAIN
Statistically significant decreases in body weight for males at the 1000 mg/kg dose were noted in all intervals from day 7 through termination. There were no noted differences in any of the female groups.

FOOD CONSUMPTION
Food consumption for males at the 1000 mg/kg dose was decreased for week 1 when compared to controls. Food consumption was significantly higher for males at the 600 mg/kg dose at weeks 10-13. Food consumption was significantly higher for females at the 600 and 1000 mg/kg dose at weeks 7-10.

HAEMATOLOGY
Non-biologically relevant but statistically significant decreases were noted in:
-Hematocrit: Males at 300, 600, and 1000 mg/kg doses; Females at 600 mg/kg dose. There is no dose response for male rats. The hematocrit count was statistically affected at all does levels; however, all treated male rats had hematocrit counts that were 95% control. Hematocrit counts were only statistically affected for the female rats at 600 mg/kg (96% of control). Hematocrit counts were not different from controls in the recovery group that was initially dosed with 1000 mg/kg/day. Hematocrit levels returned to normal in the recovery group (initially dosed with 1000 mg/kg). Since only the male rats were statistically affected, no dose response was observed, and experimental values were within 10% of controls (normal physiological range), these changes are not considered to be an adverse effect.
-Hemoglobin: Males at 300, 600, and 1000 mg/kg doses: Females 600, and 1000 mg/kg dose. Male rats exposed to 300, 600, and 1000 mg/kg had decreased hemoglobin counts of 95%, 95%, and 92.5% of control, respectively. Female rat hemoglobin was 93% of control at the 600 and 1000 mg/kg doses. Hemoglobin concentrations returned to normal in the recovery group (initially dosed with 1000 mg/kg). Since no dose response was observed in either the male or female rats, and experimental values were within 10% of controls (normal physiological range), these changes are not considered to be an adverse effect.
-Mean corpuscular hemoglobin concentration (MCHC): Males at 300, 600, and 1000 mg/kg doses: Females 600, and 1000 mg/kg dose. Only male rats in the 1000 mg/kg were statistically affected, however, the MCHC level was 97.5% of control. Female rats were statistically affected at all doses, however, no dose response was observed. The values (% control) for MCHC were 96.7%, 96.9%, 96% for the 300, 600, and 1000 mg/kg treated female rats respectively. MCHC levels returned to normal in the recovery group (initially dosed with 1000 mg/kg). Since only the female rats were statistically affected, no dose response was observed, and experimental values were within 10% of controls (normal physiological range), these changes are not considered to be an adverse effect.
- Bone Marrow – There were no adverse pathology findings of the bone marrow in any animal at any dose.
- RBC– Only male rats in the 600 and 1000 mg/kg were statistically affected. The values for RBC (% control) were 95% and 91.5% for the 600 and 1000 mg/kg groups, respectively. Female rats RBC were not significantly decreased at any test dose. RBC levels returned to normal in the recovery group (initially dosed with 1000 mg/kg). Conclusion: Since only the male rats have a significant decrease in RBC counts, it is unlikely that this change is an adverse effect or is related to the splenic hemosiderosis observed in both sexes of rats. The decrease observed in the male rats was less than 10% of control (normal physiological range) and these changes are not considered to be an adverse effect.

The hematology for the recovery period revealed a recovery trend for all of the observed decreased parameters; including a significant change in the red blood cells, hemoglobin, MCHC for both males and females.

CLINICAL CHEMISTRY
Statistically significant increases were noted in mean serum chemistry values in:
-Albumin: Females at 600 and 1000 mg/kg doses
-Creatinine: Females at 1000 mg/kg doses
-Potassium: Males at 1000 mg/kg doses
-Total Protein: Females at 600 and 1000 mg/kg doses
-Cholesterol: Females at 600 and 1000 mg/kg doses
Sporadic, statistically significant increases were also noted in albumin (males) and transferase (females) and significant decreases were noted in chloride (females) and triglycerides (males).

ORGAN WEIGHT
There was a statistically significant increase in male liver weight (600 mg/kg) and in male kidney weight (300 and 600 mg/kg). Females demonstrated a significant dose related increase in both liver and kidney weight. The relative organ weights for the recovery period revealed a recovery trend in the liver and testes.

GROSS PATHOLOGY and HISTOPATHOLOY
The most frequently noted abnormalities in surviving animals were cervical lymph node redness, abnormal small/large intestine contents, small/large intestine distention, staining of the fur, alopecia, and eye damage from eye bleeding. The most frequent abnormalities for the animals which succumbed prior to study termination were: thickened liver, small spleen, enlarged adrenals, staining of fur, emaciation, abnormal stomach/small/large intestine contents, stomach distension, and stomach mucosa thinner than normal.

Treatment related changes were noted in the livers of the females of all dosage groups consisting of centrilobular hepatocellular hypertrophy. These liver changes were noted at a sporadic and lower incidence in male rats. Hepatocellular hypertrophy was not observed in the satellite group that was allowed to recover. A dose related increase amount of brown pigment (hemosiderin) in macrophages in the splenic red pulp was observed in all animals that received 600 and 1000 mg/kg doses. This hemosiderin material was still present after recovery, but was reduced. An increased incidence of minimal to moderate hyperplasia and hypertrophy of the thyroid follicular epithelial cells was noted in all male animal dosage groups and in females exposed to 600 and 1000 mg/kg. These effects were reversed in the recovery group. In response to xenobiotics, there is a greater predisposition towards proliferate lesions (e.g. hyperplasia) of follicular cells in laboratory rodents, especially males, than in the human thyroid. Capen et al. Toxicol Pathol 1989; 17:266 as cited in Werner and Ingbar’s The Thyroid. 7th ed. Braverman and Utiger. 1991. Since these effects were reversible in the recovery group and due to the rodent specific response, this effect at the lower dose is deemed to be an adaptive response.

Recovery Group:
The relative organ weight, clinical chemistry and hematology data indicated recovery during the 28-day post exposure period. The satellite group values were similar to control values.

Liver and thyroid changes seen at termination appeared to be reversible as they were not observed in the satellite group after the 28-day recovery period. The changes seen in the spleen, urinary bladder and stomach in the main study termination were at a lower incidence and/or severity in the satellite group after the 28-day recovery period also indicating reversibility. The hematology, serum chemistry and relative organ weights also indicated reversibility of the effects.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Mortality observed at the highest dose tested. Food consumption was higher at the 600 and 1000 mg/kg doses in both sexes. Clinical chemistry and haematology parameters were affected at the 00 and 1000 mg/kg doses in both sexes.

Critical effects observed:

not specified

Conclusions:

The NOAEL for MRD-90-884 is 300 mg/kg body weight under the test conditions of this study.

Executive summary:

A 90-day subchronic study was conducted in rats to assess the toxicity of MRD-90-884. The test mixture was administered by oral gavage at a dose of 0, 300, 600, or 1000 mg/kg 7 days per week for a period of 13 weeks.  The control animals received a carrier (corn oil) dose and a satellite group was dosed at 1000 mg/kg, 7 days/week for 13 weeks and was then observed for reversibility, persistence or delayed occurrence of toxic effects for 28 days post-treatment.  Observations were made as to the nature, onset, severity, and duration of toxicological signs. There were a total of 10 animal deaths; four deaths (1 male, 3 female) at the 1000 mg/kg dose were attributed to the treatment.   There was an overall low incidence of clinical signs at the 300 and 600 mg/kg doses.  Low food consumption and emaciation were observed in animals dosed at 1000 mg/kg.  Post mortem examinations revealed a significant increase in liver and kidney weights in male and female animals as well as increase in body weight for all animals except for males in the high dose group.  Organ weight, clinical chemistry, and hematology data from the satellite recovery group indicated recovery during the 28 day recovery period.  Based on the data recorded in this study, the NOAEL for MRD-90-884 is 300 mg/kg.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study planned

Study period:

Will be completed in the timescale as indicated in the ECHA decision letter.

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : Hydrocarbons, C11-C15, aromatics, <1% naphthalene (EC# 922-153-0)
- Name of the substance for which the testing proposal will be used [if different from tested substance] : Not different

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies : There are no OECD Guideline 408 studies available on this substance to evaluate the sub-chronic toxicity endpoint. These data only exist for structural analogues.
- Available non-GLP studies : There are no ‘non-GLP’ studies available for this substance to evaluate the sub-chronic toxicity endpoint. These data only exist for for structural analogues.
- Historical human data: No human data exist for this substance to evaluate sub-chronic toxicity hazard.
- (Q)SAR : There are no recognised (Q)SAR methods available for reliable prediction of sub-chronic toxicity.
- In vitro methods : There are no in vitro methods currently accepted by Regulatory Authorities for the reliable prediction of sub-chronic toxicity.
- Weight of evidence : Currently there are insufficient data available to develop a robust weight of evidence approach for the sub-chronic toxicity.
- Grouping and read-across : This test proposal maybe used to help develop a category approach for a wider range of hydrocarbons.
- Substance-tailored exposure driven testing [if applicable] : Insufficient data available
- Approaches in addition to above [if applicable]: None applicable
- Other reasons [if applicable]: None identified

According to the adaption principles of Annex XI of the REACH Regulation, there are no existing sub-chronic toxicity data (mammalian and human) for this substance, or for similar compositions, which could subsequently be used for read-across. Consequently, a weight of evidence approach is not sufficient to address this endpoint.

Both in-vitro and in-silico approaches are not considered to be appropriate, as there are no suitable methods which are considered to be valid and reliable to address the requirements for the Sub-Chronic Toxicity (90-Days) toxicity endpoint.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- There are no short-term toxicity studies (28-days) on this substance
- There are no chronic toxicity studies on this substance

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed [if relevant]: See 'Materials and Methods' Section for further information.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Sex:

male/female

Route of administration:

oral: unspecified

Endpoint conclusion

Dose descriptor:

NOAEL

300 mg/kg bw/day

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1978/08/29-1980/01/25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-conducted study comparable to standard studies.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

GLP compliance:

no

Remarks:

prior to GLP

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 Breeding Unit
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 150-300 g
- Housing: Hanging aluminum cages with stainless steel mesh bases. Paper lined catch trays were beneath each layer of cages and cleaned daily. Animals were identified using an ear punch/notch system.
- Diet (e.g. ad libitum): PRD Labsure food was available ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 20
- Humidity (%): 50%
- Air changes (per hr): continuous airflow of 3-6 m³/min
- Photoperiod (hrs dark / hrs light): 12 hrs light/ 12 hrs dark

IN-LIFE DATES: 1978/08/29-1980/01/25

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 8 m³ stainless steel chamber.
- Method of holding animals in test chamber: cages
- Source and rate of air: air drawn from laboratory at 3-6m³/min
- Method of conditioning air: filters
- System of generating particulates/aerosols: Vapors were generated using quartz tubes with their surface temperature adjusted to 148°C, (high concentration), 130°C (medium concentration), 225°C (low concentration). The vapor entered the experiment chamber via micrometering pumps.
- Temperature, humidity, pressure in air chamber: 14-29.5°C, 35.5-73.5% humidity
- Air flow rate: 3-6m³/min
- Air change rate: continuous

TEST ATMOSPHERE
- Brief description of analytical method used: Test atmospheres were analyzed by a total hydrocarbon analyser for 10 min at intervals of 40 min during exposures. Test atmospheres were also analyzed continuously for 2 hrs during each exposure using gas chromatographs with a flame iionization detector.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Control: Nominal 0 mg/m^3, measured 0 mg/m³
Low: Nominal 450 mg/m^3, measured 470 +/- 29 mg/m³
Medium: Nominal 900 mg/m^3, measured 970 +/- 70 mg/m³
High: Nominal 1800 mg/m^3, measured 1830 +/- 130 mg/m³

Duration of treatment / exposure:

12 months

Frequency of treatment:

6 hrs per day, 5 days per week except for 8 public holidays, 2 snowdays, and exposure limited on 2 other days for unknown reasons

Remarks:

Doses / Concentrations:
0, 470 mg/m³, 970 mg/m³, 1830 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

50

Control animals:

yes, concurrent no treatment

Details on study design:

- Rationale for animal assignment (if not random): Randomized block design with each control and experimental group in one block from the same litter.
- Post-exposure recovery period in satellite groups: 4 months

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: before start of experiment, weekly for first 4 weeks, monthly thereafter

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 10 animals of each sex from the high exposure and control groups had blood drawn at weeks 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 28, and 32 of exposure. Blood was also taken from 10 animals of each sex from each exposure level at the 26 and 52 week sacrfice, and from the 4 month recovery group.
- How many animals: 10 of each sex
- Parameters checked: Repeated sampling - erythrocyte count, mean cell volume, hemoglobin concentration, leucocyte count, mean corpuscular hemoglobin concentration, and hematocrit
Terminal sampling - erythrocyte count, mean cell volume, hemoglobin, leucocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hematocrit, prothrombin time, kaolin-cephalin coagulation time, erythrocyte osmotic fragility. Reticulocytes were only counted in the high exposure and control groups at the 12 month sacrifice.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood taken from 10 animals of each sex from each exposure level at the 26 and 52 week sacrfice, and from the 4 month recovery group.
- Animals fasted: overnight
- How many animals: 10 of each sex
- Parameters checked: 12 month sacrifice - total protein, urea nitrogen, alkaline phosphatase, chloride ion, total bilirubin, total calcium, inorganic phosphate, uric acid, sodium, potassium, alanine amino transferase, aspartate amino transferase, glucose concentration, protein eleectrophoresis,
26 week sacrifice - protein, urea, alkaline phosphatase, alanine amino transferase, aspartate amino transferase, chloride, sodium, potassium, portein electrophoresis

URINALYSIS: Yes
- Time schedule for collection of urine: male and females from each exposure group at 0, 3, 6, 9, and 12 months exposure, and 3 months after exposure.
- Parameters checked: Glucose, protein, ketones, bilirubin, blood pigments, pH, nitrite, urobilinogen

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals were sacrificed by injection of sodium pentobarbitone. All external surfaces, orifices, and the throacic and abdominal cavities were then examined. The following organs were then weighed: liver, kidneys, spleen, brain, heart, testes

HISTOPATHOLOGY: Yes
Histopathology was done on the following organs: salivary gland, stomach, heart, brain, spinal cord, pituitary gland, sciatic and posterier tibial nerves, lungs, prostate, seminal vesicles, testes, ovaries, uterus, skeletal muscle, adrenal glands, thyroids with esophagus and trachea, caecum, spleen, thymus, lymph node, mammary gland, small intestine, large intestine, pancreas, liver, kidneys, urinary bladder, eye and lachrymal glands, nasal cavity, spinal cord, tongue, knee joint and femur, any lesions

Statistics:

Body and organ weights were analyzed by covariance analysis. Hematological and clinical parameters were examined using analysis of variance. Significance of any difference between treated and control goup means was tested using the Williams t test. If a monotonic dose response could not be assumed, Dunnett's test was used instead.

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):

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:

no effects observed

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:

CLINICAL SIGNS AND MORTALITY
No clinical signs attributable to exposure were observed. Five animals died during the study. 2 males and 1 female in the control group died during the exposure phase of the experiment, and 2 males in the medium exposure group died during the recovery phase. These deaths were not considered attributable to exposure to the test substance. 37 animals were removed from the study during both the exposure and recovery phase for various health reasons, most commonly sore hocks. The number of removals was similar for all exposure levels, including the control group and was not attributable to exposure to the test substance.


BODY WEIGHT AND WEIGHT GAIN
Body weight gain was decreased the first 4 weeks in high exposure males, and medium exposure females. High exposure females had decreased body weight gains for the first 12 weeks of exposure, but recovered after exposure was terminated.

HAEMATOLOGY
Though some statistically significant changes were observed, particularly in males, no biologically significant changes were observed.

CLINICAL CHEMISTRY
Minor clinical chemical changes were observed at both the 26 and 12 month sacrifice, but were within normal ranges.

URINALYSIS
No differences between exposure and control groups were observed.

ORGAN WEIGHTS
Liver and kidney weights in the high dose males were significantly increased, but there were no histopathological lesions, so this was considered a functional hypertrophy of the organs.

HISTOPATHOLOGY: NON-NEOPLASTIC
No histopathological changes that could be attributable to exposure to the test substance were detected.

Key result

Dose descriptor:

NOAEC

Effect level:

1 800 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Systemic Toxicity

Key result

Dose descriptor:

NOAEC

Effect level:

900 mg/m³ air (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduced body weight

Critical effects observed:

not specified

Conclusions:

The NOAEC for male rats was determined to be 1800 mg/m3, the highest concentration tested. The NOAEC for female rats was determined to be 900 mg/m3, due to the reduced body weight noted.

Executive summary:

This study examined the effects of 12 months of inhalation exposure of rats to a commercial mixture of C9 Aromatics. Male and female rats were exposed to concentrations of 450, 900, or 1800 mg/m³ 6 hrs/day, 5 days/week, for up to 12 months. Some of the rats were sacrificed at 26 weeks, others at 12 months, and others after a 4 month recovery period after the end of the 12 month exposure. Animals were examined for clinical signs and behaviour twice daily, and weighed weekly for the first 4 weeks, and monthly thereafter. After sacrifice, the animals were examined for clinical chemistry, hematology, urine analysis, gross pathology, histopathology, and organ weights. No deaths attributable to exposure to the test substance occurred. There was depressed weight during the first few months of the experiment in medium (900 mg/m³) and high dose males (1800 mg/m³). However, the animals quickly recovered and this effect is not considered biologically significant. High dose females (1800 mg/m³) had depressed weight gain for the first 3 months of exposure, but recovered in subsequent months and in the satellite recovery group. No other adverse effects attributable to exposure to the test substance were seen. Based on the reversibility of the reduced weight gain and the lack of any noted pathology, the NOAEC for male rats was 1800 mg/m³ and the NOAEC for female rats was 900 mg/m3.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Source of data is from peer reviewed literature. Acceptable well-documented study report which meets basic scientific principles: non-GLP.

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

Species:

other: Rats and Dogs

Strain:

other: RATS: Harlan Wistar; DOGS: Beagles

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RATS: Harlan
-Number: RATS: 25 males; DOGS: 4 males

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

25 rats and four dogs per dose were used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatographic analyses of the nominal 0.25, 0.5, and 1.0 mg/liter vapor-air mixtures yielded mean measured concentrations of 0.10, 0.22, and 0.38 mg/liter or 17, 38, and 66 ppm.

Remarks:

Doses / Concentrations:
17, 38, and 66 ppm (0.10, 0.22, 0.38 mg/L)
Basis:
analytical conc.

No. of animals per sex per dose:

RATS: 6 males/dose (25 total); DOGS: 1 male/dose (4 total)

Details on study design:

Methods

Species and number. Twenty-five male rats and four dogs per level were used. Another 20 rats, from the same week of production, were maintained for use as challenge exposure controls (naive rats). The challenge exposures were run to determine whether the 6-hr daily inhalation of a non-lethal level of hydrocarbon, whether by acclimatization or induction of enzymes, would result in the rat becoming more or less resistant than non-exposed or naive controls from the same lot of animals.

Concentrations. A control group (exposed to dilution air) plus groups exposed to three graded levels of test material were utilized for each compound.

Duration. Exposures were 6 hr/day, 5 days/wk for 13 wk.

Observations. Once each week, body weights of both species and food consumption for dogs were determined. Overall appearance and behavior were checked daily.

Procedure. At 3, 8, and 13 wk of exposure three, three, and four rats, respectively, from each group, including controls, were sacrificed. After 13 wk of exposure 10 surviving rats from each group and 20 unexposed rats of the same age were used to detect any significant differences in time to death or in occurrence of signs of distress among the groups. The groups were “challenged” simultaneously by exposure to a vapor air concentration 1.25-2 times the 4-hr rat LC50 until 25 % of the group succumbed or for no more than a 6-hr period. The remaining rats, or those surviving the subacute exposure, exclusive of the ten per level reserved for the challenge exposure were sacrificed after 13 wk and tissues were taken for histopathological interpretation following gross autopsy. The assignment of rats to each of the above groups was made by random selection before the start of the study.

Clinical and hematological schemata. These tests were performed on all dogs initially and prior to sacrifice; and on rats, prior to sacrifice at 3, 8, and 13 wk of exposure. Minimum hematological evaluation for each animal included hematocrit, total erythrocyte count, reticulocyte count, and total and differential leucocyte counts. The biochemical survey included serum alkaline phosphatase, serum glutamic oxoacetic transaminase, serum glutamic pyruvic transaminase, and blood-urea-nitrogen on the rats, and these tests plus bilirubin and blood glucose on the dogs. Initial pre-terminal electrocardiograms were performed on all dogs. Urinalyses were conduct with both species.

Autopsy and pathology schemata. Gross examinations were made of all organ system Histopathological examination included brain (three sections), respiratory (three sections minimum, based on acute exposure data results), heart, thyroid, live kidney, adrenal, spleen, pancreas, stomach and intestines, skeletal muscle, bat marrow, and peripheral nerves. The reproductive organs and eyes were not scheduled for histopathological study unless abnormalities were discovered upon gross examination. Tissues were taken from all dogs and from rats sacrificed after 3, 8, and 13 wk of inhalation of the vapor.

Observations and examinations performed and frequency:

Observations. Once each week, body weights of both species and food consumption for dogs were determined. Overall appearance and behavior were checked daily.

Clinical and hematological schemata. These tests were performed on all dogs initially and prior to sacrifice; and on rats, prior to sacrifice at 3, 8, and 13 wk of exposure. Minimum hematological evaluation for each animal included hematocrit, total erythrocyte count, reticulocyte count, and total and differential leucocyte counts. The biochemical survey included serum alkaline phosphatase, serum glutamic oxoacetic transaminase, serum glutamic pyruvic transaminase, and blood-urea-nitrogen on the rats, and these tests plus bilirubin and blood glucose on the dogs. Initial pre-terminal electrocardiograms were performed on all dogs. Urinalyses were conducted with both species.

Sacrifice and pathology:

Autopsy and pathology schemata. Gross examinations were made of all organ system. Histopathological examination included brain (three sections), respiratory tract (three sections minimum, based on acute exposure data results), heart, thyroid, live kidney, adrenal, spleen, pancreas, stomach and intestines, skeletal muscle, bone marrow, and peripheral nerves. The reproductive organs and eyes were not scheduled for histopathological study unless abnormalities were discovered upon gross examination. Tissues were taken from all dogs and from rats sacrificed after 3, 8, and 13 wk of inhalation of the vapor.

Statistics:

Statistical analysis. Based on mortality during a 14-day observation period, the most probable LC50 with its fiducial range was calculated by the Thompson method of moving averages using tables by Weil (1952) and other unpublished tables. The results of the quantitative continuous variables, such as body weight changes, were intercompared for the dosage groups and the controls by the use of the following tests: Bartlett’s homogeneity of variance, analysis of variance, rank sum, and Duncan’s multiple range. The latter was used, if F for analysis of variance was significantly high, to delineate which groups differed from the controls. If Bartlett’s test indicated heterogeneous variances, the F test was used for each group versus the control. If these individual F tests were not significant, Student’s t test was used; if significant, the means were compared by the Cochran t test or the rank sum test. Frequency data, such as incidences of mortality or of micro-pathological conditions, were intercompared by the normal deviate of chi square calculated with Yates’ correction for continuity. In all cases the fiducial limit of 0.05 was employed to delineate the critical level of significance.
In general, only criteria that differed statistically significantly from the control group are discussed. Omission of comment is indicative that no valid differences were found. An attempt has been made to round off machine calculated data but if it is obvious that the data portend fictitious accuracy the implication should be ignored.

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):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

RATS. One rat at the 0.10-mg/liter level died after 14 days. Death was attributed to a pneumonic infection as evidenced by extensive lung abscesses. None of the observations were dosage-related and are discounted for that reason.
Significant findings (Rats) - Concentration (mg/liter)
Specific gravity urine, 9 weeks, rats - 0.38 mg/liter - Not dosage-related;
Blood urea nitrogen, 3 weeks, rats 0.10 mg/liter - Not dosage related;
Red blood cell count, Packed cell volume, and Hemoglobin, 9 weeks, rats 0.38 mg/liter - Not dosage-related
Alkaline phosphatase, 9 weeks, rats 0.10 mg/liter - Not dosage-related;
Neutrophil differential, 13 weeks, rats 0.22 mg/liter - Not dosage-related;
Lymphocyte differential, 13 weeks, rats 0.22 mg/liter - Not dosage-related.

DOGS. Pre-exposure polymorphonuclear neutrophilic leucocytes for the 0.1 mg/liter dogs were slightly higher than the controls on an absolute basis, but on the basis of change from pre-exposure after 13 weeks of inhalation, there were no significant differences that were treatment related.

Key result

Dose descriptor:

NOAEC

Remarks:

Rat

Effect level:

> 0.38 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Key result

Dose descriptor:

NOAEC

Remarks:

Dog

Effect level:

> 0.38 mg/L air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Systemic Toxicity

Critical effects observed:

not specified

Conclusions:

The test material’s NOAEC > 0.38 mg/liter (66 ppm), which was the highest achievable vapor concentration.

Executive summary:

Twenty-five male rats and four dogs per level were exposed for 6 hr/day, 5 days/wk for 13 wk. Another 20 rats, from the same week of production, were maintained for use as challenge exposure controls (naive rats). The challenge exposures were run to determine whether the 6-hr daily inhalation of a non-lethal level of hydrocarbon, would result in the rat becoming more or less resistant. One rat at the 0.10-mg/liter level died after 14 days. Death was attributed to a pneumonic infection as evidenced by extensive lung abscesses. None of the observations were dosage-related and are discounted for that reason. The test material’s NOAEC > 0.38 mg/liter (66 ppm), which was the highest achievable vapor concentration.

Endpoint conclusion

Dose descriptor:

NOAEC

900 mg/m³

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral: C10-C15 Aromatics are expected to have a low order of repeated dose toxicity by the oral route of exposure.  All tests were performed in a manner similar or equivalent to currently established OECD guidelines. In a repeated dose study where the test substance, C10-C15 Aromatics, was administered via oral gavage, signs of toxicity were observed and characterized at doses of 600 mg/kg.  The severity and frequency of the responses increase with increasing doses.  Noted changes include alterations to hematology (hematocrit, hemaglobin, MCHC), changes to thyroid follicular epithelial cells, and increases in the hemosiderin in macrophages.  The effects had reversed completely after the 4-week recovery period. Based on these observations, the repeat oral dose NOAEL is 300 mg/kg for C10-C15 Aromatics.

 

Inhalation: In a 12 month repeated dose study where the read-across substance C9 Aromatics were administered via inhalation, no signs of toxicity were observed at the maximum concentration of 900 mg/m3. Based on these observations, the repeat inhalation concentration NOAEL is >900 mg/m3.

Justification for classification or non-classification

In a repeated dose study where C10-C15 Aromatics was administered via oral gavage, signs of toxicity were observed and characterized at doses of 600 mg/kg.  The severity and frequency of the responses increase with increasing doses. Noted changes include alterations to clinical chemistry, changes to thyroid follicular epithelial cells, and increases in the hemosiderin in macrophages.  Based on these observations, the repeat oral dose NOAEL is 300 mg/kg for C10-C15 Aromatics. In a 12-month repeated dose study where the read-across substance C9 Aromatics were administered via inhalation, no signs of toxicity were observed at the maximum concentration of 900 mg/m3. Based on these observations, the repeat inhalation concentration NOAEL is >900 mg/m3.

 

These findings do not warrant the classification of C10-C12 Aromatics as a repeated dose toxicant under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).