Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides, reaction products with polyethylenepolyamines and tall-oil fatty acids, humates hydrochlorides

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Substance type: UVCB
- Physical state: powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited, Margate, Kent, UK
- Age at study initiation: ca 6-7 weeks
- Weight at study initiation: 201-227 g males; 163-195 g females
- Housing: Housed in polycarbonate cages with stainless steel grid tops and solid bottoms. Animals were initially housed 2/cage by sex. Males were individually housed a few days prior to mating. After mating, males were re-housed with original cage mates.
- Diet (e.g., ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded) provided ad libitum
- Water (e.g., ad libitum): Public water supply provided ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%
- Air changes (per hr): 10 air changes per hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Gravimetric particle size distribution measurement
Treatment Group 1: Air Control
Treatment Group 2: MMAD <4.45; GSD 2.123
Treatment Group 3: MMAD <4.85; GSD 2.043
Treatment Group 4: MMAD <5.11; GSD 2.153

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Modular snout only flow through system
- Method of holding animals in test chamber: Rats were restrained in clear, tapered, polycarbonate tubes fitted with a back-stop appropriately adjusted to prevent the animals from turning in the tubes.
- Rate of air: ~20 L/min
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: Rotating brush generator device
- Temperature, humidity, pressure in air chamber: 17.0-25.2 ºC, 0.7-43.7 %, pressure not reported
- Method of particle size determination: Particle size was measured by a cascade impactor located and sealed in a port in the animals’ breathing zone; particle size distribution was determined by plotting the cumulative percentage (by mass) of particles smaller than the cut point of each impactor stage against the logarithm of each stage cut point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis.
- Treatment of exhaust air: Chamber air was exhausted via a double filter system with a fiber glass coarse prefilter and a HEPA ultimate particulate filter.

TEST ATMOSPHERE
- Brief description of analytical method used: The aerosol concentration of the test aerosols in the animals’ breathing zone was measured
gravimetrically for Groups 2 to 4 at regular intervals throughout each daily exposure period. Aerosol concentrations were also measured and assessed prior to initiation of inhalation exposure to ensure concentrations were within target ranges. The test aerosols were sampled with filter media placed in a filter holder with a sampling system in place. Filters were weighed before and after sampling, and the volume of air sampled was recorded.
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Comparison of the nominal and achieved gravimetric aerosol concentrations showed good correlation throughout the study. Overall, the system efficiency 14 to 23%, 12 to 25 % and 15 to 26% for Groups 2, 3, and 4 respectively. The achieved concentrations were 100%, 102% and 107% of target for Groups 2, 3 and 4, respectively.

Duration of treatment / exposure:

Males were treated daily 2 weeks prior to mating, throughout mating, and until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated daily 2 weeks prior to mating, throughout mating, and up to Day 19 of gestation (ca. 6 weeks for treatment).

Frequency of treatment:

6 hr per day, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/m³ air (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/m³ air (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/m³ air (nominal)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes

Details on study design:

Dose selection rationale: Doses were selected based on the results of a 14-Day Dose Range Finding Study (supporting study) with organolignite.
Rationale for animal assignment (if not random): Cages were allocated by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups.

Positive control:

The study did not include a positive control.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: One week prior to treatment and weekly thereafter
- Cage side observations included: posture/condition (i.e., prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremor, fasciculation, convulsions, biting, vocalizations, and piloerection); ease of removal from the cage; body temperature; condition of the eyes (i.e., pupillary function, miosis, mydriasis, exophthalmos, encrustation, and lacrimation) and coat; presence of salivation, and overall ease of handling.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: One week prior to treatment and daily thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: One week prior to treatment for both sexes. Thereafter, males were weighed twice weekly at the start of dosing until the end of the study. Females were weighed twice weekly at the start of dosing and daily from mating to termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: No
- Time schedule for examinations: Not reported

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: One week prior to treatment for both sexes. An additional evaluation was conducted at Week 4 for males and shortly prior to sacrifice for females
- Dose groups that were examined: All dose levels

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples were obtained at Week 4 for males and on Day 4 of lactation for females
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: First 5 males per group; first 5 females per group that reared litter to Day 3 of lactation
- Parameters examined included: red and white blood cell count; hemoglobin concentration; hematocrit; mean cell volume; red blood cell width and volume; mean cell hemoglobin and mean cell hemoglobin concentration; reticulocyte count; platelet count; absolute neutriphil, lymphocyte, monocyte, eosinophil, and basophil count; large unstained cells; activated partial thromboplastin time; fibrogen; and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples were obtained at Week 4 for males and on Day 4 of lactation for females
- Animals fasted: No
- How many animals: First 5 males per group; first 5 females per group that reared litter to Day 3 of lactation
- Parameters examined included: urea; blood urea nitrogen; total bile acids; glucose; aspartate aminotransferase; alanine aminotransferase; alkaline phosphatase; creatine phosphokinase; lactate dehydrogenase; sodium; potassium; chloride; total protein; albumin; globulin; albumin/globulin ratio; cholesterol; creatinine; total bilirubin; calcium; inorganic phosphate

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 for males and during lactation for females
- Dose groups that were examined: 5 males per group and first 5 females per group that reared litter to Day 3 of lactation
- Battery of functions tested: / grip strength / motor activity / pain perception / landing foot splay / other physical and/or functional abnormalities

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 1)
HISTOPATHOLOGY: Yes; tissues identified in table 1 (except bone marrow smears) were processed from 5 males and 5 females from the control and high-dose groups.

Other examinations:

Appropriate reproductive and developmental endpoints were evaluated. Discussion of these endpoints is provided in the Results section of the main Combined Study Report.

Statistics:

The following endpoints were analyzed using the 'F Max' test: selected body weight and food consumption data; all hematology, coagulation, and clinical chemistry; and selected functional observational battery and motor activity data. Homogeneous variances were analyzed using a parametric ANOVA. Pairwise comparisons were made using Fisher's F protected LSD method via Student's t test only if the overall F test was significant. Heterogeneous variances were initially transformed using the log or square root. If variances remained heterogeneous, then a Kruskal-wallis non-parametric ANOVA was used and pairwise compassions were made using chi squared protection.

Organ weight data were analyzed using the methods noted above and through analysis of covariance (ANCOVA) using terminal body weight as the covariate. Organ weights were also analyzed as a percentage of terminal body weight.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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):

no effects observed

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: Increased incidence of staining around the head, nose, eyes, muzzle, and ears for all treated animals. Mid- and high-dose animals also had staining on their coats. Staining is considered due to dark color of test material.

HAEMATOLOGY: Measured endpoints in females were comparable to control values for all dose levels. There was a statistically significant increase in white blood cells (p<0.001), neutrophils (p<0.01), monocytes (p,0.01), basophils (p<0.001) and large unclassified cells (p<0.05) in 0.3-mg/L males. Mid-dose males also exhibited a slight increase in basophils; however, this effect was not statistically significant and was considered to be incidental. Statistically-significant increases in the number of white blood cells (p<0.05), monocytes (p<0.05), and basophils (p<0.05) were observed in low-dose males. Because of the lack of effect observed at the mid dose, these effects were considered not to be treatment-related.

CLINICAL CHEMISTRY: In high-dose males, there was a decrease in LDH levels that were considered incidental. All other clinical chemistry parameters for both males and females at all dose levels were similar to the control values.

ORGAN WEIGHTS: At the high-dose, a statistically significant increase in both absolute and relative lung weight was observed in high-dose males and females. At the mid-dose, there was a statistically significant increase in absolute lung weight for both males and females; however, only females exhibited a statistically significant increase in relative lung weight. No effects on organ weights were observed at the low dose for both males and females.

GROSS PATHOLOGY: There was an increased incidence of dark discoloration of the lungs for all high-dose males and females and dark discoloration of the bronchial lymph node for high-dose males (10/10) and females (9/10). High-dose females also showed an increased incidence of pale foci on the lungs (5/10) and dark discoloration of the mediastinal lymph node (5/10). Mid-dose males (8/10) and females (8/10) showed an increased incidence of dark discoloration of the lungs.

HISTOPATHOLOGY: NON-NEOPLASTIC: At the high-dose, the following treatment-related findings were noted: bronchiolo-alveolar pigmented macrophages/hyperplasia; multifocal alveolar foamy macrophage accumulation; and a higher severity and incidence of mononuclear cell infiltration. In the bronchial lymph node there were pigmented macrophages and increased cellularity. In the mediastinal lymph node, there were pigmented macrophages. A microscopic examination was not conducted at 0.05 and 0.005 mg/L.

Effect levels

Dose descriptor:

NOAEC

Effect level:

300 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No local, systemic or reproductive/developmental adverse effects were observed at the high dose.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) via inhalation, the NOAEC was established at 300 mg/m3 (equivalent to 0.3 mg/L; i.e., the high dose) based on the lack of adverse effects.

Executive summary:

The purpose of this study was to assess whether inhalation exposure of rats to the test article, organolignite, was associated with any systemic toxicity, and to provide information on effects on reproduction and/or development. The study also placed an emphasis on neurological effects as a specific endpoint to identify any neurotoxic potential of the test article. 

Sprague-Dawley rats were randomised into 3 exposure groups (i.e., 5, 50, or 300 mg/m³) and one control group (air exposure only), each group contained 10 males and 10 females. The males were treated daily for 2 weeks prior to mating, then through mating and until the day prior to necropsy (ca 4 weeks of treatment). Females were treated daily for 2 weeks prior to mating, throughout mating and up to Day 19 of gestation (ca 6 weeks). Females were maintained until Days 4-7 of lactation.

 

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights), gross necropsy findings, organ weights and histopathological examinations.

 

With regards to clinical signs, there was an increase in the incidence of dark staining around the head, nose, eyes, muzzle, ears and coat in all organolignite treated groups. These signs were all observed immediately after dosing and were a direct result of being exposed to organolignite, which is dark in color, on the inhalation dosing chamber. There were no treatment related effects at any dose level on body weight gains, food consumption, ophthalmic examinations or functional observational battery parameters.

 

There were no statistically significant changes to the haematology and coagulation parameters in the females treated at 300 mg/m³, compared to the controls. The females ceased dosing in late gestation to allow littering, and therefore, it is possible that any changes to the white blood cell types had started to recover by the time blood samples were collected. There were no changes to the clinical chemistry parameters in males or females that were associated with treatment.

 

At 300 mg/m³, there was an increase in some of the white blood cell types in the males (white blood cells (p < 0.001), neutrophils (p < 0.01), monocytes (p < 0.01), basophils (p < 0.001) and large unclassified cells (p<0.05). At 5 mg/m³, there was an increase in the number of white blood cells (p < 0.05), monocytes (p < 0.05) and basophils (p < 0.05). There were no observed changes at 50 mg/m³. As there was no similar pattern at 50 mg/m³, and the increases were small, these differences were not considered to be biologically significant and were not considered to constitute an adverse effect.

 

At necropsy, dark discolouration of the lungs was noted in males and females treated at 50 and 300 mg/m³. However, this observation is considered to be associated with the dark color of the test article and is not considered to constitute an adverse effect. Other necropsy findings at 300 mg/m³ included pale foci on the lungs, dark discolouration of the bronchial lymph nodes and dark discolouration of the mediastinal lymph nodes. Lymph node discolouration is consistent with clearance. Dark discolouration of the skin was also noted. At 300 mg/m³, there was an increase in lung weights in males and females that was still statistically significant (p<0.001) after adjustment for body weight. At 50 mg/m³, there was an increase in absolute lung weights in males and females, however, after adjustment for body weight, this increase only achieved statistical significance in females (p<0.001). The increased lung weight in females was not considered to constitute an adverse effect.

 

Microscopic findings at 300 mg/m³ included pigmented macrophages in the lung, which is expected following inhalation administration of a dark pigmented material; multifocal alveolar macrophage accumulation is also associated with this phagocytosis process. At this dose level, areas of bronchiolo-alveolar hyperplasia were characterised by hypertrophic alveolar Type II pneumocyte cells forming a single layer outlining alveolar walls. However, there was no evidence of associated tissue damage such as necrosis, degeneration or fibrosis. Nor was there any evidence of focal proliferation of epithelial cells, which might be indicative of a pre-neoplastic lesion. Both bronchiolo-alveolar hyperplasia, and the slightly

higher incidence of mononuclear cell infiltration, represent a local response to the pigment and pigmented macrophages. Observed bronchiole-alveolar hyperplasia was a local pulmonary response to the test article and based on the microscopic findings was considered not to be an adverse change after 4 or 6 weeks of exposure. In conclusion, under the conditions of this study, the No Observed Adverse Effect Concentration (NOAEC) for adults was considered to be 300 mg/m³ (equivalent to 0.3 mg/L). The change in the white blood cell parameters in the males did not show a dose-response relationship and is considered a reversible effect, therefore, this effect was considered not to be adverse.