Asphalt, sulfonated, sodium salt

Endpoint summary

Description of key information

In a study conducted according to OECD Test guideline 408 and in compliance with GLP (Charles River Laboratories, 2021), Wistar Han rats were administered Asphalt, Sulfonated, Sodium Salt (SAS) at 0, 100, 300 and 1000 mg/kg bw/day by oral gavage (water vehicle) for 7 days a week for at least 90 days. A NOAEL of 1000 mg/kg bw/day was identified based on no effects in males and females.

In males, minimal to moderate statistically significant dose-related observations were seen at all doses (decreased eosinophils) and non-statistically significant decreases to lymphocytes at all doses (resulting in slightly lowered total white blood cell counts) were reported. In females, non-statistically significant decreases to eosinophils at all doses were also recorded. These effects remained after the period of recovery in both males and females. However, the effects were not considered to be adverse due to a lack of corroborative histopathological finding (in the spleen, for example) and/or the mean values remained within the range considered normal for rats of this age and strain (historical control data). Taken together, these observations were considered a chance finding.

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:

12 April 2021 to 22 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: 161 – 206 g males, 109 – 152 g females
- Fasting period before study: Not specified
- Housing: Up to five animals per sex per dosing group were housed together in polycarbonate cages (in a Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm cage - Type 2000P if animals >400g (in general >13 Wks study)). Bedding material consisted of sterilized wooden fibers (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). During locomotor activity monitoring, animals are housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water. Cages were arranged on the racks according to a Latinsquare model.
- Diet (e.g. ad libitum): Diet (pellets) was sourced from SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany and administered ad libitum except during motor activity measurements when animals did not have access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Municipal drinking water ad libitum - cages were equipped with water bottles.
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22°C
- Humidity (%): 49 to 76%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: 28 April 2021 (initiation of dosing) to 25 August 2021

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

water

Remarks:

(Elix)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a (Water)
- Concentration in vehicle: n/a
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): n/a
- Purity: n/a

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted in Weeks 1, 6 and 12 to assess accuracy and homogeneity. Formulation analyses confirmed that formulations of test item in Elix water were prepared accurately and homogenously. The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration). For the formulation of Group 2 prepared for use in Week 1, the mean concentration was 82% of target. As the difference from the target concentration was minimal and concentrations were accurate on the other occasions, this slightly lower mean concentration was considered not to have impacted the study outcome. The formulations of Groups 2 and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

Duration of treatment / exposure:

Once daily

Frequency of treatment:

Seven days a week for a minimum of 13 weeks

Dose / conc.:

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

Remarks:

Group 1 - Control, vehicle only (Elix water)

Dose / conc.:

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

Remarks:

Group 2 - SAS treatment

Dose / conc.:

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

Remarks:

Group 3 - SAS treatment

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4 - SAS treatment

No. of animals per sex per dose:

10 animals per sex per dose in the main study and a further 5 animals per sex in the recovery satellite group (Groups 1 and 4 only)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on results of a previously conducted Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test study (OECD 422; https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/12030/7/6/2) and in an attempt to produce graded responses to the test item. The high-dose level was expected to produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level was expected to produce no observable indications of toxicity.
- Rationale for animal assignment (if not random): Animals were assigned to groups at random at the discretion of the biotechnician.
- Fasting period before blood sampling for clinical biochemistry: Yes, (overnight with a maximum of 24 hours)
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: Not specified
- Section schedule rationale (if not random): not specified
- Other: n/a

Positive control:

No

Observations and examinations performed and frequency:

MORTALITY: Yes, all animals (within cage)
- Time schedule: At least twice daily beginning upon arrival through termination. Except on days of receipt and necropsy where frequency was at least once daily.

CAGE SIDE OBSERVATIONS: Yes, all Main Study and Recovery animals (animals remained in their cage, unless it was necessary to remove them for identification or confirmation of possible findings).
- Time schedule: At least twice daily; from Day 1 at 1-hour and 5-hours post dose.

DETAILED CLINICAL OBSERVATIONS: Yes, all Main Study and Recovery animals (animals were removed from their cage)
- Time schedule: Observations were made before treatment and weekly (from Week 1 through to, and including, the day of necropsy).

ARENA OBSERVATIONS: Yes, all Main Study and Recovery animals (animals removed from cage)
- Time schedule: Pretreatment and weekly; from Week 1, throughout the study. Animals were observed for clinical signs outside the home cage in a standard arena (data were collected under Test Facility Reference No. 522968 for logistic reasons and reported under Test Facility Reference No. 20283737). The time of onset, grade and duration of any observed signs were recorded.

BODY WEIGHT: Yes, all Main Study and Recovery animals.
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study. If necessary, animals were weighed more often in order to monitor their health status.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, all Main Study and Recovery animals.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, all Main Study and Recovery animals. Water consumption was monitored by visual inspection of the water bottles. If inter group differences were noted, consumption was assessed by weight.
- Time schedule for examinations: Regular basis throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes, by ophthalmoscope after application of a mydriatic agent (tropicamide 0.5%).
- Time schedule for examinations: Pretreatment Period - All Main Study and Recovery animals once (including spare animals), Dosing Period - All Group 1 and 4 Main Study animals during Week 13. If treatment-related findings were noted, the other animals (including Recovery animals) were examined. Recovery Period - Examination at End of Recovery was performed if treatment-related effects were seen at the end of treatment.

HAEMATOLOGY: Yes (except unscheduled euthanised animals)
- Time schedule for collection of blood: Sampled between 7.00 and 10.30 from the retro-orbital sinus
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight and for a maximum of 24 hours
- How many animals: All Main Study animals at End of Treatment, all Recovery animals at End of Recovery
- Parameters: see Table 1 for list of analysed parameters.

CLINICAL CHEMISTRY: Yes (except unscheduled euthanised animals)
- Time schedule for collection of blood: Sampled between 7.00 and 10.30 from the retro-orbital sinus
- Animals fasted: Yes, overnight and for a maximum of 24 hours
- How many animals: All Main Study animals at End of Treatment, all Recovery animals at End of Recovery
- Parameters: see Table 2 for list of analysed parameters.
- In addition, coagulation was assessed (prothrombin time (PT) activated partial thromboplastin time (APTT))

URINALYSIS: Yes (except unscheduled euthanised animals)
- Time schedule for collection of urine: Overnight (approximately 15-20 hours).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight for a maximum of 24 hours)
- Parameters: see Table 3 for list of analysed parameters.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during Week 12-13 of the Dosing Period. If treatment-related findings were noted in any of the tests then tests were conducted for all Recovery animals at the end of the Recovery Period.
- Dose groups that were examined: The first 5 animals per sex per group.
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex and static righting reflex), fore- and hind-limb grip strength and locomotor activity (total movements and ambulations in a 1 hour recording period under normal laboratory light conditions, using a computerized monitoring system)

IMMUNOLOGY: No

OTHER: ESTROUS STAGE DETERMINATION:
- A vaginal smear was taken to determine the stage of estrous from all the Main Study females at End of Treatment (day of necropsy) and all Recovery females at the End of Recovery (on day of necropsy).

Sacrifice and pathology:

SACRIFICE:
Main study and recovery animals surviving until scheduled euthanasia were weighed, and euthanized using isoflurane anaesthesia, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

The terminal procedures are summarized in Table 4 below.

ORGAN WEIGHT: Yes
All main study and recovery animals.
- See the checked parameters in Table 5 below for a list of weighed organs

GROSS PATHOLOGY: Yes
All main study and recovery animals, as follows:
- Full list of tissues
- See the checked parameters in Tables 4 and 5 for terminal procedures and the full list of tissues collected

HISTOPATHOLOGY: Yes
All main study and recovery animals, as follows:
- Group 1 (0 mg/kg bw/day) and Group 4 (1000 mg/kg bw/day) dose groups: Full list of tissues
- Group 2 (100 mg/kg bw/day) and Group 3 (300 mg/kg bw/day) dose groups: Gross lesions and target tissues
- Target tissues: Thyroid gland of males of Groups 2 and 3 and males of Recovery Groups 1 and 4.
- See checked parameters in Tables 4 and 5 below for terminal procedures and full list of tissues collected

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Any clinical signs noted during the Dosing Period (e.g. absent tail, fur loss, thin fur cover, skin lesions, scabs and discharge from the eyes) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.

Male No. 41 (1000 mg/kg bw/day) was euthanized for humane reasons on Day 41 due to a severe tail injury (half of its tail lost). The most significant microscopic finding in this animal was a skin lesion on the tail base (moderate ulceration with neutrophilic inflammation and
superficial bacterial colonies (cocci)), which correlated to the clinically observed skin lesion on Day 40. The skin ulceration was the cause of moribundity of this animal; there were no other microscopic findings of note recorded for this animal. This lesion was considered to have resulted from an incident unrelated to the treatment with the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals were considered to have been unaffected by treatment with the test item.

The slightly lower body weights of males and females at 1000 mg/kg bw/day on Day 91 (0.98x and 0.95x of control, respectively) were caused by a slightly lower start weight of these animals. The apparent lower body weight and body weight gain of females at 1000 mg/kg bw/day during the Recovery Period, was due to the fact that the Recovery females had a lower mean start weight (-9.4% of control). The statistically significant changes in body weight gain for females at 1000 mg/kg bw/day during the Recovery Period were considered to be unrelated to the test item since no trend was apparent regarding the direction of change.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was considered not to have been affected by treatment with the test item.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment with the test item.

The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 were similar among the groups, and occurred within the range considered normal for rats of this age and strain. The findings in Week 13 were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A (generally) dose-related decrease in eosinophil (EOS) and lymphocyte (LYMPH) counts was noted in males at all dose levels (0.64x, 0.57x and 0.52x of control for EOS and 0.83x, 0.89x and 0.77x for LYMPH (not statistically significant) at 100, 300 and 1000 mg/kg bw/day, respectively), resulting in a slightly lower total white blood cell (WBC) count (0.90x, 0.88x and 0.81x; not statistically significant). Eosinophil count remained decreased at the end of the Recovery period (0.80x). The mean values of the eosinophil and lymphocyte counts remained within the range considered normal for rats of this age and strain. These findings are considered to be test item-related.

In females at all dose levels, EOS counts were also decreased (0.59x, 0.57x, 0.53x at 100, 300 and 1000 mg/kg bw/day, respectively), although not statistically significant. Eosinophil count remained decreased at the end of the Recovery period (0.56x). Furthermore, decreases in hemoglobin (HGB) and hematocrit (HCT) levels (0.94 and 0.93x, respectively) were noted in females at 1000 mg/kg bw/day (all not statistically significant) at the end of the Dosing period, but were comparable with control values at the end of the Recovery period. These findings are considered to be test item-related.

At the end of the Recovery Period, platelet (PLT) count was slightly lower in males and females at 1000 mg/kg bw/day (0.87 and 0.90x, respectively). Additionally, reticulocyte (RETIC) count was lower (0.86x) for females at 1000 mg/kg bw/day. None of the differences reached statistical significance and as no effects were seen at the end of the Dosing Period, these changes were considered not test item-related. All other parameters were considered to be similar between treated and control animals at the end of the Recovery Period.

The apparent decrease in all WBC parameters in females at 1000 mg/kg bw/day was mainly due to one control female (No. 65) that had very high values, and these differences were therefore considered not related to the test item.

Platelet clumps were noted in two control females (Nos. 58 and 60) at the end of the Dosing Period and in one control male (No. 12) and one female at 1000 mg/kg bw/day (No. 99) at the end of the Recovery Period. As this concerned mostly control animals, no toxicological relevance was attached to this.

Remaining differences in hematology parameters, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters were considered not to have been affected by treatment in males at 100 mg/kg bw/day.

Calcium (CA) concentration was increased in males at 300 and 1000 mg/kg bw/day (1.07x and 1.08x of control, respectively). In females at 1000 mg/kg bw/day, potassium (K) and CA concentrations were also increased (both 1.05x) and in females at 300 mg/kg bw/day CA was also increased (1.06x), however without reaching statistical significance. These parameters were comparable with controls at the end of the Recovery period.

The apparent increase in aspartate aminotransferase (AST) activity in females at 1000 mg/kg bw/day was caused by a single female (No. 89) with a very high value (352 U/L). As all other individual values were within the concurrent control range, this single occurrence was considered unrelated to treatment with the test item.

At the end of the Recovery Period, TBIL concentration was high for males and females at 1000 mg/kg bw/day (1.32x and 2.65x respectively; not statistically significant). In females at 1000 mg/kg bw/day, triglycerides (TRIG) concentration was decreased (0.52x). Furthermore, statistically significantly increased chloride concentration was noted in males at 1000 mg/kg bw/day (1.01x). These findings were considered to be not related to treatment with the test item, given the minimal effect, caused mainly by a single animal (No. 100 for bilirubin) and/or these changes did not occur at the end of the Dosing Period.

Other values achieving a level of statistical significance, when compared to controls, were considered to have arisen as a result of slightly high or low control values, occurred in the absence of a dose-related distribution and/or were, given the magnitude of change, considered to be not test item related.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroxine (T4) levels were slightly increased in males at 1000 mg/kg bw/day (1.15x). Thyroid stimulating hormone (TSH) levels in males at 1000 mg/kg bw/day was also increased (1.25x) but was mainly caused by one animal (No.39: 1.430 mU/L) and therefore considered to be not test item related. TSH levels were also comparable with controls at the end of the Recovery period at 1000 mg/kg/day.

In females at all dose levels, TSH levels were increased in a dose-related manner (1.29x, 1.41x and 1.97x at 100, 300 and 1000 mg/kg bw/day, respectively), however without reaching statistical significance.

At the end of the Recovery Period, TSH was still higher in females at 1000 mg/kg bw/day (1.79x, not statistically significant). Triiodothyronine (T3) level was lower in males at 1000 mg/kg bw/day (0.78x) at the end of the Recovery Period but without any changes at the end of the Dosing Period and were therefore considered to be not test item related.

All mean hormone values remained within the range considered normal for rats of this age and strain (historical data for Wistar Han rats for the period 2018-2021 were provided).

Microscopic findings, which were regarded test item-related, were noted in the thyroid gland of males at 300 and 1000 mg/kg bw/day. At the end of treatment, increased incidence and severity of diffuse follicular cell hypertrophy with concurrent colloid alteration was recorded in 6/10 males at 300 mg/kg bw/day (minimal) and 6/9 males at 1000 mg/kg bw/day (up to mild degree).

Minimal follicular cell hypertrophy with colloid alteration was noted at the end of the treatment period in 2/10 males at 100 mg/kg bw/day. However, at this low incidence and severity a relationship to the test item was unlikely as these findings were present at generally comparable incidence in the control males (2/10 versus 1/10 in the controls).

Following the 28-Day Recovery Period at 1000 mg/kg bw/day, follicular cell hypertrophy (minimal) was regarded as not test item related as it was recorded at a comparable incidence and severity in the control males (2/5 versus 2/5 in the controls). Colloid alteration (up to mild degree) was recorded in 2/5 males but at a slightly higher degree compared with the control animals.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urine parameters of treated rats were considered not to have been affected by treatment with the test item.

The statistically significantly slight decreases in specific gravity in females at 300 and 1000 mg/kg bw/day at the end of the Dosing Period (0.99x of control for both dose levels) were considered to have arisen as a result of a higher urinary volume. These slight differences were therefore considered to be unrelated to treatment with the test item.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Higher mean liver weight (relative to body weight only) was noted in females at 300 and 1000 mg/kg bw/day at the end of the Dosing Period. Given the small magnitude of the change (8.5% and 7.5% relative to body weight, respectively), the lack of dose response and histologic correlate, these small differences were interpreted to represent biologic variability or a reflection of the slightly lower body weight (-3%, -2.4%, respectively) rather than a test item-related effect.

Any additional differences, including those that reached statistical significance at the end of the dosing or the slightly lower body weights of males and females at 1000 mg/kg bw/day on Day 91 (0.98 and 0.95x of control, respectively) were caused by a slightly lower start weight of these animals. Recovery Period (higher relative seminal vesicle weight in Main study males at 1000 mg/kg bw/day; higher relative adrenal gland and kidney weight in Recovery females at 1000 mg/kg bw/day) were considered not to be test item-related. The higher seminal vesicles weight occurred in the absence of macroscopic correlate; the higher relative adrenal and kidney weights were in line with the lower terminal body weight of Recovery females at 1000 mg/kg bw/day (-11%).

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations. All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histologic changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. In the liver of males and females at 1000 mg/kg bw/day there was a slightly higher incidence of mononuclear cell infiltrates compared to the control animals. However, the incidences and severities as recorded for the males and females at 1000 mg/kg bw/day were within the normal range of background pathology encountered in rats of this age and strain and were therefore regarded to be unrelated to the treatment with the test item. The differences were interpreted as most likely to represent normal physiologic variability of this common observation in the rat liver.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

COAGULATION: Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.

The prolonged activated partial thromboplastin time (APTT) in females at 1000 mg/kg bw/day at the end of the Recovery Period (1.53x of control, not statistically significant) was caused by two females (Nos. 98 and 100) with extreme values. As other values were within the same range as the concurrent controls and no effects were seen at the end of the Dosing Period, these two extreme values at the end of Recovery were considered a chance finding.

Any statistically significant changes in coagulation parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related, statistically significant effects

Key result

Critical effects observed:

no

Conclusions:

In a study conducted according to OECD Test guideline 408 and in compliance with GLP (reliability score 1), Wistar Han rats were administered Asphalt, Sulfonated, Sodium Salt (SAS) at 0, 100, 300 and 1000 mg/kg bw/day by oral gavage (water vehicle) for 7 days a week for at least 90 days. A NOAEL of 1000 mg/kg bw/day was identified based on no effects in males and females. In males, statistically-significant dose-related observations were seen at all doses (decreased eosinophils) and non-statistically-significant decreases to lymphocytes at all doses (resulting in slightly lowered total white blood cell counts) were reported. In females non statistically-significant decreases to eosinophils at all doses were also recorded. These effects remained after the period of recovery in both males and females. However, the effects were not considered to be adverse due to a lack of corroborative histopathological finding and/or remained within the range of historical control data.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005-08-08 to 2007-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP study conducted with methods similar to OECD Guideline 407. This study was part of a reproductive/developmental toxicity screening study. Males were the only sex of animals included for the repeat dose endpoint. Females were included in the reproduction endpoint study listed below in the "cross-reference to same study record" section. Guideline 407 states to have at least 10 animals (5 from each sex). However, even though females were not included in this study (based on the fact that they were pregnant and this can alter toxic effects), there were 10 males included in the study. Therefore, enough animals were included. Treatment was also performed up until Day 43 instead of 28 days.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Only males were included and treatment lasted for 43 days.

Principles of method if other than guideline:

This repeat dose study was part of a reproduction/developmental screening study. Females were not included in this repeat dose entry, based on the fact that they were pregnant.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate from "The Department of Health of the Government of the United Kingdom"

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD (SD) IGS BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 8 weeks
- Weight at study initiation: 278-333 g
- Fasting period before study: N/A
- Housing: Initially, all animas were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages.
- Diet (e.g. ad libitum): ad libitum (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK)
- Water (e.g. ad libitum): ad libitum (drinking water supplied from polycarbonate bottles attached to the cage)
- Acclimation period: The animals were acclimatized for 10 days, during which time their health status was assessed.
- Other: On receipt, the animals were examined for signs of ill-health or injury.


ENVIRONMENTAL CONDITIONS
- Temperature (deg. C): Temperature controls were set to achieve target values of 21+/-2 deg C. Deviations from this target were considered not to affect the purpose or integrity of the study.
- Humidity (%): Relative humidity controls were set to achieve target values of 55+/-15 %. Deviations from this target were considered not to affect the purpose or integrity of the study.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
- Other: Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidities were included in the study records.


IN-LIFE DATES: From: N/A To: N/A

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared weekly and stored at approximately +4 deg C in the dark.


DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: 0, 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg (The volume administered to each animal was based on the most recent body weight and was adjusted weekly.
- Lot/batch no. (if required): N/A
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the formulations was determined spectrophotometrically. The test substance formulations were sampled and analyzed within three days of preparation.
The test substance formulations were diluted with DMSO to give a final, theoretical test substance concentration of approximately 0.025 mg/mL.
Standard solutions of test substance were prepared in DMSO at a nominal concentration of 0.025 mg/mL.
The standard and sample solutions were analyzed spectrophotometrically using the following conditions:
Spectrophotometer: Perkin-Elmer Lambda 20 and Camspec M330
Wavelength: ~268 nm
Cell path length: 10 mm
Reference medium: DMSO
For homogeneity determination, test substance formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
For stability determinations, the test substance formulations were sampled and analyzed initially and then after storage at approximately +4degrees C in the dark for eighteen days.
Results showed the formulations to be stable for at least fourteen days and the formulations were within acceptable limits of the nominal concentration.

Duration of treatment / exposure:

Animals were treated up until Day 43, at which time they were sacrificed.

Frequency of treatment:

The test substance (or vehicle alone for control animals) was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of a preliminary range-finder.
- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomization procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: A control group, each of five males and females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.
- Section schedule rationale (if not random): N/A

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: see below
- Cage side observations included: All animals were examined for overt signs of toxicity, ill health and behavioral change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing on weekends. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of treatment and at weekly intervals thereafter


BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on day 1 (prior todosing) and then weekly until termination.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption calculated: Yes; Food consumption was recorded on a weekly basis and was reported as g/rat/day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Food efficiency was also determined: yes; This was determined by dividing the group mean body weight gain (g/rat) by the group mean food consumption (g/rat/day) times number of days.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt change.



OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A


HAEMATOLOGY: Yes
- Time schedule for collection of blood: See below.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: See below.
- Parameters examined. Investigations were performed on five non-recovery males selected from each test and control group on Day 14. In addition, assessments were performed on five non-recovery animals at termination and on Recovery animals on Day 57. Blood samples were obtained from the lateral tail vein. The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: haemoglobin, erythrocyte count, haematocrit, erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin concentration), total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count, reticulocyte count (cresyl blue stained slides were prepared but reticulocytes were not assessed), prothrombin time (assessed by Thrombomax HS with Calcium) and activated partial thromboplastin time (assessed by Actin FS using samples collected into sodium citrate solution).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: See below.
- Animals fasted: No
- How many animals: See below.
- Parameters examined. Investigations were performed on five non-recovery males randomly selected from each test and control group on Day 14. In addition, assessments were performed on five non-recovery animals at termination and on Recovery animals on Day 57. Blood samples were obtained from the lateral tail vein. The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, and total bilirubin.

URINALYSIS: Yes
- Time schedule for collection of urine: During the final week of treatment, urinalysis was performed on five selected non-recovery males from each group and on recovery males during the final week of recovery.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances, blood


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: See below.
- Dose groups that were examined: See below.
- Battery of functions tested: Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males per dose level, prior to termination, together with assessment of sensory reactivity to various stimuli.
Detailed individual clinical observations were performed for each non-recovery animal using a purpose-built arena. The following behavioral assessment parameters were observed: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, urination, defecation, transfer arousal, and tail elevation.
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory condition. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).
For forelimb/hindlimb grip strength, an automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: grasp response, vocalization, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex and blink reflex.

OTHER: N/A

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; Animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguinations on Day 43 (except for recovery males). Gross necropsy consisted of a full external and internal examination and any macroscopic abnormalities were recorded.
HISTOPATHOLOGY: Yes; The following organs, removed from the five selected non-recovery and recovery adult animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals; brain; epididymides*; heart; kidneys; liver; spleen; testes*; thymus (*=organs weighed from all animals).
Samples of the following tissues were preserved from five males from each dose group: adrenals; aorta (thoracic); bone and bone marrow (femur including stifle joint; sternum); brain (including cerebrum, cerebellum and pons); caecum; coagulating gland*; colon; duodenum; epididymides*; eyes; gross lesions; heart; ileum; jejunum; kidneys; liver; lungs (with bronchi); lymph nodes(cervical and mesenteric); muscle (skeletal); pancreas; pituitary*; prostate*; oesophagus; rectum; salivary glands (submaxillary); sciatic nerve; seminal vesicles*; skin (hind limb); spinal cord (cervical); spleen; stomach; thyroid/parathyroid; trachea; testes*; thymus; urinary bladder (*=tissues also removed from the remaining animals).
The tissues from five selected control and high dose animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 mm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues with (*) next to them from the remaining control and high dose animal were also processed.

Other examinations:

N/A

Statistics:

Statistical Methods: Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight) weekly bodyweight gain and quantitative functional performance data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Leven's test for homogeneity of variance. Where variances were shown to be homogeneous, pairwise comparisons were conducted using Dunnett's test. Where Leven's test showed unequal variances, the data were analysed using non-parametric methods: Kruskal-Wallis
ANOVA and Mann-Whitney 'U' test.
The haematology variable basophils were not analysed since consistently greater than 30% of the data were recorded as the same value.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not specified

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 unscheduled deaths during the study. No clinically observable signs of toxicity were detected in animals treated with 250, 500 or 1000 mg/kg bw/day. Episodes of respiratory pattern changes and generalised fur staining were evident throughout the treatment groups during the treatment period.


BODY WEIGHT AND WEIGHT GAIN: There were no adverse effects on bodyweights for males through the study. Recovery males treated with 1000 mg/kg showed a statistically significant increase in body weight gain during week 6 and a statistically significant reduction in body weight gain during week 7. In the absence of similar effects seen in non-recovery males, the intergroup differences were considered of no toxicological significance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No adverse effects on food consumption were detected for males throughout the treatment period.


FOOD EFFICIENCY: No adverse effects on food efficiency were detected for males throughout the treatment period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No effects.


OPHTHALMOSCOPIC EXAMINATION: N/A


HAEMATOLOGY: No treatment-related effects were detected. Males treated with 1000 mg/kg/day showed statistically significant reductions in clotting time at the terminal bleed. In the absence of any supporting data to suggest an effect of treatment, this intergroup difference was considered to be of no toxicological significance.


CLINICAL CHEMISTRY: No treatment-related effects were detected. Males treated with 1000 mg/kg/day showed a statistically significant reduction in plasma calcium concentration on Day 14. The effect continued at Day 43 and extended to males from the remaining treatment groups. Males treated with 1000 and 250 mg/kg/day also showed a statistically significant reduction in plasma inorganic phosphorus at Day 14. In the absence of a dose related response or in the absence of any supporting data to suggest an effect of treatment, these intergroup differences were considered to be of no toxicological significance. Males treated with 1000 mg/kg/day showed a statistically significant reduction in plasma urea. In the absence of any
histopathological correlates or a dose related response, the intergroup difference was considered of no toxicological importance. Recovery males treated with 1000 mg/kg/day showed a statistically significant increase in plasma creatinine levels together with a reduction in plasma inorganic phosphorus. The differences were considered to be incidental and of no toxicological importance.


URINALYSIS: No treatment-related effects were detected.


NEUROBEHAVIOUR:
-Behavioural Assessments: All inter- and intra-group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
- Functional Performance Tests: No treatment-related effects were detected. Statistical analysis of the data revealed no significant intergroup differences.
- Sensory Reactivity Assessments: No treatment-related effects were detected. All inter- and intra-group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and ages used and were of no toxicological importance.


ORGAN WEIGHTS: No treatment-related effects were detected in the organ weights measured. Statistical analysis of the data revealed no significant intergroup differences.


GROSS PATHOLOGY: No treatment-related macroscopic abnormalities were detected at terminal kill.


HISTOPATHOLOGY: NON-NEOPLASTIC: No treatment-related microscopic changes were detected.


HISTOPATHOLOGY: NEOPLASTIC (if applicable): No treatment-related microscopic changes were detected.


HISTORICAL CONTROL DATA (if applicable): N/A


OTHER FINDINGS: N/A

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No toxicologically significant changes were observed in the parameters measured.

Critical effects observed:

no

N/A

Conclusions:

The oral administration of the test substance to rats by gavage at dose levels of 1000, 500 or 250 mg/kg bw/day produced no toxicologically significant changes in the parameters measured. The No Observed Effect Level (NOEL) was therefore considered to be 1000 mg/kg bw/day.

Executive summary:

N/A

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The key study for repeated dose toxicity was conducted according to OECD Test guideline 408 and in compliance with GLP (Charles River Laboratories, 2021). Wistar Han rats were orally (gavage, vehicle water) administered Asphalt, Sulfonated, Sodium Salt (SAS) at 0, 100, 300 and 1000 mg/kg bw/day for 7 days a week for at least 90 days. The animals of the control group received the vehicle, water (Elix) alone.

A (generally) dose-related decrease in eosinophil and lymphocyte counts was noted in males at all dose levels, resulting in a slightly lower total white blood cell count. Also in females at all dose levels, eosinophil counts were decreased, although not statistically significant. In both males and females, eosinophil count remained decreased at the end of the Recovery Period. Furthermore, decreases in hemoglobin and hematocrit levels were noted in females at 1000 mg/kg bw/day (both not statistically significant) at the end of the Dosing Period, but was comparable with controls at the end of the Recovery Period. In absence of any corroborative histopathological findings (e.g., in the spleen), these test item-related changes were considered to be not adverse.

Calcium concentration was increased in males and females at 300 and 1000 mg/kg bw/day (not statistically significant in females). In addition, potassium concentration was increased in these high dose females (not statistically significant). All values had returned to control levels at the end of the Recovery Period. In absence of any histopathological correlation, these test item-related findings were considered to be not adverse.

Thyroxine (T4) levels were slightly increased in males at 1000 mg/kg bw/day. In females at all dose levels, thyroid stimulating hormone (TSH) levels were increased in a dose-related manner, however without reaching statistical significance. At the end of the Recovery Period, TSH was still higher in females at 1000 mg/kg bw/day. In absence of a clear histopathological correlation (valid for TSH only) and as the mean values remained within the historical control data range, these test item-related findings were considered to be not adverse.

Most of the males starting at 300 mg/kg bw/day presented test item-related findings in the thyroid gland consisting of follicular cell hypertrophy and colloid alteration up to a mild degree. The incidence and severity of follicular cell hypertrophy in males at 1000 mg/kg bw/day at the end of Recovery Period was regarded as not test item-related as it was present at a low degree (minimal) and at a similar low incidence in control and test item-treated males. Colloid alteration was present at a similar incidence at the end of the Recovery Period but a slightly higher degree (up to mild degree) in males at 1000 mg/kg bw/day compared with the control animals indicating nearly complete recovery. These findings were considered non-adverse based on the low severity and absence of associated degenerative, inflammatory or necrotic changes.

Supporting data are available from a combined repeat dose toxicity with reproduction/developmental toxicity screening test (OECD 422) with SAS. Female and male Sprague-Dawley rats (10 animals/sex/dose group) were dosed daily with 0, 250, 500, 1000 mg/kg bw/day for 43 days for males and 14 days for females (till day 4 post partum). No significant treatment-related effect was detected on reproduction or offspring development at the maximum dose tested. Males treated with 1000 mg/kg bw/day showed statistically significant reductions in clotting time at the terminal bleed. In the absence of any supporting data to suggest an effect of treatment, this intergroup difference was considered to be of no toxicological significance. Some statistically significant changes in blood chemical parameters were noted, but were considered to be of no toxicological significance. No significant effects were observed on necropsy.

Justification for classification or non-classification

 The No Observed Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day, the highest dose administered. Based on the study results, no classification is required according to Regulation (EC) No 1272/2008.