Asphalt, sulfonated, sodium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a reproduction study on SAS conducted under OECD 422 (“Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test”), SAS was administered daily by gavage as a solution in distilled water to three groups of ten male and ten female Sprague-Dawley Crl:CD®(SD) IGS BR strain rats for fifty-four consecutive days, at dose levels of 250, 500 and 1,000 mg/kg bw/day. A control group of ten males and ten females were dosed with distilled water alone. Pairing of animals within each dose group was undertaken on a one male:one female basis on day 15 of the study, to produce litters.  No reproductive toxicity was noted in rats dosed up to the 1,000 mg/kg bw/day (the highest dose tested) in an OECD 422 screening study. Therefore, SAS is not toxic to reproduction, including fertility.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-08-08 to 2007-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

N/A

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/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: (P) 8 weeks
- Weight at study initiation: (P) Males: 278-333 g; Females: 169-228 g
- Fasting period before study: N/A
- Housing: Initially, all animals 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. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- 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): at least 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 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 for males, recovery animals and females during maturation and on Days 0, 7, 14 and 20 of gestation, and on Days 1 and 4 of lactation).
- Lot/batch no. (if required): N/A
- Purity: N/A

Details on mating procedure:

- M/F ratio per cage: On Day 15, all animals (excluding Recovery animals) were paired on a 1 male:1 female basis within each dose group.
- Length of cohabitation: until evidence of mating (maximum of 14 days)
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation pugs, and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating. Sperm in smear referred to as day 0 of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: N/A
- Further matings after two unsuccessful attempts: N/A
- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes.
- Any other deviations from standard protocol: 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:

up to 54 consecutive days (males were killed on Day 43 and females were killed on Day 5 post partum)

Frequency of treatment:

The test substance (or vehicle alone for control animals) was administered daily (except for females during littering/parturition where applicable) by gavage using a stainless steel cannula attached to a disposable plastic syringe.

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters: N/A
- Selection of parents from F1 generation when pups were [...] days of age: N/A
- Age at mating of the mated animals in the study: [...] weeks: N/A
-Other: Each pregnant female was observed at approximately 0830, 1230, and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours on weekends and public holidays. The following was recorded for each female:
1. date of mating
2. date and time of observed start of parturition
3. date and time of observed completion of parturition
4. duration of gestation

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/sex/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.
- Other: N/A

Positive control:

No

Parental animals: Observations and examinations:

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 (except for females during parturition where applicable). During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.


DETAILED CLINICAL OBSERVATIONS: Neurobehavioral tests were performed. See below for details.
- Time schedule: N/A


BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Body weights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1, and then weekly until termination.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption calculated: Yes; During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-24. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Food consumption for recovery animals was recorded weekly until termination. Food consumption was reported as g/rat/day. Food efficiency was also determined by dividing the group mean body weight gain (g/rat) by the group mean food consumption (g/rat/day) times number of days.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


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.


OTHER:
1. Neurobehavioral: 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 and females 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.

2. Haematological and blood chemical investigations were performed on five non-recovery males and five non-recovery females randomly selected from each test and control group on Day 14 (day prior to pairing). Blood chemical and haematological 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. Animals were not fasted prior to sampling. For haematology, 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). For Blood chemistry, 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.


3. The following parameters were measured on collected urine: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances, blood.

Oestrous cyclicity (parental animals):

During mating, a vaginal smear was prepared daily for each female, and the stage of the oestrous cycle was recorded.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight and epididymides weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: N/A
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible: N/A

PARAMETERS EXAMINED
The following parameters were examined in offspring: On completion of parturition, the number of live and dead offspring was recorded. For each litter the following was recorded: number of pups born, number and sex of pups alive recorded daily and reported on Days 1 and 4 post partum, clinical condition of pups from birth to Day 4 post partum, and individual pup and litter weights on Days 1 and 4 post partum. All offspring were observed for the detachment of pinna and assessed for reflexological response to stimuli by assessing surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Yes; all animals (both those that died and ones that were sacrificed) were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguinations on Day 43.
- Maternal animals: Females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguinations on Day 5 post partum.


GROSS NECROPSY
- Gross necropsy consisted of a full external and internal examination and any macroscopic abnormalities were recorded. In addition, corpora lutea of all ovaries from pregnant females were counted at necropsy. Uterine implantation sites were counted also.

HISTOPATHOLOGY / ORGAN WEIGHTS
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; ovaries*; spleen; testes*; thymus (*=organs weighed from all animals).
Samples of the following tissues were preserved from five males and five females from each doses 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); mammary gland; muscle (skeletal); ovaries*; 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; uterus/cervix*; vagina* (*=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.

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed at 5 days of age.
- These animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


GROSS NECROPSY
- Gross necropsy consisted of a full external and internal examination, and any macroscopic abnormalities were recorded.




HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively: N/A

Statistics:

Statistical Methods: Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight) weekly bodyweight gain, litter weights, offspring bodyweights 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 non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Reproductive indices:

see below

Offspring viability indices:

see below

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No unscheduled deaths during the study. No clinically observable signs of toxicity were detected in animals of either sex 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 FOOD CONSUMPTION (PARENTAL ANIMALS): There were no adverse effects on bodyweights for males through the study or for females during the two week maturation period. 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. There were no adverse effects on bodyweight gains for females during the gestation or lactation phase of the study. No adverse effects on food consumption or food efficiency were detected for males throughout the treatment period or for females throughout the two week maturation period. There were no adverse effects on food consumption for females during the gestation or lactation phase of the study.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): N/A


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): N/A


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): N/A


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): There were no treatment-related effects on mating performance or fertility. The distribution of pre-coital intervals for treated animals was comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing. The distribution of animals with precoital intervals in excess of four days showed no treatment-related trends. Only one control female failed to achieve pregnancy. There were no significant intergroup differences in gestation length or parturition. The distribution for treated females was comparable to controls. In total there were 9, 10, 10, and 10 females at 0, 250, 500 and 1000 mg/kg, respectively, who gave birth to a live litter and successfully reared young to Day 5 of age. The mean numbers of corpora lutea observed for treated females were considered to be good and did not indicate any adverse effect of treatment.


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

GROSS PATHOLOGY (PARENTAL ANIMALS): No treatment-related macroscopic abnormalities were detected at terminal kill.


HISTOPATHOLOGY (PARENTAL ANIMALS): No treatment-related microscopic changes were detected.


OTHER FINDINGS (PARENTAL ANIMALS)
-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.
-Water Consumption: No intergroup differences were detected.
- 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.
- Blood 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. Recovery females treated with 1000 mg/kg/day showed a statistically significant reduction in plasma albumin and albumin/globulin ratio, together with an increase in plasma inorganic phosphorus. In the absence of similar treatment-related effects for non-recovery animals at the end of the dosing period, the intergroup differences were considered to be incidental and of no toxicological importance.
- Urinalysis: No treatment-related effects were detected.

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically significant changes in the parameters measured (general or reproductive).

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

VIABILITY (OFFSPRING): Prenatal losses and resultant litter size at Day 1 for treated animals were similar to controls. Postnatal survival was unaffected in all treated groups, with litter size at Day 4 again being similar to controls.


CLINICAL SIGNS (OFFSPRING): No toxicologically significant clinical findings were observed. The type and incidence of clinical observations recorded for offspring throughout the dose groups were consistent with what is normally expected of the age examined and were of no toxicological importance.


BODY WEIGHT (OFFSPRING): Mean litter weights, mean offspring bodyweights and mean bodyweight gain of the offspring were considered to have been unaffected by maternal exposure in females treated with 250, 500 or 1000 mg/kg/day.


SEXUAL MATURATION (OFFSPRING): N/A


ORGAN WEIGHTS (OFFSPRING): N/A


GROSS PATHOLOGY (OFFSPRING): The macroscopic findings observed for interim and terminal kill offspring throughout the dose groups were consistent with normally expected low incidence findings in pups of the age examined and were of no toxicological importance.



HISTOPATHOLOGY (OFFSPRING): N/A


OTHER FINDINGS (OFFSPRING): No treatment-related effects on offspring growth or development were detected. Intergroup differences in offspring maturation (as assessed by pinna unfolding) and reflexological assessment (percentage successful at surface righting) did not indicate any adverse effects of treatment at 250, 500 or 1000 mg/kg/day. Statistical analysis of the data revealed no significant intergroup differences.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect of treatment was detected on offspring development.

Critical effects observed:

no

Reproductive effects observed:

no

N/A

Conclusions:

The oral administration of sodium sulfonated asphalt to rats by gavage at dose levels of 1000, 500 or 250 mg/kg/day produced no toxicologically significant changes in the parameters measured. The No Observed Effect Level (NOEL) for parental and offspring toxicity was therefore considered to be ≥1000 mg/kg/day.

No effect of treatment was detected on reproduction or offspring development and the No Observed Effect Level (NOEL) for reproductive and developmental toxicity was also considered to be 1000 mg/kg/day.

Executive summary:

N/A

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD 422 study, no effect of treatment was detected on fertility and other reproductive parameters at doses (oral gavage) of 250, 500, and 1000 mg/kg bw/day. The no observed effect level (NOEL) for reproductive toxicity was considered to be 1,000 mg/kg bw/day (SafePharm Laboratories, 2007).

Effects on developmental toxicity

Description of key information

In a key oral (gavage) prenatal developmental toxicity study on SAS in time-mated female Wistar Han rats, conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAELs for maternal and developmental effects were greater than 1000 mg/kg bw/day (Charles River Laboratories, 2021).

In a reproduction study (including offspring development) on SAS conducted under OECD 422 (“Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test”), SAS was administered daily by gavage as a solution in distilled water to three groups of ten male and ten female Sprague-Dawley Crl:CD®(SD) IGS BR strain rats for fifty-four consecutive days, at dose levels of 250, 500 and 1,000 mg/kg bw/day. A control group of ten males and ten females was dosed with distilled water alone (SafePharm Laboratories, 2007).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Remarks:

Crl: Wistar (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: approximately 6-7 weeks old
- Weight at study initiation: approximately 161 to 206 g males and 109 - 152 g females
- Fasting period before study: No
- Housing: Animals were individually housed in polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Cages were arranged on the racks according to a Latin-square model.
- Diet (e.g. ad libitum): Animals were fed ad libitum, except during designated procedures with SM R/M-Z (from SSNIFF® Spezialdiäten GmbH) as pellets (alternate diet may have been provided on an individual animal basis as warranted and approved by the Study Director).
- Water (e.g. ad libitum): Municipal drinking water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22ºC
- Humidity (%): 49 to 76% humidity
- 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: 25th May 2021 To: 18th June 2021 (last date of necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Tap water purified by a Elix water purification system (Millipore, Bedford, MA, USA)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

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): Water (Elix)
- Concentration in vehicle: 0, 20, 60 or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL
- Lot/batch no. (if required): n/a
- Purity: n/a

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Test formulations prepared were considered homogeneous at the concentrations tested, and analysis of the accuracy revealed acceptable levels.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant arrived day 0 or 1 post-coitum
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy:
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Day 6 to day 20 post-coitum

Frequency of treatment:

Once daily

Duration of test:

14 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 - Control (Vehicle only)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 - SAS treatment

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 - SAS treatment

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4 - SAS treatment

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected to be 0, 100, 300, 1000 mg/kg bw/day, based on a previously conducted Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test study (OECD 422) with oral exposure of Asphalt, Sulfonated, Sodium Salt (SAS) in Sprague-Dawley rats.
- Rationale for animal assignment (if not random): Random
- Fasting period before blood sampling for (rat) dam thyroid hormones: No fasting
- Time of day for (rat) dam blood sampling: Between 07.00 and 09.00 from the jugular vein
- Other: n/a

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, on all animals, within their cage.
- Time schedule: Twice daily; starting on Day 6 post-coitum up to the day prior to necropsy. At 1 hour and 5 hours post dose.

DETAILED CLINICAL OBSERVATIONS: Yes, on all animals (removed from their cage)
- Time schedule: On Days 2, 6, 15 and 21 post-coitum

BODY WEIGHT: Yes, on all animals
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, on all animals.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes, 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: A regular basis throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 post-coitum
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. The thyroid gland was weighed at necropsy for all scheduled euthanasia animals, except for females that delivered their offspring early. Organ weights were not recorded for animals found dead, euthanized in poor condition or in extremis or that had delivered their offspring early. Paired organs were weighed together. Organ weight as a percent of body weight (using the body weight on Day 21 post-coitum) was calculated.The thyroid gland and macroscopic abnormalities were collected from all animals and preserved in 10% buffered formalin.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not for animals found dead, sacrificed before planned necropsy or that had started to deliver)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number and distribution of live and dead fetuses: Yes
- The sex of each fetus: Based on the anogenital distance (not for animals found dead, sacrificed before planned necropsy or that had started to deliver).

Blood sampling:

- Plasma: No
- Serum: Yes, for T3, T4 and TSH analysis
- Volume collected : 1.0 mL

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes, in all viable fetuses

Statistics:

- Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences will be reported as appropriate by dataset.
- Inferential statistical analysis: All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted. Parametric/Non-Parametric; Non-Parametric depending on the variables; Analysis of covariance (ANCOVA) the data corresponding to a response variable of interest and to a related covariate were submitted to ANCOVA, including only groups with at least three non-missing paired values. If found to be significant, pairwise comparisons were conducted using Dunnett’s test.
- Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Historical control data:

Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in the Wistar Han rat from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, mean body weight gain was decreased (10.3% lower than control; not statistically significant) between Days 18-21 post-coitum, leading to a decreased trend in overall body weight between Days 6-21 post-coitum (3.3% lower than control; not statistically significant). Mean body weight adjusted for gravid uterus weight was decreased at 1000 mg/kg bw/day (9.8% lower than control; not statistically significant). Mean body weights, body weight gain and adjusted body weight gain at 300 and 100 mg/kg bw/day were considered to be in the same range as the controls over the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, mean food consumption was slightly decreased at the start of treatment (Days 6-9 post-coitum; 8% lower than control, not statistically significant), and a low trend remained during the total treatment period (4.7% lower than control between Days 6-21 postcoitum; not statistically significant). Mean food consumption at 300 and 100 mg/kg bw/day was considered to be in the same range as the controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

Mean serum levels of Total T3, Total T4 and TSH were considered to be unaffected by treatment with the test item up to 1000 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in thyroid gland weights.

Mean gravid uterus weight at 1000 mg/kg bw/day was slightly lower (5.9% lower than control; not statistically significant). This was mostly caused by Female Nos. 71 and 79 with 3 and 5 fetuses, respectively. As all individual weights were within the range of biological variation, this was considered not test-item related. Mean gravid uterus weight at 300 and 100 mg/kg bw/day was considered unaffected by treatment with the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations in the thyroid glands. The recorded macroscopic finding of the thyroid gland in a single animal (bilateral enlargement at 1000 mg/kg bw/day) had no microscopic correlate and was within the range of background gross observations encountered in rats of this age and strain. The only remaining finding was scabbing of the skin in a single female in the control group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

All pregnant females had live fetuses. Therefore, the number of females with viable litters for evaluation was 21, 21, 21 and 19 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean percentage pre-implantation loss in the control and test groups were considered in the range of normal biological variation.
The mean percentage of post-implantation loss at 300 mg/kg bw/day was slightly higher compared to control (6.05% vs. 3.09%; not statistically significant) but remained within the range of the available Historical Control Data. This was mostly due to Female Nos. 47 and 58 with a relative high number of early resorptions compared to total number of implantations (n=3/9 and 4/9, respectively). In the absence of a dose-related effect, this was considered not test item-related.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean number of early or late resorptions in the control and test groups were considered in the range of normal biological variation.

Dead fetuses:

no effects observed

Description (incidence and severity):

The mean number of dead fetuses in the control and test groups were considered in the range of normal biological variation.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of corpora lutea at 1000 mg/kg bw/day was slightly lower (10% lower than control), although not statistically significant. This was mostly due to Female Nos. 71 and 79 with a relatively low number of corpora lutea (n=3 and n=5, respectively) compared to available Historical Control Data. Accordingly, mean numbers of implants and live fetuses at 1000 mg/kg bw/day were slightly lower (8% and 7% below control, respectively; not statistically significant). However, since treatment with the test item did not start before implantation of the conceptus was completed, these lower numbers in corpora lutea and consequently lower litter sizes were considered to reflect normal biological variation.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal body weights (male, female and combined) of all treated groups were considered unaffected by treatment with the test item.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was considered unaffected by treatment with the test item.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Mean fetal anogenital distance (both sexes) of all treated groups was considered unaffected by treatment with the test item.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External malformations were observed in one fetus of each of the low- and high-dose groups. The low-dose fetus (No. 42-R5) had no lower jaw, whereas the lower jaw of the high dose fetus (70-L5) was small. Due to single occurrence and the resemblance, these malformations were considered chance findings. There were no external variations observed in any groups.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Both fetuses of which the lower jaw was externally affected (Nos. 42-R5; low dose and 70-L5; high dose), had matching (small mandibles) and relating skull malformations. Additionally, skeletal malformations occurred in two other fetuses of the high-dose group. Fetus No. 85-R5 was also noted with lower jaw malformations (fused mandibles with a single incisor socket) despite a normal jaw externally. The malformations affecting the jaw were considered not test item-related due to the low incidence and absence of a clear dose-response. The other high-dose fetus (No. 75-L5) had lumbar vertebral malformations that were considered chance findings due to their single occurrence.

All skeletal variations occurred in the absence of a dose-related incidence trend, infrequently, and/or in control fetuses only. Therefore, they were considered not test item-related.

Visceral malformations:

no effects observed

Description (incidence and severity):

Visceral malformations were not observed in this study, and the visceral variations that were noted affected the liver (supernumerary lobes) and ureters (convoluted). The low incidences and group distribution of these findings did not indicate any test item-relationship.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related changes were noted in any of the developmental parameters investigated (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, based on the results of this prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, in time-mated female Wistar Han rats, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Asphalt, Sulfonated, Sodium Salt (SAS) were established to be greater than or equal to 1000 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005-08-08 to 2007-02-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

N/A

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:CH (SD) IGS BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: (P) 8 weeks
- Weight at study initiation: (P) Males: 278-333 g; Females: 169-228 g
- Fasting period before study: N/A
- Housing: Initially, all animals 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. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes.
- 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): at least 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 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 for males, recovery animals and females during maturation and on Days 0, 7, 14 and 20 of gestation, and on Days 1 and 4 of lactation).
- 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.

Details on mating procedure:

- M/F ratio per cage: On Day 15, all animals (excluding Recovery animals) were paired on a 1 male:1 female basis within each dose group.
- Length of cohabitation: until evidence of mating (maximum of 14 days)
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation pugs, and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating. Sperm in smear referred to as day 0 of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: N/A
- Further matings after two unsuccessful attempts: N/A
- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes.
- Any other deviations from standard protocol: N/A

Duration of treatment / exposure:

up to 54 consecutive days (males were killed on Day 43 and females were killed on Day 5 post partum)

Frequency of treatment:

The test substance (or vehicle alone for control animals) was administered daily (except for females during littering/parturition where applicable) 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/sex/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.
- Other: N/A

Maternal examinations:

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 (except for females during parturition where applicable). During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.


DETAILED CLINICAL OBSERVATIONS: Neurobehavioral tests were performed. See below for details.
- Time schedule: N/A


BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Body weights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1, and then weekly until termination.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption calculated: Yes; During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-24. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Food consumption for recovery animals was recorded weekly until termination. Food consumption was reported as g/rat/day. Food efficiency was also determined by dividing the group mean body weight gain (g/rat) by the group mean food consumption (g/rat/day) times number of days.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


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.


OTHER:
1. Neurobehavioral: 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 and females 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.

2. Haematological and blood chemical investigations were performed on five non-recovery males and five non-recovery females randomly selected from each test and control group on Day 14 (day prior to pairing). Blood chemical and haematological 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. Animals were not fasted prior to sampling. For haematology, 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). For Blood chemistry, 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.


3. The following parameters were measured on collected urine: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances, blood.
Ovaries and uterine content
Fetal examination: SACRIFICE
- The offspring were sacrificed at 5 days of age.
- These animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other: no data

Fetal examinations:

- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Soft tissue examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data

Statistics:

: Statistical Methods: Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight) weekly bodyweight gain, litter weights, offspring bodyweights 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 non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY: No unscheduled deaths during the study. No clinically observable signs of toxicity were detected in animals with 250, 500 or 1000 mg/kg bw/day.
BODY WEIGHT AND FOOD CONSUMPTION: There were no adverse effects on bodyweight gains for females during the gestation or lactation phase of the study. There were no adverse effects on food consumption for females during the gestation or lactation phase of the study.
REPRODUCTIVE PERFORMANCE: Only one control female failed to achieve pregnancy. There were no significant intergroup differences in gestation length or parturition. The distribution for treated females was comparable to controls. In total there were 9, 10, 10, and 10 females at 0, 250, 500 and 1000 mg/kg, respectively, who gave birth to a live litter and successfully reared young to Day 5 of age. The mean numbers of corpora lutea observed for treated females were considered to be good and did not indicate any adverse effect of treatment.
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: No treatment-related microscopic changes were detected.
OTHER FINDINGS:
-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.
-Water Consumption: No intergroup differences were detected.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING): Prenatal losses and resultant litter size at Day 1 for treated animals were similar to controls. Postnatal survival was unaffected in all treated groups, with litter size at Day 4 again being similar to controls.

CLINICAL SIGNS (OFFSPRING): No toxicologically significant clinical findings were observed. The type and incidence of clinical observations recorded for offspring throughout the dose groups were consistent with what is normally expected of the age examined and were of no toxicological importance.

BODY WEIGHT (OFFSPRING): Mean litter weights, mean offspring bodyweights and mean bodyweight gain of the offspring were considered to have been unaffected by maternal exposure in females treated with 250, 500 or 1000 mg/kg/day.

GROSS PATHOLOGY (OFFSPRING): The macroscopic findings observed for interim and terminal kill offspring throughout the dose groups were consistent with normally expected low incidence findings in pups of the age examined and were of no toxicological importance.

OTHER FINDINGS (OFFSPRING): No treatment-related effects on offspring growth or development were detected. Intergroup differences in offspring maturation (as assessed by pinna unfolding) and reflexological assessment (percentage successful at surface righting) did not indicate any adverse effects of treatment at 250, 500 or 1000 mg/kg/day. Statistical analysis of the data revealed no significant intergroup differences.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a key oral (gavage) prenatal developmental toxicity study on SAS in time-mated female Wistar Han rats, conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAELs for maternal and developmental effects were greater than 1000 mg/kg bw/day (Charles River Laboratories, 2021). The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on a previously conducted Combined Repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test study (OECD 422) with oral exposure of Asphalt, Sulfonated, Sodium Salt (SAS) in Sprague-Dawley rats.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), macroscopic examination, organ weights (thyroid gland), uterine contents, microscopic examination (thyroid gland), corpora lutea, implantation sites, and pre- and post-implantation loss. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that formulations of test item in water were prepared accurately and homogenously.

No maternal toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Asphalt, Sulfonated, Sodium Salt (SAS) of at least 1000 mg/kg bw/day were established.

Supporting data are available from a repeated dose reproductive screen (OECD 422), no effect of treatment was detected on development at doses (oral gavage) of 250, 500, and 1000 mg/kg bw/day, and the no observed effect level for developmental toxicity was considered to be 1,000 mg/kg bw/day (SafePharm Laboratories, 2007).

Toxicity to reproduction: other studies

Additional information

N/A

Justification for classification or non-classification

 The NOEL was determined to be 1000 mg/kg bw/day, the highest dose administered, for fertility and developmental toxicity. Therefore, SAS does not require classification for reproductive toxicity according to Regulation (EC) No 1272/2008.