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Description of key information

Read across: CAS 115733-09-0, One-generation reproductive toxicity study, rats, NOAEL > 500 mg/kg bw; no substance related effects in treated animals except post dose salivation and dark material around the nose in the mid and the highest dose. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

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Reference
Endpoint:
repeated dose toxicity: oral
Remarks:
other: one generation reproduction toxicity study
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2003-2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines to to GLP, and therefore meets the requirements for Klimisch code 1. However as this study is used in the context of a read across, Klimisch 2 is assigned.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: OECD 415
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: Sprague-Dawley Crl: CD®(SD) IGS BR rats,
- Source: Charles River Laboratories
- Age at study initiation: (P) males 5 wks, females 7 weeks
Males approximately 7 weeks of age at initiation of treatment. Females approximately 8 weeks of age at initiation of treatment.
- Weight at study initiation: (P) Males: 154-197 g; Females: 139-184 g
- Housing: Suspended wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Corn oil was added to the test substance to achieve the desired volume and then stirred for 30 minutes.
VEHICLE: Justification for use and choice of vehicle (if other than water): Corn oil
The test article was administered orally via gastric intubation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical confirmation of concentration: Homogeneity, stability and weekly dose concentration confirmation.
Duration of treatment / exposure:
F0 males - 70 days premating; mating period through completion of parturition
F0 females - 14 days premating; mating; 25 days of gestation and 20 days of lactation.
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
167 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
28 F0 rats/sex/group in control, low, mid and high dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results of a 28 day oral gavage study (according to OECD 407).
- Control and treatment groups: 28 F0 rats/sex/group in the control, low, mid and high dose groups.
- Mating: 1 male mated to 1 female from the same group until evidence of mating (presence of copulatory plug or sperm) was observed. If evidence of mating was not observed mating was discontinued after three weeks.
Observations and examinations performed and frequency:
Parental animals:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly and daily for females during gestation

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and on the day on euthanasia for males. After evidence of mating, females were weighed on gestational days 0, 7, 14 and 21 and on lactation days 1, 4, 7, 14 and 21.

Sperm parameters (Parental animals)
Parameters examined in P male parental generations:
testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology.
Sacrifice and pathology:
gross necropsy on death, organ weights and microscopic examination on termination
SACRIFICE
- Male animals: All surviving animals after completion of female parturition.
- Maternal animals: All surviving animals that delivered on lactation day 21; females that failed to deliver were sacrificed on gestation day 25.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
ANOVA for body weights, changes, food consumption semen parameters, organ weights.

Body weights, body weight changes, food consumption, semen parameters, organ weights, number of days to mating, gestation length, pup viability data, total pups delivered, pup body weights and mean live litter size were analysed by ANOVA followed, as needed, by Dunnett’s test. Count data were analysed by Chi-Square test followed by Fisher’s Exact Test for copulation and fertility indices, pup sex ratios, number of live and dead pups/group and pup survival. All analysis were two-tailed with a minimum significance level of 5%..
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:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
There were no remarkable findings in F0 males, with the exception of post dosing salivation.
In F0 females there were no remarkable findings with the exception of negative ammonium sulphide staining in two high dose and one mid dose animal.
Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant adverse effects occurred at 500 mg/kg bw (highest dose tested).
Critical effects observed:
not specified

Results of the homogeneity analysis indicate that the test article was homogeneous in the vehicle and stable for ten days when stored under ambient conditions. Concentration analysis confirmed that the test article was at the appropriate concentration in the dosing solutions.

Results

F0 males exhibited a dose related increase in post dosing salivation and dark material around the nose in the mid and high dose groups. The remaining F0 male parameters were unremarkable including: mean body weight and food consumption, mating and fertility indicies, absolute and relative organ weights, sperm evaluation parameters and macro and microscopic pathology.

The clinical signs of the Fo females were generally unremarkable. There were no toxicologically meaningful differences between the control low, mid and high dose groups with respect to F0 female mean body weights, body weight change, food consumption, mating and fertility indicies, precoital intervals or gestation length. A macroscopic finding observed in two high dose and one mid female sacrificed on post mating day 25 was a finding of negative ammonium sulfide staining in animals that failed to deliver and were euthanized on gestation day 25.

No other remarkable findings were noted in the F0 females at necropsy and no meaningful microscopic lesions were observed in any of the treated F0 females.

Conclusions:
Adverse effects did not occur in parental animals at doses up to 500 mg/kg bw/day, therefore a NOAEL of >500 mg/kg bw was identified with the help of this study.
Executive summary:

In a key 1-generation reproduction study, the calcium sulfonate read across substance (CAS 115733-09-0) was administered in corn oil via oral gavage to 28 Sprague-Dawley rats/sex at dose levels of 0, 50, 167 and 500 mg/kg bw/day (Bjorn, 2004, according to OECD 415). All F0 males were dosed for 70 days prior to mating, mating (maximum 3 weeks) and through the completion of parturition. All F0 females were dosed for up to 70 days (14 days prior to mating, during mating and gestation, and through day 20 of lactation). The animals were observed twice daily for appearance and behaviour, and a detailed clinical observation was performed weekly and daily for females during gestation. Cage site observations were performed daily approximately 30 to 120 minutes post dosing. In addition, the bodyweights were determined weekly and on the day of euthanasia for males. Females were weighed after evidence of mating on gestational days 0, 7, 14 and 21 and on lactation days 1, 4, 7, 14 and 21. Food consumption was recorded on the same days as body weights except during the mating period and during lactation. Animals were paired 1:1 for mating, after successful mating each pregnant female was caged individually. Positive evidence of mating was confirmed by the presence of sperm or a vaginal copulatory plug (day 0 of gestation). If evidence of mating was not present after three weeks, mating was discontinued. All of the surviving F0 females were allowed to deliver and rear their pups to lactation day 21.

Gross necropsies (consisting of external and internal examinations including the cervical, thoracic and abdominal viscera) were performed on death, organ weights and microscopic examinations were performed on termination. The surviving F0 dams were necropsied on lactation day 21, following a minimum of 60 days of dosing. The surviving F0 males were necropsied at the conclusion of parturition following a minimum of 96 days of dosing. F0 females that failed to deliver were necropsied on post-mating day 25 (with evidence of mating) or 25 days following the termination of the mating period (with no evidence of mating). Organ weights were determined and microscopic examinations were conducted for all surviving control and high dose F0 animals. Tissues examined microscopically included the liver, kidney, brain, right epididymides, cervix, coagulation gland, ovaries, pituitary, prostrate, seminal vesicles, testes, uterus, vagina and gross lesions. F0 animals from all groups found dead or sacrificed early were subjected to a gross necropsy and the microscopic evaluation of all tissues. Sperm was collected from all surviving F0 males and evaluated for sperm count, concentration, motility and morphology assessment. The parameters examined in P males included: testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility and sperm morphology.

No substance related effects occurred in treated animals, except for the observation of post dosing salivation and dark material around the nose in the mid and high dose groups in F0 males and the negative ammonium sulfide staining in two high dose and one mid dose F0-female. As no effects occurred at the highest dose, a NOAEL of > 500 mg/kg bw was identified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality since the key and two supporting studies are GLP compliant and are of high quality (klimisch score = 2 due to read-across)

Repeated dose toxicity: inhalation - systemic effects

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Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Repeated oral toxicity on read across substances:

In a key one-generation reproductive toxicity study, the calcium sulfonate read across substance (CAS 115733-09-0) was administered in corn oil via oral gavage to 28 Sprague-Dawley rats/sex at dose levels of 0, 50, 167 and 500 mg/kg bw/day (Bjorn, 2004, according to OECD 415). All F0 males were dosed for 70 days prior to mating, mating (maximum 3 weeks) and through the completion of parturition. All F0 females were dosed for up to 70 days (14 days prior to mating, during mating and gestation, and through day 20 of lactation). The animals were observed twice daily for appearance and behaviour, and a detailed clinical observation was performed weekly and daily for females during gestation. Cage site observations were performed daily approximately 30 to 120 minutes post dosing. In addition, the bodyweights were determined weekly and on the day of euthanasia for males. Females were weighed after evidence of mating on gestational days 0, 7, 14 and 21 and on lactation days 1, 4, 7, 14 and 21. Food consumption was recorded on the same days as body weights except during the mating period and during lactation. Animals were paired 1:1 for mating, after successful mating each pregnant female was caged individually. Positive evidence of mating was confirmed by the presence of sperm or a vaginal copulatory plug (day 0 of gestation). If evidence of mating was not present after three weeks, mating was discontinued. All of the surviving F0 females were allowed to deliver and rear their pups to lactation day 21.

Gross necropsies (consisting of external and internal examinations including the cervical, thoracic and abdominal viscera) were performed on death, organ weights and microscopic examinations were performed on termination. The surviving F0 dams were necropsied on lactation day 21, following a minimum of 60 days of dosing. The surviving F0 males were necropsied at the conclusion of parturition following a minimum of 96 days of dosing. F0 females that failed to deliver were necropsied on post-mating day 25 (with evidence of mating) or 25 days following the termination of the mating period (with no evidence of mating). Organ weights were determined and microscopic examinations were conducted for all surviving control and high dose F0 animals. Tissues examined microscopically included the liver, kidney, brain, right epididymides, cervix, coagulation gland, ovaries, pituitary, prostrate, seminal vesicles, testes, uterus, vagina and gross lesions. F0 animals from all groups found dead or sacrificed early were subjected to a gross necropsy and the microscopic evaluation of all tissues. Sperm was collected from all surviving F0 males and evaluated for sperm count, concentration, motility and morphology assessment. The parameters examined in P males included: testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility and sperm morphology.

No substance related effects occurred in treated animals, except for the observation of post dosing salivation and dark material around the nose in the mid and high dose groups in F0 males and the negative ammonium sulfide staining in two high dose and one mid dose F0-female. As no effects occurred at the highest dose, a NOAEL of > 500 mg/kg bw was identified.

In the 28 -day subacute toxicity supporting study the calcium sulfonate read across substance, (Analogue of CAS 70024-69-0), was administered via gavage to 12 Sprague-Dawley rats/sex/dose in the control and top dose groups and 6 animals Sprague-Dawley rats/sex/dose in the low and mid dose via gavage at dose levels of 0, 100, 500 or 1000 mg/kg bw/day (Wong, 1989, according to OECD 407). The control group received daily doses of peanut oil at 2.0 ml/kg, and treatment groups received the indicated dose of test material diluted in peanut oil at a dose volume of 2.0 ml/kg. The animals were treated 7 days/ week for 29 days duration with a 14 day recovery period in the control and high dose satellite recovery groups. Clinical observations were made daily. Viability checks were performed twice daily. Body weights were recorded twice weekly during treatment and weekly during recovery. Terminal body weights were recorded. Food consumption was recorded during treatment and recovery. Haematology, clinical chemistry and urinalysis parameters were evaluated at termination of treatment and recovery. Macroscopic examinations were performed on all animals. Selected organs were weighed. A range of tissues was examined microscopically.

One animal was sacrificed on day 0 and one animal was found dead on day 9, probably a result of misdosing. Stained fur was observed in high dose animals, scabbed skin occurred in one control male and high dose female displayed sneezing and abnormal respiratory sounds. No statistically significant differences were observed in mean body weights or body weight gains. Male mean cell haemoglobin concentrations were significantly decreased compared with the controls at all dose levels. However, this was not considered to be biologically significant, as there was no dose response trend. A statistically significant increase in partial thromboplastin time was observed in mid and top dose males compared with controls. Prothrombin time was significantly increased in the mid and high dose females during the treatment period, and was significantly reduced in males in the recovery group. These were within normal limits and therefore not considered to be biologically significant. A statistically significant increase in the reticulocyte count was observed in treated males in the recovery group, however, was not considered to be biologically significant. A statistically significant decrease in serum cholesterol was observed in high dose males and females and persisted in females into the recovery period. This was considered to be treatment related. Statistically significant increases were observed in alanine aminotransferase, lactic dehydrogenase, aspartate aminotransferase, sodium, phosphorus and triglycerides were observed as well as decreases in albumin and chloride. There was no dose related trend with these changes, therefore they are not considered to be treatment related. A statistically significant increase in specific gravity was observed in low dose males. Urine volume was significantly reduced in treated males in the recovery group. This was not considered to be biologically significant. No statistically significant differences were observed in organ weight, gross pathology or histopathology. The LOAEL is 1000 mg/kg bw/day, based on a decrease in mean serum cholesterol in males and females at the top dose. Based on these findings, the NOAEL is determined to be 500 mg/kg bw/day.

In another supporting study in rats (28 -day subacute oral toxicity study), a calcium sulfonate read across substance (CAS 115733-09-0) was administered via gavage at doses of 0, 50, 150, 500, and 1000 mg/kg/day to groups of 5 rats/sex/dose (Rush, 2003). This study was conducted in order to set the doses for the one generation reproductive toxicity study (Bjorn, 2004). Notable microscopic changes were limited to irritations of the nonglandular stomach at 500 and 1000 mg/kg/day in males and 125, 500 and 1000 mg/kg/day in females. The irritation effects in males and females resolved by the end of the recovery period except for one 500 mg/kg/day female, which had an ulcer with inflammation, hyperplasia, haemorrhage and oedema. In addition, non-toxicologically significant decreases in body weight gain, reduction in food consumption during certain weeks, and the described transient/reversible irritation of the nonglandular stomach were found. As the irritation effects resolved and there is no evidence of a dose-related effect on severity in females, the NOAEL for systemic toxicity is considered to be 1000 mg/kg/day which is above the limit for classification.

Repeated dermal toxicity on read across substances:

The waiving approach is supported by the weight of evidence data. The calcium sulfonate read across substance (CAS 68783-96-0) exhibited no evidence of systemic toxicity via the dermal route under the conditions of the study (Sanitised, K., 1995) when 5 Sprague Dawley CD rats/sex were exposed to the test item over a period of 28 days. A NOAEL of 1000 mg/kg was established for this study. Under the conditions of this study, dermal application of this test material resulted in no signs of overt systemic toxicity.

Another weight of evidence study (Laveglia, 1988) refers to the dermal applications of the calcium sulfonate read across substance (CAS 61789-86-4). The test item was applied to the skin of 5 male Sprague Dawley rats at a dose level of 1000 mg/kg for five days per week over a four week treatment period. The substance did not elicit related effect as determined by daily observations for physical changes and skin irritation and weekly determinations of body weights and food consumption. No treatment-related effects were noted at necropsy or microscopically. A NOAEL of greater than 1000 mg/kg bw was established.

Repeated inhalatory toxicity on read across substances:

The waiving approach is supported by the weight of evidence study (Hoffmann, 1987), in which the calcium sulfonate read across substance (CAS 61789 -86 -4), where the product as manufactured in mineral oil was further diluted 65/35 in mineral oil, was administered to assess the toxic effects by inhalation of the test item as an aerosol to 30 rats (5/sex/group) for 6 hours per day, 5 days per week for 4 weeks at target concentrations of 50, 150 and 250 mg/m³. Control animals (5/sex) received air only, while in chamber. Exposure levels were monitored gravimetrically four times per chamber per day. Particle size distribution measurements were made once each week.Physical observations for abnormal signs were made during exposure for all animals. Detailed physical examinations were conducted weekly on all animals. Body weight measurements were recorded weekly and once during the pre test period. The cumulative mean analytical exposure concentrations as determined gravimetrically were 49.5, 156 and 260 mg/m³, with an average nominal concentration of 272, 1060 and 1360 mg/m³ for the low, mid and high dose groups, respectively. Particle size distribution determinations indicated the test aerosol atmosphere was respirable to the rat. All animals survived the duration of the study. Physical observations during the exposures included red nasal discharge, matted coat and decreased activity at the two higher dose levels. No significant respiratory signs were noted during the weekly observations. However, increased incidence of dried red nasal discharge and matted coat was seen among the treated animals. Body weight measurements indicated a trend towards lower weight gain, especially in the high level males. Nevertheless, this difference was not statistically significant. Haematology and clinical chemistry parameters were generally not indicative of any test-material effect. Terminal organ weight and organ body weight ratios were clearly indicative of a respiratory effect with significant dose related increases in lung weights and ratios at the mid and high levels. Other organ weights and ratios were unremarkable.

Gross post mortem evaluations were unremarkable as well. However, a higher incidence was seen microscopically in the lungs at the mid and high levels. An accumulation of intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium were observed. Other microscopic findings were not considered test-substance related. To sum it up, treatment-related changes at all dose levels occurred. On this basis, a "No Observed Effect Level" (NOEL) could not be established. The change observed at the concentration of 50 mg/m³ was considered not to be indicative of a serious adverse effect of treatment and on this basis may be regarded as a “No Observed Adverse Effect Level” (NOAEL).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study with a nearest structural analogue is with the longest duration (at least 90 days in males and 59 days in females) and lowest NOAEL.

Justification for classification or non-classification

No classification and labelling for repeated dose effects is required for Benzenesulfonic acid, mono-C20-24 (even)-sec-alkyl derivs., para-, sodium salts (generic name: C20-24 sodium sulfonate) as the effects observed in the oral repeated dose toxicity studies with the read-across substances calcium sulfonates are limited to minimal non-adverse effects. Post dose salivation and dark material around the nose were observed in the oral one-generation study in rats (CAS 115733-09-0; Bjorn, 2004), slight reduction in food consumption and body weigh gain at the highest dose level in males in the 28 -day study in rats (1000 mg/kg bw), which significance is questionable (CAS 115773 -09 -0; Rush, 2003) and a slight reduction of serum cholesterol in the 28 -day study with rats (Analogue of CAS 70024 -69 -0; Wong, 1989), the effect which is not toxicologically significant and not relevant to humans.

Concerning possible systemic effects by dermal route of exposure, they could be ruled out because no significant dermal absorption is expected for C20 -24 sodium sulfonate due to its physico-chemical properties and the evidence from the dermal 28 -day studies with the structural analogues (CAS 68783-96-0 and CAS 61789-86-4; Sanitised, K., 1995; Laveglija, 1988), in which no systemic toxicity was observed at the highest dose levels tested.

The inhalation route is also a not relevant route of exposure for C20-24 sodium sulfonate due to its physico-chemical properties. Although treatment-related effects were observed in the inhalation study in rats with the structural analogue calcium sulfonate (CAS 61789-86-4; Hoffman, 1987), they are not severe (as defined in the ECHA guidance on CLP) and seem to be rather local effects: an accumulation of intraalveolar macrophages and hyperplasia/hypertrophy of bronchiole epithelium.

Based on these data and in accordance with EU CLP Regulation (EC) No. 1272/2008, classification and labelling is not required for systemic organ toxicity after repeated exposure (STOT-RE) for C20-24 sodium sulfonate.