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Administrative data

Description of key information

Read across: CAS 115733-09-0, One-generation reproductive toxicity study, rats, NOAEL > 500 mg/kg bw


Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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.
Key result
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 Generation:

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:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
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.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
A functional observation battery for neurotoxicity was not performed since this test was not part of the OECD 407 guideline at the time the study was performed
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 41 days
- Weight at study initiation: males, 179-215g; female 141-170g
- Housing: hanging stainless steel wire-bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23°C
- Humidity (%): 48-66%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Mixed weekly weight/volume in peanut oil
DIET PREPARATION: Rate of preparation of diet (frequency): weekly
Route of administration: oral gavage (syringe and dosing tube)

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis of dosing solutions was conducted to confirm that they were homogeneous and met the desired concentrations.
Duration of treatment / exposure:
29 day treatment duration with a 14 day recovery period (29 days of treatment followed by a 14 day recovery period in the control and high dose satellite recovery groups.)
Frequency of treatment:
7 days/week.
Remarks:
Doses / Concentrations:
gavage dose of 0 mg/kg/bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
gavage dose of 100 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
gavage dose of 500 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
gavage dose of or 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6
Control animals:
yes
Details on study design:
- Dose selection rationale: Data from a pilot two week repeated dose oral study
Control group and treatment: 6 rats/sex/group for each dose, and satellite recovery groups of 6 animals/sex for the control and 1000 mg/kg/day dose. 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
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: Yes
- How many animals: All

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight before termination
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Tests applied included; parametric ANOVA with Dunnetts post-hoc test, non parametric Kruskal-Wallis and Mann-Whitney U test, Bartletts test for equal variances, Students t test and Dixons teat for rejection of outlying values.
Body weight, food consumption, feed efficiency, haematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analysed. Mean values of all dose groups were compared to control at each time interval. Tests included parametric ANOVA with a Dunnett’s post-hoc test, non-parametric Kruskal-Wallis and a Mann-Whitney U-test, Bartlett’s test for equal variances, a Student’s t-test and Dixon’s test for rejection of outlying values.
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 examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One animal was sacrificed on Day 0 and one animal was found dead on Day 9, a result of probable 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.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences were observed in mean bodyweights or body weight gains.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A statistically significant increase in food consumption was observed in low dose males compared with controls.

FOOD EFFICIENCY
No statistically significant differences were observed in food efficiency.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not recorded

OPHTHALMOSCOPIC EXAMINATION
Not recorded

HAEMATOLOGY
Male mean cell haemoglobin concentrations were significantly decreased compared with the controls at all dose levels. However, these were 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.

CLINICAL CHEMISTRY
A statistically significant decrease in serum cholesterol was observed in high dose males and females and persisted in females into the recovery period. this are 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.

URINALYSIS
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.

NEUROBEHAVIOUR
Not recorded

ORGAN WEIGHTS
No statistically siognificant differences were observed.

GROSS PATHOLOGY
No substance related macroscopic changes were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
No substance related microscopic changes were observed.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not recorded

HISTORICAL CONTROL DATA (if applicable)
Not recorded
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: Mean serum cholesterol levels were significantly reduced in the 1000 mg/kg males and females at termination of dosing. this was still significantly reduced in 1000 mg/kg females at the end of the 14 day recovery period.
Critical effects observed:
not specified

Table 1: Average body weights and body weight gains during xx days of treatment

Dose rate (ppm)

Body Weights (g)

 

Total Weight Gain

Week 0

Week 1

Week 2

Week 3

Week 4

g               

% of control

Male

 0

195

243

286

323

352

157

181

100

196

245

298

340

374

175

191

500

200

245

289

329

362

162

181

1000

193

240

283

315

346

154

179

Female

  0

155

175

194

213

223

67

144

100

156

174

194

215

228

72

146

500

154

175

190

212

221

67

144

1000

155

174

195

212

224

69

145

 

An NOEL of 500 mg/kg/day was established for this study. No test material related mortality was observed. One low dose male was found dead on Day 9. This was attributed to a probable misdosing. A second low dose male was replaced, due to a possible misdosing, on the first day of treatment. Mean serum cholesterol levels were significantly reduced in the 1000 mg/kg males and females at termination of dosing and in the 1000 mg/kg females at the end of the 14-day recovery period. No treatment-related effects were observed on mortality, clinical observations, body weight and body weight gain, food consumption, feed efficiency, haematology, urinalysis, absolute and relative organ weights and macroscopic or microscopic pathology. Statistically significant differences from control were observed for some haematology and clinical chemistry parameters. These values were within clinically normal limits and were not associated with corresponding histopathological changes. They were not considered biologically significant. Chemical analysis of dosing solutions confirmed that they were homogeneously prepared at the desired concentrations.

Conclusions:
A NOAEL of 500 mg/kg bw/day was identified in this study.
Executive summary:

In a subchronic toxicity study calcium sulphonate was administered 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). A decrease in serum cholesterol levels occurred in the top dose group. The LOAEL is 1000 mg/kg bw/day, based on  a decrease in serum cholesterol at the top dose.  The NOAEL is 500 mg/kg bw/day. This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in rats. Little subchronic toxicity was observed over the range of doses administered in this study. Based on a reduction in mean cholesterol values in the males and females treated at the 1000 mg/kg dose level, the NOAEL was 500 mg/kg.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
Between 18 April 2002 and 31 May 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3050
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 6-7 weeks of age at initiation of treatment
- Weight at study initiation: 240-290 gr (males) and 166-206 g (females)
- Housing: individually in suspended stainless steel wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
maize oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis performed for dosing solution stability, homogeneity and concentration.
Duration of treatment / exposure:
28 days (28 days of treatment followed by 14 day recovery period in the control and high dose groups)
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
5 or 10 (control group with 10 animals, low and mid- dose groups with 5 animals each and high-dose group with 10 animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected by the sponsor based on available data from previous studies.
- Rationale for animal assignment (if not random): Random
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): Random

Control group and treatment
5 rats/sex/group for each dose, and recovery groups of 5 animals/sex for the control and 1000 mg/kg/day dose. Control group received daily doses of corn oil at 5.0 ml/kg, and treatment groups received the indicated dose of test material diluted in corn oil at a dose volume of 5.0 ml/kg.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (between approximately one-half and two hours following dosing, and daily during recovery phase.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and prior to the initiation of treatment on day -2.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Days 25 and 38 (during week 3 28-day main and recovery animals and week 5 - recovery animals)
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Animals fasted: Yes
- How many animals: All

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight prior to sacrifice
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Abbreviated battery on day -1, weeks 0, 1 and 2. Full battery on weeks 3 and 5
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (day 28 or 42).
Fresh organ weights were obtained for all animals at scheduled euthanasia and selected tissues were preserved from all rats.
All tissues and organs collected at necropsy from animals in the control and high-dose groups and the stomach from the 50, 150, and 500 mg/kg /day groups were examined microscopically.
HISTOPATHOLOGY: Yes
Statistics:
Parametric and count data were analysed by One-Way analysis of Variance (ANOVA). If significance was detected, pair-wise group comparisons proceeded using the Tukey-Kramer test. Ranked data were analysed by Kruskall Wallis non-parametric ANOVA, followed by Dunn's test. Descriptive and quanta data were analysed by fisher's Exact test. Group by group comparison was undertaken using the Chi-Square test.

Absolute and relative organ weights and clinical pathology data were analysed for homogeneity of variance using Levene's test, then Kruskal-Wallis non-parametric ANOVA, followed by Dunn's test.
Clinical signs:
no effects observed
Description (incidence and severity):
no mortality, significant clinical abnormalities or toxicological meaningful neurological changes were observed during the main or recovery phase of this study.
Mortality:
no mortality observed
Description (incidence):
no mortality, significant clinical abnormalities or toxicological meaningful neurological changes were observed during the main or recovery phase of this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Notable body weight effects were primarily limited to the 500 and 1000 mg/kg/day group males and included decreased weight gain during the third study week. Overall weight gain was app. 9 % and 6 % lower, respectively.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was also decreased in the mid dose group males durign the third study week
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically meaningsful changes in the haematology, coagulation, clinical chemistry or urinalysis parameters examined during the main and recorvery phases of this study.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality or significant clinical abnormalities were observed during the main or recovery phases of this study.

BODY WEIGHT AND WEIGHT GAIN
In the males, a statistically significant decreased bodyweight gain was observed in the top dose group. A decrease in bodyweight gain (not statistically significant) was observed in the next highest dose group. However, these decreases were less than 10%., and therefore of questionable statistical significance.

In females, no statistically significant changes were observed.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Statistically significant decreases in food consumption occurred in the second highest group males and the top dose group females.

FOOD EFFICIENCY
Not recorded.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not recorded.

OPHTHALMOSCOPIC EXAMINATION
No treatment related effects occurred.

HAEMATOLOGY
No biologically significant changes occurred.

CLINICAL CHEMISTRY
No biologically significant changes occurred.

URINALYSIS
No biologically significant changes occurred.

NEUROBEHAVIOUR
No biologically significant changes occurred.

ORGAN WEIGHTS
No biologically significant changes occurred.

GROSS PATHOLOGY
No adverse gross pathology occurred in treated animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
Notable microscopic changes in the organs and or tissues were limited to irritation of the non-glandular stomach occurred in the two highest dose group males and the three highest dose group females. This irritation appeared to be transient. Minimal oedema in the submucosa was observed in the second highest dose group males and minimal to mild oedema in the submucosa and minimal epithelial hyperplasia was observed in the highest dose group males. However, these effects were not observed after the recovery phase. In addition, no stomach abnormalities were observed in the group 1, 2 and 3 (0, 50 and 150 mg/kg day) males at the end of the treatment phase.


Minimal oedema in the submucosa, minimal haemorrhage, minimal epithelial hyperplasia, mild inflammation and a mild ulcer occurred in females in the second highest dose group. Minimal oedema in the submucosa and minimal to mild epithelial hyperplasia was observed in females in the top dose group.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not recorded.

HISTORICAL CONTROL DATA (if applicable)
Not recorded.

Other: Ophthalmology - Ocular findings were consistent with this strain and age of animal.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Critical effects observed:
not specified

Table 1: Average body weights and body weight gains during 28 days of treatment

Dose rate (ppm)

Body Weights (g)

 

Total Weight Gain

Week 0

Week 1

Week 2

Week 3

Week 4

g               

% of control

Male

 0

 268

302

331

358 

372 

104

 139

50

 268

303 

335 

359 

373 

105

 139

150

 267

301 

330 

360 

370 

103

 139

500

 263

290 

312 

327 

340 

77

 129

1000

267

298

324

340

351

83

131

Female

  0

 186

203 

216 

228 

263 

50

 141

50

 186

206 

222 

235 

239 

53

 128

150

 184

199 

214 

223 

235 

50

 128

500

 187

202 

212 

224 

228 

41

 122

1000

184

202

211

226

229

45

125

 

Results

An NOAEL (no observed adverse effect levels) for systemic toxicity of 1000 mg/kg/day was established in this study. Body weight effects were limited to the males at 500 and 1000 mg/kg/day and included decreased weight gain during the third study week. Overall weight gain was reduced, compared to control, in the 500 and 1000 mg/kg males by approximately 9 and 6% at the end of treatment. The weight gain of the 1000 mg/kg/day males was approximately 6% lower than control at the end of recovery. Statistically significant reductions in mean food consumption were observed in the 500 mg/kg/day males during week 3 of the treatment period and in the 1000 mg/kg/day females during week 2 of the treatment period. Notable microscopic changes were limited to irritation of the nonglandular stomach in the 500 and 1000 mg/kg/day males and in the 150, 500 and 1000 mg/kg/day females. This finding was transient and was not evident in the recovery animals. In the 500 mg/kg/day males, minimal oedema in the submucosa was observed in 2 of 5 animals. In the 1000 mg/kg/day males minimal to mild oedema in the submucosa and minimal epithelial hyperplasia were observed in 3 of 5 animals at the end of treatment. No stomach abnormalities were evident after recovery. In the 150 mg/kg/day females, minimal oedema in the submucosa was observed in 2 of 5 animals. In the 500 mg/kg/day females, mild edema in the submucosa, minimal hemorrhage, minimal epithelial hyperplasia, mild inflammation and a mild ulcer were observed in 1 of 5 animals. In the 1000 mg/kg/day females minimal oedema in the submucosa was observed in 1 of 5 animals and minimal to mild epithelial hyperplasia was observed in 2 of 5 animals at the end of treatment. No stomach abnormalities were evident after recovery. No stomach abnormalities were evident after recovery.

No mortality, significant clinical abnormalities, meaningful neurological changes, ophthalmoscopic changes, hematology, clinical chemistry, urinalysis, absolute and relative organ weights and macroscopic pathology were observed during treatment or recovery. Chemical analysis of dosing solutions confirmed that they were homogeneously prepared and stable at the desired concentrations. Weekly concentration analysis confirmed that the dosing solutions were prepared appropriately.

Conclusion: Oral administration of CAS 115733 -09 -0 to rats for up to 28 consecutive days did not produce mortality or notable clinical, neurological or clnical pathology abnormalities at up to 1000 mg/kg/day. Microscopic evidence of minimal irritation of the nonglandular portion of the stomach was observed in the 500 and 1000 mg/kg/day males. However, the changes in the 500 and 1000 mg/kg /day males were limited to minimal to mild edema and minimal hyperplasia and resolved by the end of the recovery phase. Therefore, the systemic no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/ day. In females, minmal irritation in the nonglandular stomach was observed in the 150 and 1000 mg/kg/day females and resolved by the end of the recovery phase. However, an ulcer with inflammation, hyperplasia, hemorrhage and edema was observed in the stomach of one 500 mg/kg/day female. Therefore the no-observed-adverse effect level (NOAEL) for local irritation for the females was considered to be 150 mg/kg/day.

Conclusions:
A NOAEL of 1000 mg/kg bw/day was identified in this study.
Executive summary:

In a subacute toxicity study was administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 50, 150, 500, or 1000 mg/kg bw/day). No dose related effects occurred. The NOAEL for systemic toxicity is 1000 mg/kg bw/day. Little subacute toxicity was observed over the range of doses administered in this study. Based on the microscopic data the Study Director concluded that the NOAEL was 1000 mg/kg/day.

Endpoint:
sub-chronic toxicity: oral
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
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic 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 study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic 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 study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated oral toxicity on read across substances:

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 14 days prior to mating, mating (maximum 3 weeks), 25 days of 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 a 28 -day subacute toxicity supporting study the calcium sulfonate read across substance, (Analogue of CAS 70024-69 -0, Wong, 1989, according to OECD 407), 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. 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.

No classification for repeated dose effects is required as the effect is limited to a slight reduction of serum cholesterol, which is not toxicologically significant and not relevant to humans. The other effects observed at the 500 mg/kg/day dose level were not considered to be adverse.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
best study available

Justification for classification or non-classification

As the magnesium and calcium sulfonate read across substances did not cause relevant significant toxicological effects after repeated oral exposure, the magnesium sulfonate target chemical is also not expected to cause significant toxicity. Therefore, the magnesium sulfonate target chemical does not meet the criteria for classification and will not require labelling, according to the European regulation (EC) No. 1272/2008.