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Description of key information

Subacute oral toxicity euivalent to OECD 407 method in male rats dosed at 0.25, 0.5 and 1 % in the
diet for 28 days, corresponding with average doses of 7.47, 74.7 and 812 mg act.ingr./kg bw.
Subchronic oral toxicity was further tested equivalent to OECD 408 method in male and female
rats at 1% in the diet for 90 days,corresponding with ca. 750 mg act. ingr./kg bw. These studies
did not reveal toxicity, therefore 1% in the diet, corresponding with ca. 750 mg act.ingr./kg bw can
be accepted as NOAEL.

A 14 -day dose-range finding toxicity study by oral gavage at 0, 100, 300 and 1000 mg/kg bw/day

was well tolerated and only resulted in increased serum Alanine aminotransferase activity in the

mid and high dosed males & females and multifocal yellow discoloration and/or thickness in the

non-glandular mucosa of the stomach 1000 mg/kg dosed males and females at necrospsy.

A Combined repeated dose toxicity with reproduction/developmental toxicity screening test by

oral gavage at 0, 100, 300 and 1000 mg/kg bw/day resulted in slight statistically significant decreases

in body weight, weight gain and food intake at 1000 mg/kg bw during the gestation and lactation period.

Statistically significant changes were also observed for clinical chemistry (increase Alanin aminotrans-

ferase at 300 & 1000 mg/kg dosed males and 1000 mg/kg dosed females; increase Alanina phos-

phatase inb the 1000 mg/kg dosed females; decreased serum cholesterol in the 1000 mg/kg dosed

females). Macroscopic thickness and discoloration were seen in the non-glandular mucosa of the

stomach of high dosed males and females at necropsy. Further post-mortem investigation is ongoing.

Preliminary results are provided; the study is not yet finalised and therefore supporting at this moment.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1969
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable. There were some deviations from the study guidelines, however these did not affect the conclusions and the validity of the study.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
only one dose per test substance
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
other: Charles River albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories, North Wilmington, Mass.
- Age at study initiation: Not provided
- Housing: individually in standard wire-bottomed steel rat cages
- Diet : standard rat ration blended with the appropriate amount of test material in a Hobart Mixer
Fresh diets were prepared each week. Each rat was offered an amount of diet sufficient for one
week ‘ad libitum’ feeding. However, checks were made periodically to ensure that the food jars were
not empty
- Water: No data provided
- Acclimation period: Not provided

ENVIRONMENTAL CONDITIONS
Not provided

IN-LIFE DATES: From: To: Not provided
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: 1.0% in the feed
Taking into account a mean body weight of 250 g and a mean food consumption of 20g/rat/day
(Derelanko M.J., 2008, The Toxicologist's Pocket Handbook, Informa).
1% in the diet = 10000 mg/kg diet corresponds with 10 mg/g diet
20 g feed/rat (250g bw)/day = 80 g feed/kg bw/day = 0.8 g active ingredient/kg bw/day = 800 mg/kg bw/day.
A higher feed intake is possible, e.g. 1000 mg/kg at higher body weight and feed intake, but from a conservative viewpoint 750 mg/kg bw is taken.

DIET PREPARATION
- Rate of preparation of diet (frequency):Fresh diets were prepared each week
- Mixing appropriate amounts with (Type of food): standard rat ration

DIET PREPARATION
- Rate of preparation of diet (frequency):Fresh diets were prepared each week
- Mixing appropriate amounts with (Type of food): standard rat ration
- Storage temperature of food: Not provided

VEHICLE No Vehicle

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
1 other: % Basis: nominal in diet
No. of animals per sex per dose:
For Aerosol TR: 20male +20 female at 1 % dietary level and 20 male +20 female as control
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 15, 30, 45, 60, 75 and 90.

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

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 84 days
- Anesthetic used for blood collection No data
- Animals fasted: Yes (fasted serum glucose concentration)
- How many animals: 5 rats of each sex (=10) and 10 control
- Parameters checked:
Hematocrit Value
Erythrocyte Count
Hemoglobin Concentration
Total Leukocyte Count
Differential Leukocyte Count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 84 days
- Animals fasted: Yes(fasted serum glucose concentration)
- How many animals: 5 rats of each sex (=10) and 10 control
- Parameters checked:
Blood Urea Nitrogen (BUN) Concentration
Serum Alkaline Phosphatase (SAP) Activity
Serum Glutamic-Pyruvic Transaminase (SGPT) Activity
Fasted Serum Glucose Concentration


URINALYSIS: Yes
- Time schedule for collection of urine: after 84 days
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked:
Glucose Concentration
Albumin Concentration
Microscopic Elements Examination
pH
Specific Gravity


NEUROBEHAVIOURAL EXAMINATION: Yes , but in general, not specific
- Time schedule for examinations: abnormal reactions and death were recorded daily during the investigation
- Dose groups that were examined: control and 1.0% dose
- Battery of functions tested: sensory activity / grip strength / motor activity / other:No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes , no findings
HISTOPATHOLOGY: Yes , no findings
Statistics:
Statistical analyses were conducted upon the absolute organ weights and their corresponding ratios to the weight of the body. An Analysis of
Variance was conducted first and any significant effects disclosed by that treatment were further studied by “t” –tests.
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 examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
elevation of SGPT and SAP in males
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects for Aerosol TR
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 other: % in the diet
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: See remark
Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: based on mean test article intake
Critical effects observed:
not specified

Table 2. Body Weight Datta - Summary of Mean Values

 

 

Body Weight (grams)      

 

Total Weight Gain (grams/rat)

Group

Sex

Day 0

Day 15

Day 30

Day 45

Day 60

Day 75

Day 90

 

Control

M

122

178

254

340

403

435

466

344

 

F

101

154

188

212

239

273

280

179

1%

M

121

175

250

338

392

415

455

334

 

F

101

151

184

216

239

263

269

168

Conclusions:
The comparisons of final body weights and total weight gains revealed no statistically significant differences between test and control animals.
No outstanding differences in food consumption were noted between test rats and control rats.
No deaths or abnormal behavioral reactions were noted among any of the animals employed in the study.
No outstanding differences between test and control rats were noted with respect to any of the blood parameters studied.
(With the exception of an elevation of both SGPT and SAP values among males fed Aerosol TR,) all data obtained from test rats were not different than those from control animals.
No significant differences between the urine of test rats and control rats were observed.
No outstanding differences between test and control rats were noted at the time of gross pathological examination.
The only statistically significant difference noted was smaller absolute liver weights among male rats fed 1.0% Aerosol IB.
There were no significant differences between the tissues of test and control rats observed upon histopathological examination.

Executive summary:

Six groups of 40 albino rats (20 male, 20 female, Charles River strain) plus 1 control group (20 male + 20 female) were fed with 1% of various test items mixed into the diet. The various test items were category members of the Sulfosuccinates Diester Group including Butanedioic acid,sulfo- 1,4 -ditridecyl ester, sodium salt. After 84 days hematological values, blood chemical values and urinalysis values were measured for all animals. Tissues were examined pathologically at the conclusion of the 90-days test period. Organ to body weight and organ to brain weight ratios were calculated. No significant differences in clinical blood chemistry studies and absolute organ weights have been detected, except a slight increase in SGPT (serum glutamic pyruvic transaminase) and SAP (serum alkaline phosphatase). Body weights, organ to body weight ratios, hematologic studies and urinalysis were not different between test and control animals. No deaths or abnormal behavioral reactions occurred; no gross pathological findings were noted. Category members at 1% in the diet (10000 ppm equivalent to 750 mg/kg bw/day) for 90 days in rats did not result in any relevant changes in the subchronic toxicity study. The NOAEL was therefore considered to be 750 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 2

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

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

Additional information

Subacute toxicity
A 28 -day study with dietary feeding of a registered substance containing 97 -98% active ingredient was available in male rats dosed at 100, 1000 and 10000 ppm in the diet (corresponding with 7.47, 74.7 and 812 mg act.ingr./kg bw/day on average) (Tusing, 1953). Animals dosed at 1000 and 10000 ppm levels showed a slightly greater retardation in growth than rats at 100 ppm. Food consumption for all groups was comparable to the controls during the 28-day period. One control animal died after 4 days of feeding. Gross autopsy revealed some incidence of intestinal irritation at all levels. Various other findings were observed at necropsy, however a respiratory infection was present, therefore the interpretation of the study is difficult. The study was considered to be supporting, and further 28 -day testing was waived based on the availability of 90 -day toxicity data.

A supporting 14 day dose range finding toxicity was conducted in order to determine the dose levels for a subsequent OECD No. 422 study. Male and female Wistar rats (8-9 weeks old animals) were treated by oral gavage at dose levels at 0, 100, 300 and 1000 mg/kg bw/day (Hargitai, 2020a). There was no test item-related mortality, clinical signs, body weight & weight gain or food intake changes. No test item-related effects were seen for haematology parameters up to 1000 mg/kg bw in males and females, however serum Alanine aminotransferase activity was significantly increased in the mid and high dosed males & females. Further test item-related macroscopic findings (multifocal yellow discoloration and/or focal/multifocal/diffuse thickness) were seen in the non-glandular mucosa of the stomach of high dosed males and females at necropsy. No test item-related effects were observed on organ weights.

A Combined repeated dose toxicity with reproduction/developmental toxicity screening test in Wistar rats was conducted under GLP conditions with the registered substance by oral gavage administration at dose levels at 0, 100, 300 and 1000 mg/kg bw/day (Hargitai, 2020b). The study is still in the reporting phase; there were only preliminary results. There was no test item-related mortality, clinical signs, body weight & weight gain (mating period) or food intake changes (mating period). Slight statistically significant decreases in body weight, weight gain and food intake were observed in females of the 1000 mg/kg bw during the gestation and lactation period. No test item-related effects were seen for haematology & urinalysis parameters up to 1000 mg/kg bw in males and females, however statistically significant changes were observed for clinical chemistry (increased Alanine aminotransferase at 300 & 1000 mg/kg dosed males & 1000 mg/kg dosed females; increased Alkaline phosphatase in the 1000 mg/kg dosed females; decreased serum cholesterol in the 1000 mg/kg dosed females). Further test item-related macroscopic findings (thickness and discoloration) were seen in the non-glandular mucosa of the stomach of high dosed males and females at necropsy. Further post-mortem investigation is ongoing.

Subchronic and chronic toxicity
A key 90 -day study was available for the registered substance in which 40 albino rats (20 males, 20 females) were fed with 1% of various test items mixed into the diet. In the same study, various members of the Sulfosuccinates Diester Group were tested (Plank et al, 1969). After 84 days hematological values, blood chemical values, urinalysis values were measured for all animals. Tissues were examined pathologically at the conclusion of the 90-days test period. Organ to body weight and organ to brain weight ratios were calculated. No significant differences in clinical blood chemistry studies and absolute organ weights have been detected, except a slight increase in SGPT (serum glutamic pyruvic transaminase) and SAP (serum alkaline phosphatase). Body weights, organ to body weight ratios, hematologic studies and urinalysis were not different between test and control animals. No deaths or abnormal behavioral reactions occurred; no gross pathological findings were noted. Administration of category members at 1% in the diet (10000 ppm equivalent to ca.750 mg act. ingr./kg body weight/day on average basis) for 90 days in rats did not result in any relevant changes in the subchronic toxicity study. The NOAEL was therefore considered to be worst case 750 mg/kg bw/day.

The validity of the study was supported by additional audits on the raw data and histopathological evaluation. Although deficiencies were detected compared to current standards, the study was concluded to be valid and reliable.

In a supporting study, groups of 10 (5 male & 5 female) Wistar rats were treated for 6 months at concentrations of 0.25, 0.5, 0.75, 1.0 and 1.25 g/kg diet, corresponding to doses of 190, 370, 550, 750 and 870mg/kg bw/day. Occasional spells of diarrhea occurred in some animals, particularly at the higher doses. Neither the total red cell, total white cell, nor the differential counts of rats was affected by the continued administration . The dose level of 750 mg/kg was confirmed as NOAEL (Literature, Benaglia et al. 1943).

Other studies were also available from literature in various species (Literature, Benaglia et al. 1943 and Case et al., 1977) in dogs, rabbits and Rhesus monkeys. The other species were considered to be less appropriate due to the gastrointestinal tensioactive local irritation by which systemic effects could not be fully evaluated.

 

General assessment and conclusion
- The fact that no relevant target organ changes were seen up to highest concentration of 1% in diet for 90 days, allows to conclude that corresponding intake of 750 mg/kg bw is NOAEL.

- Further information supporting the safety of the test substance is provided in the read across justification for the Diester category, showing that all substances in the group had a NOAEL of at least 750 mg/kg bw (justification with data matrix separately attached in Section 13).

 

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

Repeated dose toxicity: via oral route - systemic effects (target organ)digestive: duodenum; digestive: liver

Justification for classification or non-classification

As there were no changes observed in the repeated dose toxicity studies with the test item up to 1% in the diet (corresponding to ≥ 750 mg act.ingr./kg bw/day on average basis), classification is not warranted according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008).