Registration Dossier

Administrative data

Description of key information

- OECD 422: NOAEL 150 mg/kg for females, 500 mg/kg for males; NOAEL (pups) 150 mg/kg bw/day (2015)

- OECD 408; GLP; Wistar rats; 150, 450, 1400 mg/kg bw/day; NOAEL (sys) 450 mg/kg bw/day; NOEL (loc) <150 mg/kg bw/day (2020)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Apr 2019 - May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Jun 2018
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Lot/batch number of test material: 0018983927
- Expiration date of the lot/batch: 01 May 2020
- Purity: 52.5%
- Physical state/ appearance: liquid/ brownish, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (RT), avoid temperatures <10 and >40 °C
- Stability under test conditions: stability in drinking water for a period of 7 days at room temperature as well as at the temperature of a refrigerator confirmed
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: males: mean 182.3 g, females: mean 141.3 g
- Fasting period before necropsy: about 16 to 20 h
- Housing: 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: The supplier assayed the food used in the study for chemical and microbiological contaminants. The drinking water is regularly assayed for chemical contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (light: 6 am to 6 pm)

IN-LIFE DATES: From: April 09, 2019 To: Jul 23, 2019 (males), Jul 24, 2019 (females)
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard. The test substance was administered daily by gavage for 3 months.
Vehicle:
water
Remarks:
deionized water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: produced at least weekly
- Administration volume: 10 ml/kg bw
- Storage temperature of preparation: room temperature.

VEHICLE : deionized water
- Justification for use and choice of vehicle (if other than water): water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and concentration control analyses of test-substance preparations were performed at the beginning and towards the end of the administration period in all concentrations. Each 3 samples were taken from the lowest and highest concentration for homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control analysis. The method used to analyze the content was HPLC.
Duration of treatment / exposure:
3 months
Frequency of treatment:
once daily
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Based on purity: 79 mg/kg bw/day active ingredient
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
Based on purity: 236 mg/kg bw/day active ingredient
Dose / conc.:
1 400 mg/kg bw/day (nominal)
Remarks:
Based on purity: 735 mg/kg bw/day active ingredient
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose rational
The aim of the study was to assess the toxicological profile of Reaction products of fatty acids, C18 (unsaturated) alkyl with sulfur trioxide, potassium salts including the target organs and the “no observed adverse effect level” (NOAEL) after 3-month administration by gavage.

Selection of doses:
The following doses were selected:
150 mg/kg bw/d (corresponds to 79 mg/kg bw/d active ingredient)
450 mg/kg bw/d (corresponds to 236 mg/kg bw/d active ingredient)
1400 mg/kg bw/d (corresponds to 735 mg/kg bw/d active ingredient)

Purity of the test substance is 52.5%, corresponding to Reaction products of fatty acids, C18 (unsaturated) alkyl with sulfur trioxide, potassium salts

The dosage of the high dose group was selected based on results of an OECD 422 (project no. 85R0449/14C067), where signs of systemic toxicity were observed in female parental animals at 500 mg/kg bw/d. However, no signs of general systemic toxicity in male parental animals up to 500 mg/kg bw/d were revealed. Taking into account the longer treatment period in the OECD 408 and considering the relative low effects in the OECD 422, the dosage for the OECD 408 was raised by a factor of about three.

- Rationale for animal assignment (if not random): random
Positive control:
not included
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily for signs of toxicity and before and within 2 hours as well as within 5 hours after treatment; twice for mortality

DETAILED CLINICAL OBSERVATIONS: Yes (in an open field)
- Time schedule: prior to the start of the administration period and weekly thereafter
- Parameters checked: Abnormal behavior when handled, Fur, Skin, Posture, Salivation, Respiration, Activity/ arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos, Assessment of the feces discharged during the examination (appearance/ consistency), Assessment of the urine discharged during the examination, Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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
- Time schedule: from day -4 to 0 and thereafter weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- monitored by daily visual inspection of the water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the beginning and at the end of the administration period, i.e. study day 91
- Dose groups that were examined: control and 1400 mg/kg bw/day

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day of necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids, HDL-cholesterol, LDL-cholesterol, ALT, AST, ALP, GGT, total triiodothyronine (T3), total thyroxine (T4), Thyroid stimulating hormone (TSH)

URINALYSIS: Yes
- Time schedule for collection of urine: On day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, protein, glucose, ketones, urobilinogen, bilirubin, blodd, specific gravity, sediment, color/turbidity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period as well as in all animals left at the end of the recovery period
- Dose groups that were examined: all animals
- Battery of functions tested: home cage observations/ open field observations / sensory activity / grip strength / motor activity

IMMUNOLOGY: No

ESTROUS CYCLE (females only):
- Time schedule for examination: on the day of sacrifice
- Vaginal smears were prepared
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- the exsanguinated animals were necropsied and assessed by gross pathology. Final body weight was taken from anesthetized animals.
- Organ weights from all animals: Adrenal glands (fixed), Brain, Epididymides, Heart, Kidneys, Liver, Ovaries (fixed), Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Spleen, Seminal vesicles including coagulating glands (fixed), Testes, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
- Organs examined for control and 1000 ppm on all animals: Adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, esophagus, eyes with optic nerve, female mammary gland, femur with knee joint, heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (mesenterial and axillary), nose (nasal cavity, level III), ovaries, pancreas, parathyroid glands, peyer'patches, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (cervical-, thoracic-, lumbar cord), spleen, stomach, testes, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina
- Organs examined for 150 and 450 mg/kg bw/day: lungs, kidneys, lymph nodes (mesenteric and axillary), nose (nasal cavity, level III)
- all gross lesions: all animals affected per group
Statistics:
Dunnett's test, Kruskal-Wallis test, Wilcoxon test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): Soft feces in 10/10 males from study day 66 onwards; Respiration sounds in 3/10 males and females on each one or two days after treatment; Semiclosed eyelids in 1/10 males and 2/10 females on each one day after treatment; Nose discharge in 1/10 males on one day

Furthermore, one female of 1400 mg/kg bw/d had an injury of the right eye (first seen on study day 48) resulting in the loss of this eyeball on study day 52. This finding was considered as accidental and not treatment-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- 150 mg/kg bw/day (79 mg/kg bw/day active ingredient): one male was found dead on day 66 showing any abnormal clinical signs before. The death of this animal was considered to be caused by a malignant lymphoma based on the microscopic findings
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): body weight change in males from administration day 0 to 91 was -9.7% lower than in the current control resulting in a nonsignificant
decrease of the corresponding body weight of -5.5%. These findings were considered as treatment-related but not adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): increase of 12%, day 35 to 42 and day 42 to 49 in males; decrease -10.7%, study day 77 to 84 in females
- 450 mg/kg bw/day (236 mg/kg bw/day active ingredient): decrease -10.9%, day 77 to 84 and -11%, day 84 to 91 in females
- 150 mg/kg bw/day (79 mg/kg bw/day active ingredient): increase of 16.8% in females

It cannot be excluded that the slight decreases of food consumption were treatment-related. However, based on the degree of decrease and the short temporary occurrence, these findings were considered as non-adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): One female had an injury of the right eye (first seen on study day 48) resulting in the loss of this eyeball on study day 52 with a closed eyelid on study day 91. It was assessed as not treatment-related.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): Decreased hemoglobin and hematocrit values in both sexes; Decreased red blood cell (RBC) counts in females; Decreased absolute reticulocyte counts in males; Increased absolute neutrophil counts in females (normocytic, normochromic anemia)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): Increased LDL-cholesterol and total bilirubin values in both sexes; Decreased HDL-cholesterol values in males; Increased inorganic phosphate levels in males
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): Increased incidences of transitional epithelial cells in combination with increasesd pH values and blood (erythrocytes, although not statistically significant) in the urine of females
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): Decreased motor activity in females during two intervals (1 and 2) and the overall interval

Home cage observations:
No test substance-related adverse effects were observed.

Open field observations:
Soft feces were observed in 4 males of test group 1400 mg/kg bw/d, which represented all males of the test group with defecation during the observation period. No test substancerelated adverse effects were observed in females.

Sensory-motoric test/ reflexes:
No test substance-related effects were observed.

Quantitative parameters:
No test substance-related adverse effects were observed

Motor activity measurement:
Single interval 2 was decreased in females of 150 mg/kg bw/d. Females of 450 mg/kg bw/d showed a decreased overall interval, but no significant alteration in any single interval.
The alteration of motor activity in females of 150 mg/kg bw/day observed in one interval only was considered as incidental and not related to treatment based on its isolated occurrence and
minor deviation from the current control having no impact on the motor activity over all intervals.
For the finding in females of 450 mg/kg bw/day, it could not be excluded that this finding was treatment-related but since there was no single interval showing a significant difference to the
control, this alteration was assessed as non-adverse.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): Increase in absolute and relative kidney weight in male (+9%/+18%) and female (+20%/+20%) animals
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- 150 mg/kg bw/day (79 mg/kg bw/day active ingredient): The animal that died revealed an enlarged liver and spleen as well as a red discoloration of the skull. This correlated to findings in microscopy

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 1400 mg/kg bw/day (735 mg/kg bw/day active ingredient): Minimal to severe degeneration/regeneration and pigment storage (all animals) in kidney tubules in 9 males and 9 females; Acute inflammation or olfactory epithelium regeneration in the nasal cavity of six male and eight female animals; Macrophage aggregates in the lungs of two males and one female animal
- 450 mg/kg bw/day (236 mg/kg bw/day active ingredient): Acute inflammation or olfactory epithelium regeneration in the nasal cavity of five male and seven female animals
- 150 mg/kg bw/day (79 mg/kg bw/day active ingredient): Acute inflammation or olfactory epithelium regeneration in the nasal cavity of four male and six female animals; Macrophage aggregates in the lungs of one female animal
Details on results:
HEMATOLOGY
In males of test group 3 (1400 mg/kg bw/d) mean corpuscular volume (MCV) and absolute basophil counts were significantly decreased. The same was true for absolute reticulocyte counts in males of test group 2 (450 mg/kg bw/d). However, all values were within historical control ranges (males, MCV 47.8-51.3 fL; absolute basophils 0.01-0.04 Giga/L; absolute reticulocytes 116.2-159.1 Giga/L). In females of test group 1 (150 mg/kg bw/d) absolute basophil counts were significantly increased, but the change was not dose-dependent. Therefore, the mentioned changes in this paragraph were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
In males of test group 2 (450 mg/kg bw/d) total bilirubin levels were already significantly higher compared to controls and they were above the historical control range (males, total bilirubin 1.03-1.92 μmol/L). However, this was the only changed clinical pathology parameter among these individuals and therefore, it was regarded as treatment-related but non-adverse.
The following significant changes were regarded as incidental and not treatment-related, because the values were within historical control ranges: increased creatinine values in males of test group 3 (1400 mg/kg bw/d); decreased glucose values in males of test groups 2 and 3 (450 and 1400 mg/kg bw/d); increased inorganic phosphate levels in males of test group 2; increased triglyceride and urea values and decreased chloride values in females of test group 3 (males, creatinine 23.4-34.2 μmol/L; glucose 5.46-6.98 mmol/L; inorganic phosphate 1.42- 1.82 mmol/L; females, triglycerides 0.44-0.82 mmol/L; urea 4.42-8.10 mmol/L; chloride 97.0- 104.2 mmol/L).
In males and females of test groups 1, 2 and 3 (150, 450 and 1400 mg/kg bw/d) no treatmentrelated alterations of T3, T4 and TSH levels were observed.

URINALYSIS
In females of test group 2 (450 mg/kg bw/d) pH values were already significantly higher compared to controls. However, this was the only changed clinical pathology parameter among these individuals and therefore, it was regarded as maybe treatment-related but non-adverse. In males and females of test group 3 (1400 mg/kg bw/d) significantly decreased urine volume was measured. In males of this test group urine specific gravity was significantly increased and urine pH values were significantly lower compared to controls. The mentioned changes in this paragraph were most probably due to a decreased fluid income in the kidneys and the excretion of compound metabolites in the urine, but they reflect the normal adaptation of the renal function. Therefore, they were regarded as treatment-related, but non-adverse.

PATHOLOGY (see tables 1 and 2)
The terminal body weight in males of test group 3 (1400 mg/kg bw/d) was within historical control data and therefore not regarded to be adverse. Nevertheless, it was regarded to be treatment-related decreased. The decrease in thymus weight in males of test group 2 and 3 (450 and1400 mg/kg bw/d) did not have a microscopic finding that could explain the weight decrease. It was therefore most likely regarded to be a consequence to the body weight decrease in test group 3 (1400 mg/kg bw/d), whereas in test group 2 (450 mg/kg bw/d) it was within historical control values and not related to treatment.
The relative liver weight increase in females of test group 3 (1400 mg/kg bw/d) did not have a correlation in absolute liver weight, did not have a microscopic correlate and was within historical control values. It was therefore regarded to be incidental. The changes in relative weights of epididymides and seminal vesicle were within historical control values and did not show any microscopic findings that could explain the weight increase and were therefore regarded to be incidental and/or related to the body weight reduction in test group 3 (1400 mg/kg bw/d) animals.

HISTOPATHOLOGY
Treatment-related findings were observed in kidneys, lungs and nasal cavity with incidences and grading according to table 3.
In the kidneys in the cortical area, mainly affecting the proximal tubules, there was degeneration/regeneration and pigment storage. The pigment was located in the cytoplasm within proximal tubular cells and had a black to gold-brownish finely granular appearance. In the vicinity of the pigment storage, there was an increase in eosinophilia of tubular cells, loss of nuclei and occasional detritus within the lumen (degeneration) and an increase in basophilia of the tubular cells with enlarged, vesicular nuclei (regeneration). In females there was predominantly degeneration rather than regeneration. These findings were regarded to be treatment related.
In contrast to regularly observed numbers of macrophages in control animals (homogeneous
eosinophilic cytoplasm, sometimes pigment storage, quiescence nuclei) in treated animals there were increased macrophage aggregates that differed: active nuclei, often elongated, light eosinophilic cytoplasm, sometimes vacuolated. These findings were regarded to be treatment related.
In the nasal cavity two findings were observed, which were regarded to represent one continuous lesion at different stages with regard to time. The term “Inflammation” was used, if serous exudate was intermingled with neutrophils. Almost exclusively the ventral half of nasal cavity was affected and also in paranasal sinus exudate was observed. Mainly the olfactory epithelium was affected to a lower degree the respiratory epithelium showed also signs of inflammatory stress. In some animals plant particles interpreted as food particles were observed within the exudate.
In case of the finding “Regeneration” mainly olfactory epithelium was affected, respiratory epithelium only in few animals. The typical findings were loss of epithelial height, no regular cellular orientation, infiltrates of neutrophils, occasional vacuole formation in respiratory epithelium, no exudate anymore. These findings were regarded to be treatment related.
In contrast to the loosely arranged intra-alveolar macrophages observed regularly in the lungs, few animals revealed closely packed aggregates of macrophages filling the complete alveolus. This finding was regarded to be treatment related.
The male animal No. 13 of test group 1 (150 mg/kg bw/d) that died revealed a malignant lymphoma which was the cause of death in this animal. Infiltration of the lymphoma led to the observed macroscopic findings (e.g. enlarged liver and spleen, discoloration of muscle at the skull) but was regarded to be incidental and unrelated to treatment.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
other: corresponding to 735 mg/kg bw/day active ingredient
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
< 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: corresponding to 79 mg/kg bw/day active ingredient
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
other: respiratory system
Organ:
lungs
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
1 400 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
1 400 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
1 400 mg/kg bw/day (nominal)
System:
immune system
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
no

Table 1: Absolute organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(150)

2

(450)

3

(1400)

1

(150)

2

(450)

3

(1400)

Final body weight

101%

97%

93%*

 

 

 

Kidneys

98%

100%

109%*

104%

99%

120%**

Thymus

92%

87%*

58%**

 

 

 

* : p <= 0.05, **: p <= 0.01

Table 2: Relative organ weights

 

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(150)

2

(450)

3

(1400)

1

(150)

2

(450)

3

(1400)

Epididymides

106%*

102%

111%*

 

 

 

Kidneys

98%

103%

118%**

103%

100%

120%**

Liver

 

 

 

101%

103%

109%*

Seminal vesicle

103%

111%

118%*

 

 

 

Thymus

92%

90%

63%**

 

 

 

* : p <= 0.05, **: p <= 0.01

Table 3: Histopathology

Kidneys

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(150)

2

(450)

3

(1400)

0

(0)

1

(150)

2

(450)

3

(1400)

No. of animals

10

10

10

10

10

10

10

10

Degeneration/

regeneration, tubular

0

0

0

9

0

0

0

8

·      Grade1

 

 

 

2

 

 

 

6

·      Grade2

 

 

 

4

 

 

 

1

·      Grade3

 

 

 

2

 

 

 

1

·      Grade4

 

 

 

1

 

 

 

 

Pigment storage,

tubular

0

0

1

10

0

0

8

10

·      Grade1

 

 

1

 

 

 

7

2

·      Grade2

 

 

 

4

 

 

1

1

·      Grade3

 

 

 

6

 

 

 

6

·      Grade4

 

 

 

 

 

 

 

1

Mesenteric lymph node

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(150)

2

(450)

3

(1400)

0

(0)

1

(150)

2

(450)

3

(1400)

No. of animals

10

10

10

10

10

10

10

10

Aggregates increased, macrophage (m)f

0

0

1

8

0

1

3

9

·      Grade1

 

 

1

4

 

 

3

4

·      Grade2

 

 

 

4

 

 

 

4

·      Grade3

 

 

 

 

 

1

 

1

Nasal cavity

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(150)

2

(450)

3

(1400)

0

(0)

1

(150)

2

(450)

3

(1400)

No. of animals

10

10

10

10

10

10

10

10

Inflammation (m)f

0

3

3

2

0

4

4

4

·      Grade1

 

1

 

 

 

1

 

 

·      Grade2

 

 

2

 

 

2

 

2

·      Grade3

 

2

1

2

 

1

3

1

·      Grade4

 

 

 

 

 

 

1

1

Regeneration (m)f

0

1

2

4

0

2

3

4

·      Grade1

 

 

1

1

 

2

1

1

·      Grade2

 

 

1

2

 

 

1

1

·      Grade3

 

1

 

1

 

 

1

2

Lungs

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(150)

2

(450)

3

(1400)

0

(0)

1

(150)

2

(450)

3

(1400)

No. of animals

10

10

10

10

10

10

10

10

Macrophage

aggregates

0

0

0

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Conclusions:
The administration of the test substance by gavage to male and female Wistar rats for 3 months caused signs of systemic and local toxicity.
The findings of systemic toxicity were observed in males and females of test group 3 (1400 mg/kg bw/d corresponding to 735 mg/kg bw/d of the active ingredient). Therefore, under theconditions of the present study the no observed adverse effect level (NOAEL) for systemic toxicity was 450 mg/kg bw/d for male and female rats corresponding to 236 mg/kg bw/d of the
active ingredient. The local toxicity has been observed in all test groups without a clear dose response in the nasal cavity. Therefore, the no observed adverse effect level (NOAEL) for local toxicity was below 150 mg/kg bw/d corresponding to 79 mg/kg bw/d of the active ingredient.
Executive summary:

The test substance was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0, vehicle control), 150 (test group 1), 450 (test group 2) and 1400 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months. Based on the purity of the test substance being 52.5%, the corresponding dose levels of the test substance were 79 (test group 1), 236 (test group 2), and 735 mg/kg bw/d (test group 3).

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. In addition, the rats were daily examined for any clinically abnormal signs before and within 2 hours as well as within 5 hours after treatment. Moreover, detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Furthermore, in female animals estrous cycle was determined on the day of sacrifice. Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals were

sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

Substance-related findings were observed at all treated doses. At 1400 mg/kg bw/day (corresponds to 735 mg/kg bw/d active ingredient), soft feces in 10/10 males from study day 66 onwards, respiration sounds in 3/10 males and females on each one or two days after treatment, semiclosed eyelids in 1/10 males and 2/10 females on each one day after treatment, nose discharge in 1/10 males on one day, decreased motor activity in females during two intervals (1 and 2) and the overall interval, decreased hemoglobin and hematocrit values in both sexes, decreased red blood cell (RBC) counts in females, decreased absolute reticulocyte counts in males, increased absolute neutrophil counts in females, increased LDL-cholesterol and total bilirubin values in both sexes, decreased HDL-cholesterol values in males, increased inorganic phosphate levels in males, increased incidences of transitional epithelial cells and blood (erythrocytes) in the urine of females, increase in absolute and relative kidney weight in male (+9%/+18%) and female (+20%/+20%) animals, minimal to severe degeneration/regeneration and pigment storage (all animals) in kidney tubules in 9 males and 9 females, acute inflammation or olfactory epithelium regeneration in the nasal cavity of six male and eight female animals and macrophage aggregates in the lungs of two males and one female animal were observed.

In test group 450 mg/kg bw/day (corresponds to 236 mg/kg bw/d active ingredient), an acute inflammation or olfactory epithelium regeneration in the nasal cavity of five male and seven female animals was reported. At 150 mg/kg bw/day (corresponds to 79 mg/kg bw/d active ingredient), an acute inflammation or olfactory epithelium regeneration in the nasal cavity of four male and six female animals and macrophage aggregates in the lungs of one female animal were observed.

The administration of the test substance by gavage to male and female Wistar rats for 3 months caused signs of systemic and local toxicity.

The findings of systemic toxicity were observed in males and females of test group 3 (1400 mg/kg bw/d corresponding to 735 mg/kg bw/d of the active ingredient). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) for systemic toxicity was 450 mg/kg bw/d for male and female rats corresponding to 236 mg/kg bw/d of the active ingredient.

The local toxicity has been observed in all test groups without a clear dose response in the nasal cavity. Therefore, the no observed adverse effect level (NOAEL) for local toxicity was below 150 mg/kg bw/d (corresponding to 79 mg/kg bw/d of the active ingredient).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2015 - August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according GLP and OECD guideline, well documented
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Name of test substance: Octadecanoic acid, sulfo-, potassium salt
CAS No.: 67968-63-2
Tes substance no.: 14/0449-1
Batch identification: 0012127444
Purity: 51.92% (project No. 14L00285)
Homogeneity: given (visually)
Date of production: 23 Jun 2014
Storage conditions: room temperature
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation:
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
suspension
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): water
- Storage temperature of food: RT

- applied as a suspension
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- according GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
50, 150, 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
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: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina
Statistics:
Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Urinalysis parameters: WILCOXON-test (one-sided)

Food consumption: DUNNETT-test (twosided)

fertility indices: FISHER'S EXACT test

Proportions of affected pups per litter with necropsy observations: WILCOXON-test

Weight parameters: KRUSKAL-WALLIS test




Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
Test group 500 mg/kg bw
CLINICAL EXAMINATIONS
• decreased food consumption during premating (up to -9%), gestation (up to -20%), and
lactation (not statistically significant -10%) in females
• decreased body weight during lactation (up to -10%)
• piloerection shown by 3 females during lactation

REPRODUCTIVE PERFORMANCE
• no test substance-related adverse findings

CLINICAL PATHOLOGY
• no test substance-related adverse findings

PATHOLOGY
• no test substance-related adverse findings

The other test substance groups (50 and 150 mg/kg bw) did not show any findings at all.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: no effects
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
clinical signs
Critical effects observed:
not specified
Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of octadecanoic acid, sulfo-, potassium salt to Wistar rats revealed no signs of general systemic toxicity in male parental animals up to 500 mg/kg bw/d. Signs of systemic toxicity were observed in female parental animals at 500 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 500 mg/kg bw/d in male parental animals and 150 mg/kg bw/d in female parental animals.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
well documented test

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test substance (51.92% in drinking water) was given daily as solutions to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage.

The nominal dose levels of the used water-based formulation of the test item were 96, 289 and 963 mg/kg bw/d corresponding to 50, 150, and 500 mg/kg bw/d. Control animals were dosed daily with the vehicle only (drinking water). The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined regularly once weekly, males during 2 weeks of premating and females before and after the mating period, as well as in dams during gestation (days 0 - 7, 7 - 14, 14 - 20) and lactation (days 1 - 4).

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0 and 4.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4 and their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2and examined macroscopically for external and visceral findings at necropsy.

Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period.

Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The following test substance-related adverse effects/findings were noted:

In test group 963 mg/kg bw/day (corresponding to 500 mg/kg bw/day test substance) for F0 parental animals, decreased food consumption during premating (up to -9%), gestation (up to -20%), and lactation (not statistically significant -10%) in females, decreased body weight during lactation (up to -10%) and piloerection shown by 3 females during lactation were observed. Reproductive performance, clinical pathology, and pathology revealed no test substance-related adverse findings. In F1 pups, decreased live birth index (64.2%),increase number of stillborn pups (35.8%) leading to 88.9% dams with stillborn pups,increased perinatal loss (36.7%),decreased viability index (73%),increased number of pups found dead (4 pups),increased number of pups missing (cannibalized, 13 pups),decreased pup weights of both sexes at postnatal day 1 (-23.0%),decreased pup weight change between postnatal day 1 and 4 in pups of both sexes

(-28.5%) were bases on a decreased pup weight change in male pups of -25.4%, and in female pups of -24.1% (not statistically significant),increased number of runts (10 males and 12 females),increased number of pups with post mortem autolyzes,increased number of pups with discolored liver, andincreased number of pups with empty stomach were reported.

In test group 289 mg/kg bw/day(corresponding to 150 mg/kg bw/day test substance) in F0 parental animals, no test substance-related adverse findings were observed. In F1 pups, no test substance-related adverse findings were noted.

In test group 96 mg/kg bw/day(corresponding to 50 mg/kg bw/day test substance) in F0 parental animals and F1 pups, no test substance-related adverse findings were observed.

Under the conditions of this Combined Repeated Dose Toxicity Study with the

Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed no signs of general systemic toxicity in male parental animals up to 500 mg/kg bw/d. Signs of systemic toxicity

were observed in female parental animals at 500 mg/kg bw/d. Thus, the no observed

adverse effect level (NOAEL) for general systemic toxicity was 500 mg/kg bw/d in male parental animals and 150 mg/kg bw/d in female parental animals.

The no observed adverse effect level (NOAEL) for reproductive performance and fertility was 500 mg/kg bw/d in male and female Wistar rats. The NOAEL for developmental toxicity in the F1 progeny was 150 mg/kg bw/d (2015).

The test substance was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0, vehicle control), 150 (test group 1), 450 (test group 2) and 1400 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months. Based on the purity of the test substance being 52.5%, the corresponding dose levels of the test substance were 79 (test group 1), 236 (test group 2), and 735 mg/kg bw/d (test group 3).

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. In addition, the rats were daily examined for any clinically abnormal signs before and within 2 hours as well as within 5 hours after treatment. Moreover, detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Furthermore, in female animals estrous cycle was determined on the day of sacrifice. Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals were

sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

Substance-related findings were observed at all treated doses. At 1400 mg/kg bw/day (corresponds to 735 mg/kg bw/d active ingredient), soft feces in 10/10 males from study day 66 onwards, respiration sounds in 3/10 males and females on each one or two days after treatment, semiclosed eyelids in 1/10 males and 2/10 females on each one day after treatment, nose discharge in 1/10 males on one day, decreased motor activity in females during two intervals (1 and 2) and the overall interval, decreased hemoglobin and hematocrit values in both sexes, decreased red blood cell (RBC) counts in females, decreased absolute reticulocyte counts in males, increased absolute neutrophil counts in females, increased LDL-cholesterol and total bilirubin values in both sexes, decreased HDL-cholesterol values in males, increased inorganic phosphate levels in males, increased incidences of transitional epithelial cells and blood (erythrocytes) in the urine of females, increase in absolute and relative kidney weight in male (+9%/+18%) and female (+20%/+20%) animals, minimal to severe degeneration/regeneration and pigment storage (all animals) in kidney tubules in 9 males and 9 females, acute inflammation or olfactory epithelium regeneration in the nasal cavity of six male and eight female animals and macrophage aggregates in the lungs of two males and one female animal were observed.

In test group 450 mg/kg bw/day (corresponds to 236 mg/kg bw/d active ingredient), an acute inflammation or olfactory epithelium regeneration in the nasal cavity of five male and seven female animals was reported. At 150 mg/kg bw/day (corresponds to 79 mg/kg bw/d active ingredient), an acute inflammation or olfactory epithelium regeneration in the nasal cavity of four male and six female animals and macrophage aggregates in the lungs of one female animal were observed.

The administration of the test substance by gavage to male and female Wistar rats for 3 months caused signs of systemic and local toxicity.

The findings of systemic toxicity were observed in males and females of test group 3 (1400 mg/kg bw/d corresponding to 735 mg/kg bw/d of the active ingredient). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) for systemic toxicity was 450 mg/kg bw/d for male and female rats corresponding to 236 mg/kg bw/d of the active ingredient.

The local toxicity has been observed in all test groups without a clear dose response in the nasal cavity. Therefore, the no observed adverse effect level (NOAEL) for local toxicity was below 150 mg/kg bw/d (corresponding to 79 mg/kg bw/d of the active ingredient) (2020).

Justification for classification or non-classification