Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 July 2020 to 21 January 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl
EC Number:
953-378-2
Molecular formula:
[(CnH2n+1)NH3+].[(CxH2x+1)-C6H4-S03-]
IUPAC Name:
Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl
Test material form:
liquid: viscous
Details on test material:
Chemical registery number : 953-378-2
Chemical name : Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 40 to 46 days
- Weight at study initiation: Males: 190 to 250 g. Females: 126 to 179 g
- Housing: Three or four animals of the same sex per cage. Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals
- Diet: Teklad 2014C diet ad libitum
- Water: Potable water from the public supply ad libitum
- Acclimation period: 12 days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Method of preparation: The test item was initially warmed in a water bath at 35°C until fully melted.
A series of formulations at the required concentrations were prepared by the addition of individual weighings of the test item with the required volume of the vehicle whilst magnetically stirring. The formulations were then transferred into final containers using a syringe, whilst magnetic stirring continued.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2 to 8°C) and protected from light.
Homogeneity and stability: The formulations prepared at 1 and 200 mg/mL were found to be homogeneous and stable at ambient temperature (15 to 25°C) for one day and refrigerated (2 to 8°C) storage for 15 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 13 of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
225 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels selected for this study were based on the results of preliminary toxicity study in which three groups of five male and five female Sprague-Dawley rats received 100, 300 or 1000 mg/kg/day for up to two weeks.
- Fasting period before blood sampling for clinical biochemistry: Yes
Positive control:
Not included

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each cage: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment and Week 12
- Dose groups that were examined: Groups 1 and 4

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 10 animals/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 10 animals/sex/dose

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: All animals
- Battery of functions tested: Approach response, Pinna reflex, Auditory startle reflex, Tail pinch response, Grip strength, Motor Activity

OTHER:
- Thyroid Hormone Analysis (all animals)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs that were due to treatment were confined to piloerection at 225 mg/kg/day, with a few males receiving 75 mg/kg/day being transiently affected. In the males given 75 mg/kg/day piloerection occurred approximately one to two hours post dose and had resolved by the end of the working day but at 225 mg/kg/day it was more persistent, being present immediately after dosing through to the end of the working day and was, on a few occasions, evident in the following morning before dose administration.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Six animals died or were killed prematurely but all were consequences of dose administration errors and were not attributable to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gain was low for males receiving 75 or 225 mg/kg/day.
There was no effect on body weights during the first six weeks of treatment for females receiving 75 or 225 mg/kg/day but their subsequent weight gain to the end of the treatment period (Week 6 to 13) was markedly lower than the controls.
Body weight was unaffected at 25 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was low for males receiving 75 or 225 mg/kg/day, with the extent of the reduction of food intake at 225 mg/kg/day tending to increase as the treatment period progressed. Low food consumption from Week 3 and 7 at 225 and 75 mg/kg/day, was reported on females. Food consumption was unaffected at 25 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematological examination in Week 13 revealed slightly high erythrocyte counts in females receiving 75 mg/kg/day and in males and females receiving 225 mg/kg/day and a reduction of hemoglobin concentration at 225 mg/kg/day in both sexes. Associated with these findings was a dose-related reduction of mean cell hemoglobin and mean cell volume at 75 and 225 mg/kg/day in both sexes, low mean cell hemoglobin concentration in females at these doses, and an increase in red cell distribution width in males and females receiving 225 mg/kg/day. Neutrophil count was slightly high in males and females receiving 75 mg/kg/day and was markedly high in males and females receiving 225 mg/kg/day. In addition, lymphocyte, basophil, monocyte and large unstained cell counts were high in males and females receiving 75 or 225 mg/kg/day, with a small increase of monocyte count occurring in females receiving 25 mg/kg/day. As a consequence, there was a dose-related increase of the total leucocyte counts of males and females receiving 75 or 225 mg/kg/day. Prothrombin time and activated partial thromboplastin time were slightly reduced in males receiving 225 mg/kg/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related biochemical changes in the blood plasma after 13 weeks comprised: a dose related increase of alanine and aspartate amino transferase activities in males and females receiving 75 or 225 mg/kg/day; high urea concentrations in males receiving 75 or 225 mg/kg/day that associated with a small increase of creatinine concentration; high glucose concentrations in females receiving 225 mg/kg/day; reduced high density lipoprotein concentrations in males and females receiving 75 or 225 mg/kg/day and increased low density lipoprotein concentrations in males receiving 75 mg/kg/day and in males and females receiving 225 mg/kg/day; slightly low total cholesterol concentrations in females receiving 75 or 225 mg/kg/day; slightly low sodium concentrations and high potassium concentrations in females receiving 225 mg/kg/day; a severe reduction of total protein concentration in females receiving 225 mg/kg/day; males receiving 75 or 225 mg/kg/day also had low total protein concentration (due to a reduction in both the albumin and, in particular, the globulin fractions, resulting in an increase of the albumin to globulin ratios of these animals) and females receiving 75 mg/kg/day had slightly low total protein concentration.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Only two of the nine females receiving 225 mg/kg/day were showing normal oestrous cycles in the four day period before necropsy in Week 13. This was considered to be secondary to the decrease in bodyweight gain and general poor clinical condition of these animals.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity responses were unaffected by treatment. Forelimb grip strength was low in Week 12 in males and females receiving 225 mg/kg/day, with the males also having reduced hindlimb grip strength. The motor activity assessment in Week 12 revealed that the low (cage floor activity) and high (rearing activity) beam break scores were low for males receiving 225 mg/kg/day and there was also a slight reduction of total low beam breaks by females receiving 225 mg/kg/day. These findings were attributed to the poor general condition of these animals as a consequence of the marked and progressive effects of treatment on body weight.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Analysis of organ weights after 13 weeks revealed high liver weights in males and females given 225 mg/kg/day, high adrenal gland weights in males given 75 or 225 mg/kg/day and high spleen weights at all doses and in both sexes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic examination after 13 weeks indicated thickening, distension, abnormal content and color in the intestines (duodenum, jejunum and, to a slightly lesser incidence, ileum) in males and females given 75 or 225 mg/kg/day, with pale or clear luminal fluid present in some of these animals. At 25 mg/kg/day there were thickening of the duodenum and ileum in one male, thickening of the jejunum in two males and one female, and abnormal color in two males. The mesenteric lymph nodes were enlarged in males and females given 25, 75 or 225 mg/kg/day, the incidence of which increased with increasing dose and for males given 75 mg/kg/day and males and females given 225 mg/kg/day some of these lymph nodes also appeared pale. Pale areas were observed in the lungs of two males and two females given 75 or 225 mg/kg/day and in one male given 25 mg/kg/day, though one control female was similarly affected.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology after 13 weeks revealed moderate to marked aggregates of macrophages in the villi of the duodenum and jejunum of males and females administered 75 or 225 mg/kg/day, and the same finding occurred at a lower severity in the jejunum of males and females administered 25 mg/kg/day. A low incidence and severity of this finding was also present in the ileum of males and females at 225 mg/kg/day. In the mesenteric lymph node there were moderate to severe multifocal macrophages present at all doses and in both sexes. In the liver, minimal to slight multifocal lymphocytic/macrophagic inflammatory cell foci were seen at all doses and in both sexes, at a similar incidence and severity across all groups. In the lungs there were aggregates of alveolar macrophages present in control and treated animals, but at a higher incidence at 225 mg/kg/day.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The serum concentration of thyroxine was reduced in males receiving 75 or 225 mg/kg/day and the thyroid stimulating hormone concentrations was increased in males receiving 225 mg/kg/day. However, in the absence of any physiological response in the thyroid glands and as there were no similar findings in females, these findings were considered to be of no toxicological importance.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Oral administration (by gavage) of Amines, tallow alkyl, dodecylbenzene sulfonate to Sprague Dawley rats for 13 weeks caused a clear non-specific response to the toxicity of the test material at 75 and 225 mg/kg/day in both sexes (low body weight gain and food intake and some findings at the behavioral investigations). Many of the other findings at these doses were attributable to stress. There were increased numbers of macrophages in the liver, mesenteric lymph nodes and intestinal tract at all doses that were attributed to a rate-limited clearance of high doses of a high molecular weight test material. All treatment-related findings at 25 mg/kg/day were consequences of non-adverse macrophage clearance of a high molecular weight test material and, consequently, the no-observed-adverse-effect level (NOAEL) in this study was considered to be 25 mg/kg/day.
Executive summary:

The purpose of this study was to assess the systemic toxic potential of the test item (Amines, tallow alkyl, dodecylbenzene sulfonate) when administered orally (by gavage) to Sprague-Dawley (Crl:CD(SD)) rats for 13 weeks.


Three groups, each comprising 10 males and 10 females, received doses of 25, 75 or 225 mg/kg/day. A similarly constituted control group received the vehicle (corn oil) at the same volume-dose as the treated groups.


During the study, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, organ weight, macropathology and histopathology investigations were undertaken.


 


Clinical signs that were due to treatment were confined to piloerection at 225 mg/kg/day, with a few males receiving 75 mg/kg/day being transiently affected. In the males given 75 mg/kg/day piloerection occurred approximately one to two hours post dose and had resolved by the end of the working day but at 225 mg/kg/day it was more persistent, being present immediately after dosing through to the end of the working day and was, on a few occasions, evident in the following morning before dose administration.


Six animals died or were killed prematurely but all were consequences of dose administration errors and were not attributable to treatment.


Bodyweight gain and food intake was low for males receiving 75 or 225 mg/kg/day, with the extent of the reduction of food intake at 225 mg/kg/day tending to increase as the treatment period progressed. There was no effect on body weights during the first six weeks of treatment for females receiving 75 or 225 mg/kg/day but their subsequent weight gain to the end of the treatment period (Week 6 to 13) was markedly lower than the controls and this associated with low food consumption from Week 3 and 7 at 225 and 75 mg/kg/day, respectively. Body weight and food consumption were unaffected at 25 mg/kg/day.


Sensory reactivity responses were unaffected by treatment. Forelimb grip strength was low in Week 12 in males and females receiving 225 mg/kg/day, with the males also having reduced hindlimb grip strength. The motor activity assessment in Week 12 revealed that the low (cage floor activity) and high (rearing activity) beam break scores were low for males receiving 225 mg/kg/day and there was also a slight reduction of total low beam breaks by females receiving 225 mg/kg/day. These findings were attributed to the poor general condition of these animals as a consequence of the marked and progressive effects of treatment on body weight.


There were no treatment-related ophthalmoscopic findings.


The hematological examination in Week 13 revealed slightly high erythrocyte counts in females receiving 75 mg/kg/day and in males and females receiving 225 mg/kg/day and a reduction of hemoglobin concentration at 225 mg/kg/day in both sexes. Associated with these findings was a dose-related reduction of mean cell hemoglobin and mean cell volume at 75 and 225 mg/kg/day in both sexes, low mean cell hemoglobin concentration in females at these doses, and an increase in red cell distribution width in males and females receiving 225 mg/kg/day. Neutrophil count was slightly high in males and females receiving 75 mg/kg/day and was markedly high in males and females receiving 225 mg/kg/day. In addition, lymphocyte, basophil, monocyte and large unstained cell counts were high in males and females receiving 75 or 225 mg/kg/day, with a small increase of monocyte count occurring in females receiving 25 mg/kg/day. As a consequence, there was a dose-related increase of the total leucocyte counts of males and females receiving 75 or 225 mg/kg/day. Prothrombin time and activated partial thromboplastin time were slightly reduced in males receiving 225 mg/kg/day.


Treatment-related biochemical changes in the blood plasma after 13 weeks comprised: a dose-related increase of alanine and aspartate amino-transferase activities in males and females receiving 75 or 225 mg/kg/day; high urea concentrations in males receiving 75 or 225 mg/kg/day that associated with a small increase of creatinine concentration; high glucose concentrations in females receiving 225 mg/kg/day; reduced high density lipoprotein concentrations in males and females receiving 75 or 225 mg/kg/day and increased low density lipoprotein concentrations in males receiving 75 mg/kg/day and in males and females receiving 225 mg/kg/day; slightly low total cholesterol concentrations in females receiving 75 or 225 mg/kg/day; slightly low sodium concentrations and high potassium concentrations in females receiving 225 mg/kg/day; a severe reduction of total protein concentration in females receiving 225 mg/kg/day; males receiving 75 or 225 mg/kg/day also had low total protein concentration (due to a reduction in both the albumin and, in particular, the globulin fractions, resulting in an increase of the albumin to globulin ratios of these animals) and females receiving 75 mg/kg/day had slightly low total protein concentration.


Only two of the nine females receiving 225 mg/kg/day were showing normal estrous cycles in the four-day period before necropsy in Week 13. This was considered to be associated with the decrease in bodyweight gain and general poor clinical condition of these animals.


The serum concentration of thyroxine was reduced in males receiving 75 or 225 mg/kg/day and the thyroid stimulating hormone concentrations was increased in males receiving 225 mg/kg/day. However, in the absence of any physiological response in the thyroid glands and as there were no similar findings in females, these findings were considered to be of no toxicological importance.


Analysis of organ weights after 13 weeks revealed high liver weights in males and females given 225 mg/kg/day, high adrenal gland weights in males given 75 or 225 mg/kg/day and high spleen weights at all doses and in both sexes.


The macroscopic examination after 13 weeks indicated thickening, distension, abnormal content and color in the intestines (duodenum, jejunum and, to a slightly lesser incidence, ileum) in males and females given 75 or 225 mg/kg/day, with pale or clear luminal fluid present in some of these animals. At 25 mg/kg/day there were thickening of the duodenum and ileum in one male, thickening of the jejunum in two males and one female, and abnormal color in two males. The mesenteric lymph nodes were enlarged in males and females given 25, 75 or 225 mg/kg/day, the incidence of which increased with increasing dose and for males given 75 mg/kg/day and males and females given 225 mg/kg/day some of these lymph nodes also appeared pale. Pale areas were observed in the lungs of two males and two females given 75 or 225 mg/kg/day and in one male given 25 mg/kg/day, though one control female was similarly affected.


Histopathology after 13 weeks revealed moderate to marked aggregates of macrophages in the villi of the duodenum and jejunum of males and females administered 75 or 225 mg/kg/day, and the same finding occurred at a lower severity in the jejunum of males and females administered 25 mg/kg/day. A low incidence and severity of this finding was also present in the ileum of males and females at 225 mg/kg/day. In the mesenteric lymph node there were moderate to severe multifocal macrophages present at all doses and in both sexes. In the liver, minimal to slight multifocal lymphocytic/macrophagic inflammatory cell foci were seen at all doses and in both sexes, at a similar incidence and severity across all groups. In the lungs there were aggregates of alveolar macrophages present in control and treated animals, but at a higher incidence at 225 mg/kg/day.