Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs.-, compd. with 1-aminopropane-2-ol

Endpoint summary

Description of key information

In the key repeated dose oral toxicity study (Yoneyama et al. 1976), male and female rats were exposed to the read across substance (Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts) in drinking water daily for 9 months. Test doses were 85, 145 and 430 mg/kg bw/d plus the control. Eight to nine animals of each sex were exposed per group. Body weight was suppressed in the highest dose group only. Significant decreases in transaminase activity and renal Na,K-ATPase were seen in the 145 mg/kg bw/d group. No significant haematological or organ weight changes were noted. Based on enzyme activity, the resultant LOAEL and NOAEL values were 145 and 85 mg/kg bw/day, respectively.

The second key repeated dose oral toxicity study is for the read across substance MIPA. In a combined repeated dose oral toxicity study with a reproduction/developmental screening study (according to OECD guideline 422 and under GLP), Wistar rats (12/sex/dose) were administered the hydrochloride salt of MIPA (CAS No. 7780-04-3) at 0, 100, 300 or 1000 mg/kg bw/day by oral gavage (BASF AG, 2008). The duration of treatment covered a 2-week premating period and mating period in both sexes, approximately 2 weeks post-mating in males, and the entire gestation period and 4 days of lactation in females, resulting in a total of 38 and 45 exposure days for males and females, respectively. Clinical pathology examinations revealed slightly, but statistically significant reduced hemoglobin and hematocrit values, which are indicative of a mild anemic process, in high dose males. Other (minor) clinical and pathological findings appear to be incidental and not dose-related. Thus, the NOAELs for general, systemic toxicity were established 300 mg/kg bw/day for males and 1000 mg/kg bw/day for females (the highest dose tested), respectively.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable study, followed scientific principles/standards, pre-dates GLP

Remarks:

Published studies

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

The purpose of this study is to determine the sub chronic toxicity of LAS in Wistar JCL rats, focusing on the liver and kidneys. Male and female rats were maintained on either test diets (0, 0.6 and 1.8%) or drinking water (0, 0.07 and 0.2%) for 9 months. 0.6 and 1.8% rats in drinking water study exhibited severe weight loss so LAS administration was stopped after 2 weeks. Mortality, clinical observations and body weights were recorded during the study. All the surviving animals were humanely euthanized at end of 9 months and gross necropsy, hematological, serum biochemical tests, enzyme tests on the liver and kidneys were performed and organs weights were measured. No histopathology was performed.

GLP compliance:

no

Remarks:

(pre-dates GLP)

Limit test:

no

Species:

rat

Strain:

other: Wistar JCL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Male and female rats were obtained from Shizuoka Agricultural Cooperative Association for
Laboratory Animals
- Age at study initiation: 4-weeks old
- Weight at study initiation: 100 - 124 g (male rats), 82 - 100 g (female rats)
- Fasting period before study: No
- Housing: 5 animals were housed per cage.
- Diet: CE-2 food (from CLEA Japan); ad libitum
- Water: ad libitum
- Acclimation period: 1 week before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 25 ± 1°C
- Humidity: 50 - 60%
- Air changes: Not reported
- Photoperiod: 12 hours dark /12 hours light

IN-LIFE DATES: Not reported

Route of administration:

other: oral: drinking water and feed

Details on route of administration:

LAS was administered to animals by mixing 0.6 and 1.8% in CE-2 food (CLEA Japan) and dissolving to 0.07 and 0.2% in drinking water.

Vehicle:

other: Test substance was administered either in diet or drinking water

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

9 months

Frequency of treatment:

Continuous in diet or drinking water (ad libitum)

Dose / conc.:

0.6 other: %

Remarks:

in diet

Dose / conc.:

1.8 other: %

Remarks:

in diet

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

in diet (corresponding to 0.6% dose level)

Dose / conc.:

900 mg/kg bw/day (nominal)

Remarks:

in diet (corresponding to 1.8% dose level)

Dose / conc.:

0.07 other: %

Remarks:

in drinking water

Dose / conc.:

0.2 other: %

Remarks:

in drinking water; 0.6 and 1.8% dose group (equivalent to 857.14 and 2571.43 mg/kg bw/day) were also included, however due to severe weight loss so LAS administration was stopped after 2 weeks.

No. of animals per sex per dose:

Feeding study (mixed in diet): 8 animals/sex/dose
Drinking water study: 9 animals/sex/dose

Control animals:

yes, concurrent vehicle

yes, plain diet

Positive control:

No

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption: Yes
- Compound intake: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 9 months, blood was collected from each animal after euthanizing.
- Anesthetic used for blood collection: No
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters: White blood cells (WBC), red blood cells (RBC), Hemoglobin (Hgb), Hematocrit (Hct), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 9 months, blood was collected from each animal after euthanizing.
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters: Glutamate oxaloacetate transaminase (GOT), glutamate pyruvic transaminase (GPT), glucose content, urea nitrogen, total cholesterol, albumin, alkaline phosphatase (ALP) and cholinesterase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
- Liver enzyme tests: Glucose 6-phosphatase (G6Pase), lactase dehydrogenase (LDH) and G6P-DH activity
- Kidney enzyme tests: G6Pase, LDH, GPT, GOT, ALP, acid phosphatase (ACP), Na, K-ATPase, and malate dehydrogenase (MDH)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (However, no details in study report in mentioned)
Gross findings were obtained after euthanizing animals and organs were removed for organ weight measurements.

ORGAN WEIGHTS: Brain, heart, lungs, liver, spleen, kidneys, adrenal gland, testes, ovary, uterus, and appendix
HISTOPATHOLOGY: No

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Both female and male rats (drinking water study) exhibited a slight redness at the tips of their facial fur, and coarse fur all over their bodies.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was significant decrease in body weight gain in males and females of 1.8% dose group fed with diet and in males of 0.2% dose group consuming LAS contained water.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Increased water consumption was observed in males of 1.8% dose group fed with diet and 0.2% dose group consuming LAS contained water.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A significant reduction in WBC was observed in 0.6% (diet) male rats and in MCV and MCH was observed in 1.8% (diet) female rats compared to controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were significant alteration in cholesterol [decrease; all doses, except female rats of 0.07% dose group (drinking water)], GPT [0.6% dose group (diet) females)], GOT [1.8% dose group (diet) males], albumin [1.8% (diet) males] , ALP levels [male and female rats fed with 1.8% LAS-diet] and cholinesterase levels [in male rats fed with 1.8% LAS-diet].

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males of 1.8% (diet), the absolute liver and testes weight was significantly decreased and the relative testes and liver weight was significantly increased and both absolute and relative spleen weight were also significantly reduced.
In females of 1.8% (diet), absolute and relative liver and caecum weight was significantly increased.
There was also significant decrease in absolute and relative heart weight in females of 0.6% (diet) and 0.07% (drinking water) dose groups.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Liver enzyme tests:
Dietary study: G6Pase activity was reduced in 1.8% dose group males and females, G6P-DH activity was reduced in 0.6 and 1.8% dose group males and females, LDH activity was reduced in 0.6, 1.8% dose group. GOT and GPT activities were clearly reduced in males, while in females GPT activity was reduced only in 1.8% dose group and GOT activity was reduced in 1.8% dose group animals.
Drinking water study: LDH activity was reduced in 0.2% dose group males. GOT and GPT activities were clearly reduced in males and GOT activity was increased in 0.07 and 0.2% dose group animals.

Renal enzyme tests:
Dietary study: A significant difference was also observed in G6Pase activity. In females, G6Pase, Na, K-ATPase, and LDH activity were significantly reduced in 1.8% dose group (diet) animals
Drinking water study: In males, Na, K-ATPase activity was significantly reduced in 0.2% dose group. A significant difference was also observed in G6Pase activity.

Details on results:

CLINICAL SIGNS: Both female and male rats consuming LAS-containing water exhibited a slight redness at the tips of their facial fur, and coarse fur all over their bodies.

BODY WEIGHT AND WEIGHT CHANGES: There was significant decrease in body weight gain in male and females of 1.8% dose group fed with diet and in males of 0.2% dose group consuming LAS contained water.

WATER CONSUMPTION AND COMPOUND INTAKE: Increased water consumption was observed in males of 1.8% dose group fed with diet and 0.2% dose group consuming LAS contained water.

HAEMATOLOGICAL FINDINGS: A significant reduction in WBC was observed in 0.6% (diet) male rats compared to controls, and a significant reduction in MCV and MCH was observed in 1.8% (diet) female rats compared to controls.

CLINICAL BIOCHEMISTRY: Except female rats of 0.07% dose group (drinking water), a significant reduction or a reduction in cholesterol was observed in male and female rats of all dose groups compared to controls. GPT was significantly reduced in 0.6% dose group (diet) females, and reduced in females of other treatment groups. GOT was significantly reduced in 1.8% dose group (diet) males, and reduced in both females and males of other treatment groups. Albumin was significantly reduced in 1.8% (diet) males and reduced in males of 0.2% dose group (drinking water) and females of 1.8% dose group (diet). There was significant increase in ALP levels in male and female rats fed with 1.8% LAS-diet and cholinesterase levels in male rats fed with 1.8% LAS-diet.

ORGAN WEIGHT: In males of 1.8% (diet), the absolute liver and testes weight was significantly decreased and the relative testes and liver weight was significantly increased and both absolute and relative spleen weight were also significantly reduced. In females of 1.8% (diet), absolute and relative liver and caecum weight was significantly increased. There was also significant decrease in absolute and relative heart weight in females of 0.6% (diet) and 0.07% (drinking water) dose groups.

LIVER ENZYME TESTS: G6Pase activity was reduced in 1.8% dose group (diet) males and females, G6P-DH activity was reduced in 0.6 and 1.8% dose group (diet) males and females, where the percentage reduction was greater in 1.8% dose group (diet) animals. LDH activity was clearly reduced in 0.6, 1.8% dose group (diet), and 0.2% dose group (drinking water) males, but reduced in only 1.8% dose group (diet) females. GOT and GPT activities were clearly reduced in males, while in females GPT activity was reduced only in 1.8% dose group (diet) animals, and GOT activity was increased in 0.07 and 0.2% dose group (drinking water) animals but reduced in 1.8% dose group (diet) animals.

RENAL ENZYME TESTS: In males, Na, K-ATPase activity was significantly reduced in 0.2% dose group (drinking water) animals, and also reduced in other male treatment groups. A significant difference was also observed in G6Pase activity, where the reduction observed was associated with an increase in amount consumed. In females, G6Pase, Na, K-ATPase, and LDH activity were significantly reduced in 1.8% dose group (diet) animals, and G6Pase and LDH activity were also reduced in other treatment groups.

Key result

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

(dietery study)

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

haematology

organ weights and organ / body weight ratios

water consumption and compound intake

other: Liver and kidney enzyme levels

Remarks on result:

other: Adverse effects were observed at all dose levels

Key result

Dose descriptor:

NOAEL

Effect level:

85 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

(drinking water study)

Sex:

male/female

Basis for effect level:

clinical biochemistry

other: Liver and kidney enzyme levels

Remarks on result:

other: Based on significant decreases in the activities of glutamate-oxalate transaminase and lactate dehydrogenase in males and significant decrease in renal Na,K-ATPase in males and females at 145 mg/kg bw/day (0.2% drinking water).

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

145 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

145 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Administration of LAS to Wistar JCL rats by test diets at dose levels of 0, 0.6 and 1.8% for 9 months (focusing on the liver and kidneys) revealed an LOAEL of 0.6% (300 mg/kg bw/day in diet), based on adverse effects at all dose levels.
Administration of LAS to Wistar JCL rats in drinking water at dose levels of 0, 0.07 and 0.2% for 9 months (focusing on the liver and kidneys) revealed an NOAEL of 0.07% (85 mg/kg bw/day in drinking water), based on significant decreases in the activities of glutamate-oxalate transaminase and lactate dehydrogenase in males and significant decrease in renal Na,K-ATPase in males and females at 145 mg/kg bw/day (0.2% drinking water).

Executive summary:

The 9 months sub-chronic oral toxicity study of LAS was performed in Wistar JCL rats, focusing on the liver and kidneys.

 

4 weeks old male and femaleWistar JCL rats(obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals) with body weight range100 - 124 g (males), 82 - 100 g (females)were used in the study. 5 animals were housed in each cage and maintained under controlled environmental conditions (temperature: Average of25 ± 1°C, humidity:50 - 60%, and 12 hours light /12 hours dark).CE-2diet (from CLEA Japan) and water were providedad libitum.

 

The animals were administered daily with the LAS at following dose levels for 9 months:

 

Mixed in diet: 0, 0.6 and 1.8% (equivalent to 0, 300 and 900 mg/kg bw/day); 8 animals/sex/dose

 

Dissolved in drinking water: 0, 0.07 and 0.2 % (equivalent to 0,85 and 145mg/kg bw/day); 9 animals/sex/dose

 

Rats in 0.6 and 1.8% dose group of drinking water study exhibited severe weight loss so LAS administration was stopped after 2 weeks

 

Clinical observations, water consumption, food consumption and body weights were recorded weekly. At the end of study, blood was collected for estimation of haematological and clinical chemistry parameters.Gross findings were observed after euthanizing animals and organs were removed for organ weight measurements. Organ weights for brain, heart, lungs, liver, spleen, kidneys, adrenal gland, testes, uterus, and appendixwere recorded. Liver and kidney enzymes were also analysed. No histopathology was performed.

 

No mortality was observed throughout the study. Both female and male rats consuming LAS-containing water exhibited a slight redness at the tips of their facial fur, and coarse fur over their bodies. There was significant decrease in body weight gain in male and females of 1.8% dose group fed with diet and in males of 0.2% dose group consuming LAS contained water.Increased water consumption was observed in males of 1.8% dose group fed with diet and 0.2% dose group consuming LAS contained water.

 

A significant reduction in WBC was observed in 0.6% (diet) male rats compared to controls, and a significant reduction in MCV and MCH was observed in 1.8% (diet) female rats compared to controls. A marked reduction in cholesterol was observed in male and female rats of all dose groups [except female rats of 0.07% dose group (drinking water)] compared to controls. This indicate hepatocyte damage. GPT was significantly reduced in 0.6% dose group (diet) females, and reduced in females of other treatment groups. GOT was significantly reduced in 1.8% dose group (diet) males, and reduced in both females and males of other treatment groups. Albumin was significantly reduced in 1.8% (diet) males and reduced in males of 0.2% dose group (drinking water) and females of 1.8% dose group (diet). There was significant increase in ALP levels in male and female rats fed with 1.8% LAS-diet and cholinesterase levels in male rats fed with 1.8% LAS-diet.

 

In males of 1.8% (diet), the absolute liver and testes weight was significantly decreased and the relative testes and liver weight was significantly increased and both absolute and relative spleen weight were also significantly reduced. In females of 1.8% (diet), absolute and relative liver and caecum weight was significantly increased. There was also significant decrease in absolute and relative heart weight in females of 0.6% (diet) and 0.07% (drinking water) dose groups.

 

Liver enzymes were markedly reduced in 1.8% fed rats, due to impaired liver function, indicates reduced enzyme synthesis and direct enzyme inhibition by LAS or its metabolites. Renal G6Pase and Na, K-ATPase activity decreased, indicating kidney impairment.

 

Administration of LAS to Wistar JCL rats bytest diets at dose levels of 0, 0.6 and 1.8% for 9 months (focusing on the liver and kidneys) revealed an LOAEL of 0.6% (300 mg/kg bw/day in diet), based on adverse effects at all dose levels.

Administration of LAS to Wistar JCL rats in drinking water at dose levels of 0, 0.07 and 0.2% for 9 months (focusing on the liver and kidneys) revealed an NOAEL of 0.07% (85 mg/kg bw/day in drinking water), based onsignificant decreases in the activities of glutamate-oxalate transaminase and lactate dehydrogenase in males and significant decrease in renal Na,K-ATPase in males and females at 145 mg/kg bw/day (0.2% drinking water).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD Test Guideline 422 - Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar (Crl:WI(Han))

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Research Models and Services, Germany Gmbh
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 267.4-309.0 g (males) and 186.7-217.7 g (females)
- Number of animals: 96 (12 per sex per dose group)
- Housing: Individually in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm2), with the following exceptions: for the overnight mating the females were put into the cages of the males; from day 18 p.c. until day 4 p.p. the pregnant animals and their litters were housed in Makrolon type M III cages (floor area about 800 cm2). The M III cages were also supplied by Becker & Co.. Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy.
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse/rat ¿GLP¿ (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: tap water

Details on oral exposure:

- Dosing solution pH 6.0-7.5
- Dose volume: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The solutions were analyzed twice and determined to be 0, 1.61, 5.11, 15.73 g/100mL using a content 68.9g/100g, which was determined by potentiometric titration and 0, 1.62, 4.67, 15.60 g/100 mL using 69.0 g/100g, also determined by potentiometric titration.

Duration of treatment / exposure:

- Males: 38 days
- Females: 45 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0 (Vehicle), 100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal

Remarks:

Doses / Concentrations:
0, 1.53, 4.59, 15.29 g isopropanolamine HCl/100 mL assuming 65.4 g isopropanolamine HCl/100 g
Basis:
other: calculated

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

MATING PROCEDURES
-Dose selection rationale: based on range finding study
-At least 13 days after the beginning of treatment, males and females from the same dose group were mated overnight in a ratio of 1:1
-The day of detection of sperm in the vaginal smear was designated day 0 of gestation
-Females were allowed to litter and rear their pups until day 4 after parturition

Positive control:

None

Observations and examinations performed and frequency:

EXAMINATIONS:
- Mortality: twice daily (once on weekends and holidays) on all animals
- Clinical observations: once a day on all animals, detailed observations before test substance administration and weekly thereafter on all animals
- Food consumption and body weight: determined once per week with the following exceptions: not during mating for males or females and on day 0, 7, 14, and 20 post coitum for females and on days 0 and 4 postpartum for females with litter
- Pups were weighed on days 1 and 4 postpartum
-Hematology and clinical chemistry: hematology (5 animals/sex/group) with EDTA-K3 as anticoagulant: Leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes
- Prothrombin time (Hepato Quick's test)
- Clinical chemistry (5 animals/sex/group): alanine aminotransferase, aspartate aminotrasferase, alkaline phosphatase, gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium
- Urinalysis: on day 38 (males) and 45 (females) volume, color, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment
- Functional observational battery and motor activity measurement: 5/group on day 36 for males and on day 43 for females
- Blood samples: from retroorbital venous plexus of 5 fasted F0 animals/group under isoflurane anesthesia on day 38 (males) and day 45 (females) followed by necropsy (including pups) under CO2 anesthesia

Sacrifice and pathology:

Organs examined at necropsy (F0 animals only)
- Organ weight: liver, kidneys, adrenal glands, testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thymus, spleen, brain, heart
- Microscopic: for all animals in the control and high dose group: trachea, lungs, liver, kidneys, spleen, adrenal glands, heart, brain, spinal cord (cervical, thoracic and lumbar), sciatic nerve, thyroid/parathyroid glands, testes, epididymides, ovaries, oviducts, uterus, vagina, prostate gland, seminal vesicles, coagulation glands, thymus, lymph nodes (mandibularr and mesenteric), stomach (forestomach and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, bone marrow (femur)
- Microscopic: any organs which had gross pathological lesions at necropsy in any dose group
- Microscopic: for all males animals: liver
- Testes, epididymides and ovaries of animals that were killed as scheduled were fixed in Bouin's solution
- The number of implantation sites was determined by the method of Salewski, 1964 using uteri stained with 10% ammonium sulfide

Organs examined at necropsy (F1 pups):
- None, gross examination only, including those pups that did not survive to sacrifice

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used) were evaluated by simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means. Feces, rearing, grip strength of forelimbs and hindlimbs, landing footsplay test, and motor activity were evaluated by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). 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 (two-sided) for the equal medians. Clinical pathology parameters, except reticulocytes and differential blood count were analyzed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians. Urinalysis, except volume, color, turbidity and specific gravity were evaluated by pair-wise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions. Weight of the anesthetized animals and absolute and relative organ weights were analyzed using KRUSKAL-WALLIS and WILCOXON test.

Details on results:

FUNCTIONAL OBSERVATIONAL BATTERY AND MOTOR ACTIVITY ASSESSMENT:
- No compound-related effects

FERTILITY/REPRODUCTIVE PERFORMANCE:
- No compound-related effects

F1 pups:
CLINICAL EXAMINATIONS (DAYS 0-4 POSTPARTUM):
- No adverse effects/findings

TOXIC EFFECTS BY DOSE LEVEL:
1000 mg/kg bw/day:
F0 parental animals:
CLINICAL EXAMINATIONS/CLINICAL PATHOLOGY/URINALYSIS/PATHOLOGY
- Transient salivation in all males and most females after treatment from week 1 on
- Urine discoloration in all males and females for the entire study period
- Statistically significant decrease (p<= 0.01) in hemoglobin (-5%) and hematocrit (-6%) values in the peripheral blood of males
- Statistically significant increase in urea concentrations (+35%) in the serum of males
- Statistically significantly increase in albumin (+6%) in the serum of females
- Decreased (not significant) urine volume (-34%,-53%) with a subsequent increase (+2%,+4%) in specific gravity in males and females, respectively, without any changes in gross or microscopic parameters.
- Statistically significant increase in absolute and relative liver weights in males with mild to minimal diffuse hepatocellular hypertrophy in 9/12 males, but no changes in liver enzymes, possibly adaptive
- Statistically significant increase in absolute and relative liver weights in females, without histological findings or changes in liver enzymes, but probably test substance-related and possibly adaptive
- Statistically significant increase in absolute and relative adrenal gland weights in males, not associated with any microscopic findings
- Statistically significant increase in absolute and relative thymus weights in females, not associated with any microscopic findings

300 mg/kg bw/day:
F0 parental animals
CLINICAL EXAMINATIONS/CLINICAL PATHOLOGY/URINALYSIS/PATHOLOGY
- Transient salivation in all males after treatment from week 1 on.
- Statistically significant (p<=0.05) increase in hemoglobin (+6%) and hematocrit (+7%) values in the peripheral blood of females, not dose-related
- Statistically significant decrease in cholesterol in males, incidental
- Urine discoloration in all males and females from week 1 on.
- Statistically significant increase in body weight gain during premating in females, incidental
- Statistically significant increase in absolute and relative thymus weights in females, not associated with any microscopic findings
- Statistically significant increase in absolute and relative brain weights in females, incidental
- Statistically significant increase in relative kidney weights in females, incidental

100 mg/kg bw/day:
F0 parental animals
CLINICAL EXAMINATIONS/CLINICAL PATHOLOGY/URINALYSIS/PATHOLOGY
- Statistically significant (p<=0.05) increase in hemoglobin (+6%) and hematocrit (+7%) values in the peripheral blood of females, not dose-related
- Statistically significant increase in body weight gain during premating in females, incidental

Dose descriptor:

NOAEL

Remarks:

for general systemic toxicity

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: some indications of a mild anemic process

Dose descriptor:

NOAEL

Remarks:

for general systemic toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: no effects at highest dose

Critical effects observed:

not specified

Absolute Weights	Male			Female
Group	1	2	3	1	2	3
Liver	+3%	-3%	+22%*	-4%	-4%	+17%*
Brain	-	-	-	0%	+4%*	-1%
Adrenal Glands	+4%	+4%	+19%*	-	-	-
Thymus	-	-	-	0%	+28%*	+26%*
*values were statistically significant different, P = 0.05

.

Relative Weights	Male			Female
Group	1	2	3	1	2	3
Liver	+3%	+1%	+23%*	-2%	0%	+16%*
Brain	-	-	-	+2%	+8%	-2%
Adrenal Glands	0%	+5%	+15%*	-	-	-
Thymus	-	-	-	+3%	+33%*	+25%**
Kidneys	-	-	-	+3%	+9%*	+4%
*values were statistically significant different, P = 0.001, (**p= 0.05)

Conclusions:

- Only the findings of decreased hemoglobin and hematocrit at 1000 mg/kg bw/day in males are considered compound-related
- The other clinical and pathological findings appear to be incidental and not dose-related
- The NOAEL for general, systemic toxicity is 300 mg/kg bw/day for the F0 parental males based upon some indications of a mild anemic process and 1000 mg/kg bw/day for the F0 parental females.

- The NOAEL for reproductive performance and fertility is 1000 mg/kg body weight/day for the F0 parental rats
- The NOAEL for general, systemic toxicity is 300 mg/kg body weight/day for the F0 parental males based on some indications of a mild anemic process and 1000 mg/kg body weight/day for the F0 parental females
- The NOAEL for developmental toxicity in the F1 progeny is 1000 mg/kg body weight/day

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

85 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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

Link to relevant study records

Reference

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable study, followed scientific principles/standards, pre-dates-GLP.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

The purpose of this study is to determine the chronic toxicity (transdermal) of C10 - C13 linear alkylbenzene sulfonic acid magnesium salt (LAS-Mg) in CRJ-SD rats. Male and female rats were transdermally administered with 0 (water), 3% PEG, 0.5, 1.0 and 5.0% of test substance dissolved in 3% polyethyleneglycol (PEG, MW: 200) for 26 weeks. General symptoms and body weight of animals were recorded on a daily-basis and food intake were recorded weekly. Urinalysis and hematological examinations were performed in week 5 and 13 of the study. At the end of study, urinalysis, haematological and serum biochemistry were also done. All the surviving animals were humanely euthanized after 26 weeks and organs (liver, kidneys, spleen, adrenal glands, lungs, heart, thymus, reproductive organ, pituitary, brain and thyroid) were collected for recording absolute and relative organ weights. All the collected organs were examined under the microscope for histopthalogical changes.

GLP compliance:

no

Remarks:

(pre-dates GLP)

Limit test:

no

Species:

rat

Strain:

other: CRJ-SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Male and female rats were obtained from Charles River Laboratories Japan, Inc.
- Age at study initiation: Approximately 4 weeks
- Weight at study initiation: Not reported
- Fasting period before study: Not reported
- Housing: Animals were housed in cages with 2 animals/cage.
- Diet: Solid feed (CE-2, CLEA Japan, Inc.); ad libitum
- Water: Tap water; ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1°C
- Humidity: 55 ± 5%
- Air changes: Not reported
- Photoperiod: Not reported

IN LIFE DATES: Not reported

Type of coverage:

not specified

Vehicle:

other: 3% polyethyleneglycol (PEG, MW: 200)

Details on exposure:

TEST SITE
- Area of exposure: Shaved backs of animals
- % coverage: 3 x 3 cm2 area in the middle of the backside
- Type of wrap if used: Not reported
- Time intervals for shavings or clippings: Weekly

REMOVAL OF TEST SUBSTANCE
- Washing: Not reported
- Time after start of exposure: Not reported

TEST MATERIAL
- Amount(s) applied: 0.1 mL
- Concentration: 0.5, 1.0 and 5.0% of C10-C13 LAS-Mg in 3% polyethyleneglycol

VEHICLE
- 3% polyethyleneglycol (PEG, MW: 200)

USE OF RESTRAINERS FOR PREVENTING INGESTION: No

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

Once daily for 26 weeks (excluding Sundays)

Dose / conc.:

0.5 other: %

Dose / conc.:

1 other: %

Dose / conc.:

5 other: %

No. of animals per sex per dose:

20 animals/sex/dose group

Control animals:

other: 2 Control animals groups were applied with either PEG or distilled water.

Details on study design:

- Dose selection rationale: Not reported
- Rationale for animal assignment: Not reported
- Rationale for selecting satellite groups: No satellite groups were used in the study

Positive control:

Not included

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for general symptoms.

DERMAL IRRITAION: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed daily.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: Amount of food intake was measured once per week.

FOOD EFFICIENCY: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During weeks 5, 11 and 26
- Anaesthetic used for blood collection: Yes. The animals were anesthetized with sodium pentobarbital
- Animals fasted: Not reported
- How many animals: During weeks 5 and 11, blood was collected from 10 animals/sex from PEG group, control group and 5.0% group and in week 26, blood was collected from all animals.
- Parameters checked: Red blood cell count, white blood cell count, hemoglobin level and hematocrit level were measured, and white blood cell fractions were calculated by Giemsa-staining the blood smear samples.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At end of study i.e. 26 weeks
- Animals fasted: Not reported
- How many animals: All animals
- Parameters checked: s-GOT, s-GPT, alkaline phosphatase, total protein, albumin, glucose, cholesterol, bilirubin, creatinine, urea nitrogen, inorganic phosphates and Cl-, Na+, K+, Ca2+ and Mg2+

URINALYSIS: Yes
- Time schedule for collection of urine: During weeks 5, 11 and 26
- Metabolism cages used for collection of urine: No
- Animals fasted: Not reported
- How many animals: During weeks 5 and 11, urine was collected from 10 animals/sex from PEG group, control group and 5.0% group and in week 26, urine was collected from all animals.
- Method: Lab sticks, Urobilistix and iktstix method (Semi-quantitative investigation)
- Parameters: pH, protein, glucose, ketone bodies, occult blood, urobilinogen and bilirubin

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were euthanized after blood sampling and organs were immediately collected.

TISSUE COLLECTED FOR ORGAN WEIGHTS AND HISTOPATHOLOGY: Liver, kidneys, spleen, adrenal glands, lungs, heart, thymus, reproductive organ, pituitary, brain and thyroid

HISTOPATHOLOGY: Above collected organs and stomach, duodenum, ileum, cecum, pancreas, bone marrow and skin were fixed in 10% formalin, embedded in paraffin and underwent H&E staining before being examined under the microscope

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In half of the animals of highest tested dose, mild redness of test site was observed one week after starting to apply it, but this disappeared after one week with desquamation. This did not occur again thereafter. Among female animals, all animals in the 5.0% group and one animal from the 1.0% group developed mild redness after one month since the start of study, but these went away after one week. After two, three months, half the animals in the 5.0% group exhibited mild redness, and it was seen sporadically thereafter.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal from the control group (Week 16) and one male animal from the 5.0% group (Week 23) died during the study, but the cause remains unknown after finding no abnormalities through the necropsies.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Male animals in the 0.5% group exhibited mild weight suppression from Week 11, but this had recovered by Week 16. At the end of the experiment, slight weight suppression was observed in male animals from the 0.5% group.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

5 weeks interim analysis: There were significant increase in segmented neutrophils and significant decrease in white blood cell count for male animals in PEG group and increase in hematocrit levels in male animals in 5.0% dose group
13 weeks interim analysis: level: Significant increase in red blood cells and hemoglobin levels were observed in males of 5.0% dose group and increased white blood cell count was also observed for male and female animals in the PEG and 5.0% groups. In white blood cell fractions, decrease in eosinophils was observed for female animals in the 5.0% group.
6 month analysis: Significant decrease in white blood cell count were observed for female animals in the 5.0% group, as well as for both male and female animals in the 1.0% group. Significant increase in lymphocytes for male animals in the PEG group and significant decrease in monocytes for male animals in the 5.0 and 0.5% groups were also observed.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

PEG group: Significant decrease was observed in s-GOT levels for female animals and in sodium and s-GPT levels for all animals.
5.0% dose group: There was significant reduction in glucose in females, in calcium levels in males and sodium and s-GPT in all animals. Elevated levels of A/G ratio and cholesterol were also observed in females.
1.0% dose group: There is significant decrease in glucose in females and sodium levels in all animals and increase in urea nitrogen in females of this group.
0.5% dose group: There is significant decrease in sodium levels in all animals of this group and increase in urea nitrogen in females of this group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Similar levels of all tested parameters (protein, occult blood, Urobilinogen and pH) were observed in treated animals as compare to control animals in 13 as well as 26 weeks interim analysis.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were decrease in actual and relative liver weights of 0.5% (low) dose group. No dose dependency was observed for this effect.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Liver stasis was observed for one male animal in the 1.0% group, while blood spots and fading color were observed for another male animal in the same group. Furthermore, white serpentine bulges were observed for three male animals each in the 5.0 and 0.5% groups, as well as in four male animals in 1.0% group and one male animal in the 0.5% group.
- Kidney edema was observed for one female each in the control and 0.5% groups.
- Yellow discoloration was observed in pancreas for one male animal in the control group.
- Lungs stasis was observed for one male animal in the 1.0% group.
- Heart hypertrophy was observed for one male animal in the 1.0% group.
- Blood spots in thymus were observed for two male animals each in the control, PEG and 1.0% groups, as well as in four male animals in the 5.0 and 0.5% groups.
- Testicular atrophy was observed for one male in the 1.0% group, while ovarian edema was observed for one female animal each in the control and 1.0% groups.
- Pituitary edema was observed for one male animal in the 1.0% group.
- Stomach hematoma was observed in the cardia for one male animal in the 1.0% group, while an ulcer of 1 mm in diameter was observed for another male animal in the same group. Edema was observed in the stomach for one male animal in the control group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Control group:
- Sinusoid bleeding was observed for one male and one female animal each, while focal necrosis was observed for one female animal.
- Renal tubular hyaline casts were observed for seven male animals and one female animal, while interstitial small round cell infiltration and renal tubular epithelial cell swelling were observed for two male and one female animals
- Fibrosis of islets of Langerhans observed for one male animal
- Interstitial myocarditis was observed for one male and one female animal each.
PEG group:
- Sinusoid bleeding was observed for three male animals, focal necrosis was observed in one male animal and Kupffer cell migration was observed for one female animal
- Renal tubular hyaline casts were observed for five male animals and one female animal, while interstitial small round cell infiltration and renal tubular epithelial cell swelling were observed for one male and one female animal each
- Adrenal gland: A hemorrhagic foci was observed in the stratum corneum for one male animal.
- Fibrosis of islets of Langerhans observed for one male animal.
5.0% dose group:
- Sinusoid bleeding was observed for five male animals, while focal necrosis was observed for two male animals. Kupffer cell migration was observed for one female animal.
- Renal tubular hyaline casts were observed for three male and female animals each, while interstitial small round cell infiltration was observed for two male animals. Renal tubular calcification was observed for another male animal.
- Fibrosis of islets of Langerhans observed for one male animal.
- Interstitial myocarditis was observed for two male animals and one female animal.
1.0% dose group:
- Sinusoid bleeding was observed for five male animals, while focal necrosis was observed for another male animal.
- Renal tubular hyaline casts were observed for five male and one female animals, interstitial small round cell infiltration was observed for one male and one female animal each, and renal tubular calcification was observed for two female animals
- Fibrosis of islets of Langerhans observed for four male animals
- Interstitial myocarditis was observed for one male animal.
- Decline in sperm production was observed for one male animal.
0.5% dose group:
- Sinusoid bleeding was observed for four male animals and one female animal, while large lipid droplets were observed for one male animal. Kupffer cell migration was observed for one female animal.
- Renal tubular hyaline casts were observed for five male and two female animals, interstitial small round cell infiltration was observed for one male animal and another male animal exhibited renal tubular epithelial cell swelling. Furthermore, renal tubular calcification was observed for one female animal.
- Fibrosis of islets of Langerhans observed for three male animals.
- Interstitial myocarditis was observed for three male animals.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- In half of the animals of highest tested dose, mild redness of test site was observed one week after starting to apply it, but this disappeared after one week with desquamation. This did not occur again thereafter.
- Among female animals, all animals in the 5.0% group and one animal from the 1.0% group developed mild redness after one month since the start of study, but these went away after one week.
- After two, three months, half the animals in the 5.0% group exhibited mild redness, and it was seen sporadically thereafter.
- One male animal from the control group (Week 16) and one male animal from the 5.0% group (Week 23) died during the study, but the cause remains unknown after finding no abnormalities through the necropsies.

BODY WEIGHT AND WEIGHT GAIN
- Male animals in the 0.5% group exhibited mild weight suppression from Week 11, but this had recovered by Week 16.
- At the end of the experiment, slight weight suppression was observed in male animals from the 0.5% group.

FOOD CONSUMPTION AND COMPOUND INTAKE
- No significant difference was noted in males or females between the control and test substance groups.

FOOD EFFICIENCY
- No effects were observed in test substance treated animals as compare to control animals

HAEMATOLOGY
- 5 weeks interim analysis: There were significant increase in segmented neutrophils and significant decrease in white blood cell count for male animals in PEG group and increase in hematocrit levels in male animals in 5.0% dose group
- 13 weeks interim analysis: level: Significant increase in red blood cells and hemoglobin levels were observed in males of 5.0% dose group and increased white blood cell count was also observed for male and female animals in the PEG and 5.0% groups. In white blood cell fractions, decrease in eosinophils was observed for female animals in the 5.0% group.
- 6 month analysis: Significant decrease in white blood cell count were observed for female animals in the 5.0% group, as well as for both male and female animals in the 1.0% group. Significant increase in lymphocytes for male animals in the PEG group and significant decrease in monocytes for male animals in the 5.0 and 0.5% groups were also observed.

CLINICAL CHEMISTRY
- PEG group: Significant decrease was observed in s-GOT levels for female animals and in sodium and s-GPT levels for all animals.
- 5.0% dose group: There was significant reduction in glucose in females, in calcium levels in males and sodium and s-GPT in all animals. Elevated levels of A/G ratio and cholesterol were also observed in females.
- 1.0% dose group: There is significant decrease in glucose in females and sodium levels in all animals and increase in urea nitrogen in females of this group.
- 0.5% dose group: There is significant decrease in sodium levels in all animals of this group and increase in urea nitrogen in females of this group.

URINALYSIS
Similar levels of all tested parameters (protein, occult blood, Urobilinogen and pH) were observed in treated animals as compare to control animals in 13 as well as 26 weeks interim analysis.
ORGAN WEIGHTS
- There were decrease in actual and relative liver weights of 0.5% (low) dose group. No dose dependency was observed for this effect.

GROSS PATHOLOGY
- Liver stasis was observed for one male animal in the 1.0% group, while blood spots and fading color were observed for another male animal in the same group. Furthermore, white serpentine bulges were observed for three male animals each in the 5.0 and 0.5% groups, as well as in four male animals in 1.0% group and one male animal in the 0.5% group.
- Kidney edema was observed for one female each in the control and 0.5% groups.
- Yellow discoloration was observed in pancreas for one male animal in the control group.
- Lungs stasis was observed for one male animal in the 1.0% group.
- Heart hypertrophy was observed for one male animal in the 1.0% group.
- Blood spots in thymus were observed for two male animals each in the control, PEG and 1.0% groups, as well as in four male animals in the 5.0 and 0.5% groups.
- Testicular atrophy was observed for one male in the 1.0% group, while ovarian edema was observed for one female animal each in the control and 1.0% groups.
- Pituitary edema was observed for one male animal in the 1.0% group.
- Stomach hematoma was observed in the cardia for one male animal in the 1.0% group, while an ulcer of 1 mm in diameter was observed for another male animal in the same group. Edema was observed in the stomach for one male animal in the control group.

HISTOPATHOLOGY
Control group:
- Sinusoid bleeding was observed for one male and one female animal each, while focal necrosis was observed for one female animal.
- Renal tubular hyaline casts were observed for seven male animals and one female animal, while interstitial small round cell infiltration and renal tubular epithelial cell swelling were observed for two male and one female animals
- Fibrosis of islets of Langerhans observed for one male animal
- Interstitial myocarditis was observed for one male and one female animal each.
PEG group:
- Sinusoid bleeding was observed for three male animals, focal necrosis was observed in one male animal and Kupffer cell migration was observed for one female animal
- Renal tubular hyaline casts were observed for five male animals and one female animal, while interstitial small round cell infiltration and renal tubular epithelial cell swelling were observed for one male and one female animal each
- Adrenal gland: A hemorrhagic foci was observed in the stratum corneum for one male animal.
- Fibrosis of islets of Langerhans observed for one male animal.
5.0% dose group:
- Sinusoid bleeding was observed for five male animals, while focal necrosis was observed for two male animals. Kupffer cell migration was observed for one female animal.
- Renal tubular hyaline casts were observed for three male and female animals each, while interstitial small round cell infiltration was observed for two male animals. Renal tubular calcification was observed for another male animal.
- Fibrosis of islets of Langerhans observed for one male animal.
- Interstitial myocarditis was observed for two male animals and one female animal.
1.0% dose group:
- Sinusoid bleeding was observed for five male animals, while focal necrosis was observed for another male animal.
- Renal tubular hyaline casts were observed for five male and one female animals, interstitial small round cell infiltration was observed for one male and one female animal each, and renal tubular calcification was observed for two female animals
- Fibrosis of islets of Langerhans observed for four male animals
- Interstitial myocarditis was observed for one male animal.
- Decline in sperm production was observed for one male animal.
0.5% dose group:
- Sinusoid bleeding was observed for four male animals and one female animal, while large lipid droplets were observed for one male animal. Kupffer cell migration was observed for one female animal.
- Renal tubular hyaline casts were observed for five male and two female animals, interstitial small round cell infiltration was observed for one male animal and another male animal exhibited renal tubular epithelial cell swelling. Furthermore, renal tubular calcification was observed for one female animal.
- Fibrosis of islets of Langerhans observed for three male animals.
- Interstitial myocarditis was observed for three male animals.

Key result

Dose descriptor:

NOAEL

Effect level:

5 other: %

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No significant adverse effect was seen at any dose level.

Remarks on result:

other: Based on no growth, functional and morphological abnormalities, other than the localized effects at any tested dose levels.

Conclusions:

Transdermal administration of C10-13 magnesium linear alkylbenzene sulphonate (LAS-Mg; purity: 96.9%) to CRJ-SD rats at dose levels of 0 (water), 3% PEG, 0.5, 1.0 and 5.0% of test substance dissolved in 3% polyethyleneglycol (PEG, MW: 200) for 26 weeks revealed no observed adverse effect level (NOAEL) of 5.0%, based no toxicologically relevant changes at any dose level.

Executive summary:

A 26-weeks repeated dose toxicity study (transdermal) was conducted in male and female CRJ-SD rats to evaluate the chronic effects of C10-13 magnesium linear alkylbenzene sulphonate (LAS-Mg; purity: 96.9%).

Approximately 4 weeks old male and females animals were housed in cages (2 animals/cage). Solid feed (CE-2, CLEA Japan, Inc.) and tap water were provided ad libitum. The animals were maintained under standard laboratory conditions (temperature: 22 ± 1°C; relative humidity: 55 ± 5%). Prior to the treatment, animals were acclimatized under laboratory conditions for 1 week.

The test substance was administered daily at 5 dose levels of i.e. 0 (water), 3% PEG, 0.5, 1.0 and 5.0% of test substance dissolved in 3% polyethyleneglycol (PEG, MW: 200) for 26 weeks with 20 animals/sex/dose group.

General symptoms and body weight of animals were recorded on a daily-basis and food intake were recorded weekly. Urinalysis and hematological examinations were performed in week 5 and 13 of the study.  At the end of study, urinalysis, haematological, serum biochemistry were also done. All the surviving animals were humanely euthanized after 26 weeks and organs (liver, kidneys, spleen, adrenal glands, lungs, heart, thymus, reproductive organ, pituitary, brain and thyroid) were collected for recording absolute and relative organ weights.  All the collected organs (including stomach, duodenum, ileum, cecum, pancreas, bone marrow and skin) were then fixed in 10% formalin, embedded in paraffin and underwent H&E staining before being examined under the microscope.

One male animal from the control group (Week 16) and one male animal from the 5.0% group (Week 23) died during the study, but the cause remains unknown after finding no abnormalities through the necropsies. There were mild redness of test site at Week 1 (in 5.0% dose) and after one month of start of study (in the 5.0% group and one animal from the 1.0% group), but these went away after one week. There was slight weight suppression in male animals from the 0.5% group at the end of the experiment. No toxicologically significant changes were observed in food efficiency of treated animals when compared to controls.

There were no toxicologically relevant changes to the urine, hematological and serum biochemical findings.

No changes were seen in the organ weights of treated animals, and gross necropsy reveal only white serpentine bulges being seen in the livers of male animals in the control and test groups. Histopathological findings were non-specific and had no dose-dependency.

Transdermal administration of C10-13 magnesium linear alkylbenzene sulphonate (LAS-Mg; purity: 96.9%) to CRJ-SD rats at dose levels of 0 (water), 3% PEG, 0.5, 1.0 and 5.0% of test substance dissolved in 3% polyethyleneglycol (PEG, MW: 200) for 26 weeks revealed no observed adverse effect level (NOAEL) of 5.0%, based no toxicologically relevant changes at any dose level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral Administration

Six studies are available to document the potential effects from repeated oral exposures to read across substances.

In the key study (Yoneyama et al. 1976), male and female rats were exposed to Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts in drinking water daily for 9 months. Test doses were 85, 145 and 430 mg/kg bw/d plus the control. Eight to nine animals of each sex were exposed per group. Body weight was suppressed in the highest dose group only. Significant decreases in transaminase activity and renal Na,K-ATPase were seen in the 145 mg/kg bw/d group. No significant haematological or organ weight changes were noted. Based on enzyme activity, the resultant LOAEL and NOAEL values were 145 and 85 mg/kg bw/day, respectively.

In a supporting study (Yoneyama et al. 1972), male and female rats were exposed to Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts in the diet daily for 6 months. The doses were 40, 115, 340 and 1030 mg/kg bw/d plus the control group. Significant diarrhea, suppressed growth, increased cecal weight, and degeneration of renal tubes were observed in the highest dose group. Similar but less severe signs were seen in other doses with the exception of the lowest dose of 40 mg/kg bw/d, which showed no adverse effects related to exposure. The resultant LOAEL and NOAEL values were 115 and 40 mg/kg bw/day, respectively.

In another supporting study (Ito et al. 1978), male and female rats were exposed to Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts via gavage daily for 28 days at three dose levels plus the control (0, 125, 250 and 500 mg/kg bw/d). Body weight gain was suppressed, some serum biochemical measures were different from the controls, and some organ weights were either decreased (spleen, heart, thymus) or increased (liver) in either the male or female high dose groups. No mortalities or histopathological abnormalities were observed. The resultant LOAEL and NOAEL values were 250 and 125 mg/kg bw/day, respectively, based on serum-biochemical differences from the controls.

The same authors (Ito et al. 1978) also conducted a study using Benzenesulfonic acid, C10-13-alkyl derivs., magnesium salts at doses of 0, 75, 150 and 300 mg/kg bw/d and following the same protocol. The resultant LOAEL and NOAEL values were 300 and 150 mg/kg bw/day, respectively, based on body weight gain, hematology, serum-biochemical and organ weight differences from the controls.

The same authors (Ito et al. 1978) also conducted a 26-weeks repeated dose toxicity study (oral) using male and female CRJ-SD rats to evaluate the chronic effects of Benzenesulfonic acid, C10-13-alkyl derivs., magnesium salts (purity: 96.9%). The test substance was administered daily at 4 dose levels of i.e. 0, 75, 150 and 300 mg/kg bw/day using a metallic gastric sonde to CRJ-SD rats with 20 animals/sex/dose group. One male animal in 300 mg/kg bw/day dose group died (in week 21) from weakness due to significant weight loss involving diarrhea and loss of appetite. There were wetting in the area surrounding animals’ mouths after dosing, and a few animals had abnormal airway sounds. Soft stools were seen in animals after one month, then recovered in few days. There was slight body weight suppression in the male animals of 300 mg/kg bw/day dose group. No toxicologically significant changes were observed in food efficiency of treated animals when compared to controls. At end of dosing period, haematological findings revealed significant reduction in hematocrit levels for female animals in 300 mg/kg bw/day dose group. There was significant reduction in bilirubin, protein, albumin and calcium levels in male animals and significant reduction in s-GPT, glucose, calcium and magnesium levels in female animals of 300 mg/kg bw/day dose group. There was also significant reduction in s-GOT, s-GPT, alkaline phosphatase, protein, albumin, sodium and calcium levels in male animals and significant reduction in chloride levels in female animals of 150 mg/kg bw/day dose groups. Changes were also observed in protein levels in urine in treated male animals as compare to control groups. Necropsy revealed atrophy and bloating of the liver and spleen, respectively. Significant increased relative liver weights (males), thymus weights (males) and pituitary (males) weights were observed in 150 mg/kg bw/day dose group animals as compare to control. Reduction in relative heart weights (females), kidneys weights (males) and adrenal gland weights (females) were also observed in highest dose treated group. Significant increased relative thymus weights were observed in male animals as compare to control and decreased relative heart weights in females and relative kidneys weights in males of 150 mg/kg bw/day dose group. Significant dip in relative adrenal glands weights of females was seen in low dose group. Histopathological examinations revealed Kupffer cell migration (at 150 mg/kg bw/day dose group), focal necrosis (at 75 mg/kg bw/day dose group) and sinusoid bleeding in the liver. In the kidneys, renal tubular epithelial cell swelling, hyaline casts and interstitial small round cell infiltration were observed (at 300 mg/kg bw/day dose group). Fibrosis of islets of Langerhans (at 75 mg/kg bw/day dose group) was also observed. However, histopathological effects seen in liver and pancreas were not dose dependent. Oral administration of the test substance to CRJ-SD rats at dose levels of 0, 75, 150 and 300 kg/kg bw/day for 26 weeks revealed no observed adverse effect level (NOAEL) of 150 mg/kg bw/day, based on weight suppression, mortality, fluctuations in hematological and serum biochemistry, altered organ weights and histopathological alterations in kidney at 300 mg/kg bw/day.

In the final study (Yoneyama et al. 1976), a 9 month sub-chronic oral toxicity study of Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts was performed in SLC-ICR mice, focusing on the liver and kidneys. 4 week old male and female SLC-ICR mice were administered LAS daily at following dose levels for 9 months: 1) Mixed in diet: 0 and 0.6% (equivalent to 0 and 780 mg/kg bw/day); 9 males in 0.6% fed group and 8 males in control group; 8 females in control as well as 0.6% fed group or 2) Dissolved in drinking water: 0, 0.07, 0.2 and 0.6 (equivalent to 0,133, 380 and 1140 mg/kg bw/day); 9 animals each in test substance treatment groups and 8 animals in control group. Mice in 1.8% dose group of both feeding and drinking water study exhibited severe weight loss so administration was stopped after 2 weeks. No mortality was observed throughout the study. There was significant inhibition of weight increase in both males and females of 0.6% dose group consuming Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts water compared to controls, while there was a significant increase in body weight compared to controls in males of 0.6% dose group fed with Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts in diet and 0.07% dose group consuming Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts water. Individual daily food consumption was higher in all treatment groups compared to controls. Water consumption was reduced in males of 0.6% dose group (drinking water study) and in females of 0.6% dose group (dietary study) and 0.2% dose group (drinking water study) and increased in males of 0.6% dose group (dietary study), 0.07 and 0.2% dose groups (drinking water study) and female of 0.07% dose groups (drinking water study). In males of 0.6% dose group (dietary study) and 0.07% dose group (drinking water study), the absolute liver weight was significantly increased, in males of 0.6% dose group (dietary study), 0.2 and 0.6 dose groups (drinking water study), the relative liver weight was significantly increased, and in 0.07% (drinking water) males the relative liver weight was increased. Relative lung, heart and kidney weights were also increased in male mice consuming 0.07% Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts water. In females, the relative liver weight was significantly increased in all treatment groups. Relative spleen, heart and kidney weights were also increased in female mice consuming 0.6% Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts water. There were reductions in LDH activity in male mice of 0.6% group fed with Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts diet and GOT activity in male mice of 0.6% group consuming Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts water were significant. GOT activity in female mice of 0.2% group (drinking water study) was also significantly reduced. G6Pase was significantly reduced in males and female mice of 0.6% group consuming Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts water compared to controls, and also reduced in other treatment groups. No NOAEL can be identified for dietary study in mice, as only one dose level for Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts was fed to animals throughout the study. Administration of Benzenesulfonic acid, C10-13-alkyl derivs., sodium salts to SLC-ICR mice by either test diets (0 and 0.6) or drinking water (0, 0.07, 0.2 and 0.6) for 9 months (focusing on the liver and kidneys) revealed an NOAEL of 0.07% (133 mg/kg bw/day in drinking water), based on observed adverse effects on organ weight and liver and kidney enzymes. Repeated administration of the test substance impaired renal and liver function.

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

Highest NOAEL below lowest LOAEL from three long term studies = 85 mg/kg bw

For the additional read across substance MIPA, in a combined repeated dose oral toxicity study with a reproduction/developmental screening study in rats, performed according to OECD guideline 422, a NOAEL for general systemic toxicity of 300 mg/kg bw/day was established for males, based on some indications for a mild anemic process. For females, the NOAEL was 1000 mg/kg bw/day, the highest dose tested.

Dermal Administration

In a supporting study (Ito et al. 1978), male and female rats were transdermally exposed to the read across substance (Benzenesulfonic acid, C10-13-alkyl derivs., magnesium salts) in 3% polyethylene glycol (PEG, MW: 200) for 26 weeks. Test doses were 0 (water), 3% PEG, 0.5, 1.0 and 5.0% of test substance dissolved in 3% PEG. No toxicologically relevant changes were observed at any dose level. The no observed adverse effect level (NOAEL) is 5.0%, the highest dose tested.

Justification for classification or non-classification

Effects in long-term repeated dose studies conducted on the read across substances and lasting up to 9 months (subchronic studies) are related to enzymatic, body and organ weight reductions but not mortality. As a result the substance does not need to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.