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Description of key information

Oral

No information is available on the repeated dose toxicity of LAS TEA. The endpoint was addressed with read across data from LAS Na and TEA.

LAS Na:

Male and female rats were exposed to LAS Na (125, 250, 500 mg/kg bw/day) orally by gavage daily for 28 days. The results showed suppressed body weight gain, differences in some serum biochemical measures when compared to the controls, and decreased (spleen, heart, thymus) or increased (liver) organ weights in the animals of the highest dose level. The resultant LOAEL and NOAEL values were 250 and 125 mg/kg bw/day, respectively (Ito et al., 1978).

In a 6-month toxicity test male and female rats were exposed to LAS Na (CAS 69669-44-9) in the diet daily:40, 115, 340, 1030 mg/kg bw/day. Diarrhea, suppressed growth, increased cecal weight, and degeneration of renal tubes characterized the highest dose group. Similar but less severe signs were seen in other doses with the exception of the lowest dose of 0.07%, which showed no adverse effects related to exposure to LAS. The resultant LOAEL and NOAEL values were 115 and 40mg/kg bw/day, respectively (Yoneyama et al., 1972).

In a 9-month toxicity study male and female rats were exposed to LAS Na (CAS 69669-44-9; 85, 145, 430 mg/kg bw/day) in drinking water daily. Body weight was suppressed in the highest dose. Significant decreases in transaminase activity and renal Na,K-ATPase was seen in the second group. The resultant LOAEL and NOAEL values were 145 and 85 mg/kg bw/day, respectively (Yoneyama et al., 1976).

TEA:

 In a 90-day toxicity study TEA was administered to male and female rats in the feed at dose levels of 0, 250, 500, and 1000 mg/kg bw/day for 91 days. No treatment-related effects were observed in the animals.The NOAEL is 1000 mg/kg bw/day (TSCATS, 1989).

Dermal

No information is available on the repeated dose toxicity of LAS TEA. The endpoint was addressed with read across data from TEA.

TEA:

In a 90-day dermal toxicity study, Fischer rats were treated with 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day, 5 days/week. Application of 2000 mg/kg bw resulted in a significant decrease in body weight gain, and grossly visible crusts at the site of application were noted in males and females administered 1000 or 2000 mg/kg bw. NOAELs for local effects were determined to be 125 and 250 mg/kg bw/day for males and females, respectively. The NOAEL's for systemic effects were established to be 125 and 500 mg/kg bw/day for males and females, respectively, based on kidney effects (Battelle, 1987a).

In a second dermal toxicity study, this time with mice, TEA (0, 250, 500, 1000, 2000 or 4000 mg/kg bw/day) was applied daily on the skin for 90 days. The NOAEL for local effects was determined to be 250 mg/kg bw/day (acanthosis at the application site). NOAEL's for systemic effects were established to be 1000 and 250 mg/kg bw/day for males and females, respectively, based on kidney effects.

The local effects should be mainly attributed to triethanolamine (free amine) and not to the protonated amine. Therefore, such effects are most probably not relevant for LAS TEA.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Does not meet important criteria of today's standard methods
Qualifier:
no guideline followed
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:
Wistar
Remarks:
JCL
Sex:
male/female
Details on test animals 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
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:
unchanged (no vehicle)
Remarks:
Test substance was administered either in diet or drinking water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Nine months
Frequency of treatment:
Continuous in diet or drinking water (ad libitum)
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 (Table 1).
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 (Table 1).
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 cho
linesterase 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 (Table 2).
There was also significant decrease in absolute and relative heart weight in females of 0.6% (diet) and 0.07% (drinking water) dose groups (Table 2).
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):
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:
NOAEL
Effect level:
85 mg/kg bw/day (nominal)
Based on:
act. ingr.
Remarks:
Drinking water
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 lactat e 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
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Remarks:
Dietary study
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
water consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
other: Liver and kidney enzyme levels
Remarks on result:
other: Adverse effects were observed at all dose levels
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

Table 1: Body weight gain, Food, water and sample consumption of Rats on administration of LAS for 9 months

 Dose group  No. of Rats Initial BW (g)  Final BW (g)  BW gain (%)  Food (g/rat/day)  Water (g/rat/day)  Sample (g/kg bw/day) 
 0 8M  113.1±1.156   421.6±8.542   372.6±5.889  15  23  -
 0.6  8M   110.6±0.925   413.5±7.356   373.8±6.380  16  23 0.234
 1.8  8M   109.2±2.218   341.1±8.008   312.5±6.518**  14  33 0.747
 0.07  8M   110.6±1.572   411.0±5.255   371.9±6.908  17  29 0.051
 0.2  8M   108.3±1.900   385.1±5.453   356.1±6.346**  15  33  0.148
               
 0  8F   92.3±1.592   212.6±4.508   230.6±5.420  10  18  -
 0.6  8F   92.1±1.663   216.1±7.705    234.3±5.753  10  17  0.287
 1.8  8F   90.6±1.463   184.8±4.278    204.3±5.791**  9  18  0.969
 0.07  8F   92.7±1.770   206.0±3.555    222.4±4.478  11  20  0.082
 0.2  8F   91.6±1.893   209.3±5.383    228.4±3.857  12  18  0.173

*p<0.05 **p<0.01

Table 2: Organ weight of male and female rats on administration of LAS for 9 months

 Dose group  Brain (g) / (g/100g)  Heart (g) / (g/100g)   Lung (g) / (g/100g)   Liver (g) / (g/100g)   Spleen (g) / (g/100g)   Kidner (R) (g) / (g/100g)   Kidney (L) (g) / (g/100g) Adrenal (R) (mg) / (mg/100g)   Adrenal (L) (mg) / (mg/100g) Testis/Ovary (R) (g) / (g/100g)    Testis/Ovary (L) (g) / (g/100g)    Uterus (g) / (g/100g)  
 0 M  1.987±0.011 /  0.463±0.008   1.026±0.018 /  0.239±0.005    1.263±0.033 /  0.294±0.008  12.7±0.341 /  2.9±0.039  0.698±0.014 /  0.162±0.003    1.162±0.040 /  0.270±0.008   1.184±0.041 /  0.275±0.007  18.3±1.117 /  4.2±0.217   18.1±0.934 /  4.2±0.225   1.591±0.023 /  0.370±0.006   1.643±0.027 /  0.382±0.005  
 0.6 M   1.982±0.011 /  0.473±0.007   0.971±0.022* /  0.231±0.004    1.269±0.042 /  0.302±0.005    12.9±0.310 /  3.0±0.049  0.682±0.019 /  0.162±0.002     1.210±0.026 /  0.290±0.006   1.214±0.032 /  0.289±0.007   19.6±1.900 /  4.7±0.435  17.6±1.602 /  4.2±0.416     1.563±0.019 /  0.373±0.004   1.585±0.027 /  0.378±0.007   -  
 1.8 M   1.827±0.131 /  0.521±0.037   0.825±0.026* /  0.235±0.003    1.040±0.025* /  0.295±0.002    11.5±0.352* /  3.2±0.043*   0.501±0.163** /  0.143±0.003**  0.977±0.022** /  0.279±0.007   0.982±0.017** /  0.281±0.007    17.5±1.370 /  5.0±0.481  17.0±0.654 /  4.8±0.146    1.455±0.027** /  0.415±0.004**    1.491±0.033** /  0.425±0.004**  -  
 0.07 M   1.995±0.016 /  0.471±0.006   0.981±0.031 /  0.232±0.008    1.287±0.021 /  0.304±0.007    13.2±0.258 /  3.0±0.052   0.682±0.009 /  0.161±0.002    1.221±0.018 /  0.288±0.005   1.254±0.012 /  0.296±0.005*   18.6±0.680 /  4.4±0.156   19.2±1.024 /  4.5±0.262   1.606±0.022 /  0.379±0.006    1.566±0.056 /  0.370±0.015  -  
 0.2 M   1.881±0.108 /  0.478±0.028   0.975±0.183 /  0.247±0.003    1.154±0.037* /  0.293±0.007    11.6±0.184 /  2.9±0.042   0.595±0.013** /  0.150±0.002   1.124±0.025 /  0.286±0.007   1.154±0.031 /  0.293±0.008   18.4±0.914 /  4.6±0.232   17.5±1.309 /  4.4±0.354   1.483±0.059 /  0.377±0.016     1.589±0.013 /  0.404±0.005  -  
                           
 0 F   1.853±0.023 /  0.846±0.016   0.647±0.013 /  0.294±0.003    0.870±0.026 /  0.397±0.012    6.2±0.150 /  2.8±0.051   0.441±0.016 /  0.201±0.006    0.650±0.021 /  0.296±0.006   0.650±0.021 /  0.296±0.008   24.8±1.381 /  11.2±0.443   25.7±1.385 /  11.7±0.487   29.3±2.738 /  13.4±1.225    31.2±4.007 /  14.2±1.857    0.687±0.037 /  0.313±0.017   
 0.6 F   1.859±0.011 /  0.845±0.027   0.595±0.019* /  0.269±0.006**   0.810±0.032 /  0.367±0.015   6.6±0.355 /  2.9±0.061   0.425±0.010 /  0.193±0.009    0.648±0.023 /  0.292±0.004   0.655±0.026 /  0.296±0.008   25.8±1.563 /  11.7±0.820   24.8±1.641 /  11.3±0.863   23.3±3.504 /  10.8±1.740    32.8±3.120 /  14.9±1.380    0.660±0.033 /  0.301±0.020    
 1.8 F   1.788±0.031 /  0.954±0.015**  
 0.455±0.008 /  0.242±0.002
  0.761±0.028 /  0.405±0.009  7.1±0.195** /  3.8±0.082**    0.351±0.012** /  0.187±0.004   0.580±0.015 /  0.309±0.003   0.586±0.021* /  0.312±0.007    19.6±1.981* /  10.3±0.924  21.3±1.487* /  11.3±0.609    25.3±2.853 /  13.4±1.417     31.5±2.322 /  16.7±1.165     0.588±0.066 /  0.312±0.034   
 0.07 F   1.854±0.019 /  0.844±0.016   0.611±0.011* /  0.277±0.004**   0.801±0.027 /  0.365±0.014   6.3±0.139 /  2.8±0.063   0.424±0.007 /  0.193±0.004    0.669±0.017 /  0.304±0.006   0.677±0.010 /  0.308±0.005  22.0±1.619 /  10.0±0.720    21.0±1.763 /  9.8±0.790   30.0±1.490 /  13.6±0.740    31.7±2.300 /  12.2±1.200     0.729±0.051 /  0.331±0.022   
 0.2 F   1.691±0.112 /  0.810±0.053   0.593±0.015 /  0.287±0.004   0.817±0.040 /  0.393±0.021   6.1±0.220 /  2.9±0.071   0.428±0.014 /  0.205±0.006    0.651±0.015 /  0.312±0.006   0.670±0.020 /  0.321±0.008*   23.2±1.495 /  11.1±0.677   24.3±1.154 /  11.6±0.431   28.4±1.633 /  13.6±0.821    28.6±1.900 /  13.7±0.830    0.644±0.032 /  0.308±0.014   
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 a 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 a NOAEL of 0.07% (85 mg/kg bw/day in drinking water), based on adverse effects at all dose levels, 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.

Four week old male and female Wistar 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. Five animals were housed in each cage and maintained under controlled environmental conditions (temperature: Average of 25 ± 1°C, humidity: 50 - 60%, and 12 hours light /12 hours dark).CE-2 diet (from CLEA Japan) and water were provided ad 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) male 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, KATPase 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 on adverse effects at all dose levels, 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).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable study, followed scientific principles/standards, pre-dates GLP
Remarks:
This study is assigned a reliability score of 2 because the original report was not available for review. However, the study was evaluated by IPCS prior to inclusion in their criteria document.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study is to determine the sub-acute toxicity of C10-13 sodium linear alkyl benzene sulfonate (LAS-Na) in CRJ-SD rats. LAS-Na was orally administered at 0 (distilled water), 125, 250 and 500 mg/kg bw to male and females rats for 29 and 30 days, respectively. General symptoms of toxicity and the body weights were recorded daily while the food consumption was measured twice a week. Urinalysis was also performed during the study. At the end of study, blood was collected for estimation of haematological and clinical chemistry parameters. After collection of blood samples, the animals were humanely euthanized, gross necropsy was performed and organs weights were determined.
GLP compliance:
no
Remarks:
Pre-dates GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: Not reported
- Fasting period before study: Not reported
- Housing: 3 male and 4 female animals per cage.
- Diet: Solid feed (CE-2, CLEA Japan, Inc.); ad libitum
- Water: Tap water; ad libitum
- Acclimation period: After 1 week pre-housing, the animals were used in the experiment.
ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2°C
- Humidity: 55 ± 5%
- Air changes: Not reported
- Photoperiod: Not reported
Route of administration:
oral: gavage
Details on route of administration:
The test substance was administered orally through a metallic gastric sonde.
Vehicle:
not specified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in distilled water prior to dosing.
- DOSE VOLUME: 5 mL/kg bw
- Concentration in vehicle: 0, 250, 500, and 1000 mg/mL respectively for administration of doses 0, 125, 250 and 500 mg/kg bw.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
29 days for males and 30 days for females
Frequency of treatment:
Once daily
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
In distilled water (corresponding to 250 mg/mL)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
In distilled water (corresponding to 500 mg/mL)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
In distilled water (corresponding to 1000 mg/mL)
No. of animals per sex per dose:
Test group: 12 animals/sex/dose group
Control group: 15 male/group and 16 female/group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose levels for this study were selected based on the results of acute toxicity study conducted with LAS-Na in rats. 1/3 of the LD50 levels of LAS-Na, i.e. 500 mg/kg bw, was selected as the highest dose. The other two doses were set at common ratios of 2, whereby the medium and low doses were 250 (1/6 LD50) and 125 (1/12 LD50) mg/kg bw, respectively.
Positive control:
No.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption: Yes
- Time schedule for examinations: Twice/week

FOOD EFFICIENCY: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 1 month, blood was collected was collected from the carotid artery using a cannula.
- Anesthetic used for blood collection: Yes, light ether anesthesia was used.
- Animals fasted: Not reported
- How many animals: All surviving animals
- Parameters: Hemoglobin, hematocrit, red and white blood cell count were measured using an autoanalyzer SMA-4A.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 1 month, blood was collected from each animal and centrifuged (at 3000 rpm for 15 minutes) to obtain the serum samples for analysis.
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: s-GOT, s-GPT, alkaline phosphatase, total protein, albumin, glucose, cholesterol, bilirubin, creatinine, urea nitrogen, inorganic phosphates and ions (Na+, K+, Ca2+, Mg2+ and Cl-)

URINALYSIS: Yes
- Time schedule: Not reported
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: pH, proteins, sugar, ketone bodies and occult blood

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: The wet weight of liver, kidneys, spleen, adrenal glands, lungs, heart, thymus, reproductive organ, pituitary and the brain were measured. Based on this, the relative weights were calculated.

HISTOPATHOLOGY: No
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw, animals exhibited the action of wetting the area around the mouth and nose, and rubbing it against the cage. Many animals exhibited diarrhea from the start of dosing as well as a suppressed state, several hours after dosing. Soft stool was observed in some animals dosed at 125 and 250 mg/kg bw, however, the animals showed recovery at low and mid doses.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals of all dose groups and female animals of high dose group (i.e. 500 mg/kg bw) exhibit body weight suppression.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals showed a dose dependent decline in the feed consumption.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Decreased feed efficacy was observed for male animals at 500 mg/kg bw (Table 1)The oragon.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Effects at 125 mg/kg/bw: Decreased total proteins (only females), albumin (only females), Ca2+ levels (only males), and increased cholesterol (only males).
Effects at 250 mg/kg bw: Decreased s-GPT (both males and females), total proteins (only females), albumin (only females), Na+ (only males), K+ (only males), Ca2+ (both males and females), and Mg2+ (only males).
Effects at 500 mg/kg bw: Decreased s-GOT (only females), total proteins (only females), albumin (only females), glucose (only females), Ca2+ (both males and females), and increased alkalinephosphatase (only males) and urea nitrogen (only females).
Urinalysis findings:
no effects observed
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):
The organ weight findings data are presented in Table 2.
The relative liver weight of female animals was significantly increased at 500 mg/kg bw (p<0.01). Apart from this, increase in relative weight of adrenal glands (p<0.05 for right side; p<0.01 for left side), testes (p<0.05) and brain (p<0.05) was also observed in male animals at 500 mg/kg bw. Decrease in the relative weight of liver (only females; at 125 mg/kg bw, p<0.05), right side kidney (only females; at 250 and 500 mg/kg bw, p<0.05), spleen (only males; at 500 mg/kg bw, p<0.05), heart (both males and females at 500 mg/kg bw, p<0.01; males at 250 mg/kg bw, p<0.05; females at 125 mg/kg bw, p<0.05) and thymus (only females; at 500 mg/kg bw, p<0.01) was also observed. Although, some significant effects were observed at the low and mid dose yet they were not dose-dependent.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 500 mg/kg bw, the animals showed hypertrophy in the stomach wall of the proventriculus, as if the process of inflammation and recovery had repeated over and over. Therefore, formation of new capillaries was observed on the peritoneal side. There were no additional changes relative to the control group although, events of hydronephrosis, bleeding plaques on the thymus and pneumonia were observed including the control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS: At 500 mg/kg bw, animals exhibited the action of wetting the area around the mouth and nose, and rubbing it against the cage. Many animals exhibited diarrhea from the start of dosing as well as a suppressed state, several hours after dosing. Soft stool was observed in some animals dosed at 125 and 250 mg/kg bw, however, the animals showed recovery at low and mid doses.

MORTALITY: No mortality was observed at any dose level.

BODY WEIGHT AND WEIGHT CHANGES: Body weight suppression was observed in animals (both males and females) belonging to the high dose group (i.e. 500 mg/kg bw).

FOOD CONSUMPTION AND COMPOUND INTAKE: Animals showed a dose dependent decline in the feed consumption. Decreased feed efficacy was observed for male animals at 500 mg/kg bw.

CLINICAL BIOCHEMISTRY: Few significant effects were observed in the biochemical parameters mainly at the mid and high dose level.

URINALYSIS: Results of urinalysis (pH, proteins, sugar, ketone bodies and occult blood) in the test animals were comparable to the control group.

ORGAN WEIGHT: The relative liver weight of female animals was significantly increased at 500 mg/kg bw (p<0.01). Apart from this, increase in relative weight of adrenal glands (p<0.05 for right side; p<0.01 for left side), testes (p<0.05) and brain (p<0.05) was also observed in male animals at 500 mg/kg bw. Decrease in the relative weight of liver (only females; at 125 mg/kg bw, p<0.05), right side kidney (only females; at 250 and 500 mg/kg bw, p<0.05), spleen (only males; at 500 mg/kg bw, p<0.05), heart (both males and females at 500 mg/kg bw, p<0.01; males at 250 mg/kg bw, p<0.05; females at 125 mg/kg bw, p<0.05) and thymus (only females; at 500 mg/kg bw, p<0.01) was also observed. Although, some significant effects were observed at the low and mid dose yet they were not dose-dependent.

GROSS PATHOLOGY: At 500 mg/kg bw, the animals showed hypertrophy in the stomach wall of the proventriculus, as if the process of inflammation and recovery had repeated over and over. Therefore, formation of new capillaries was observed on the peritoneal side. There were no additional changes relative to the control group although, events of hydronephrosis, bleeding plaques on the thymus and pneumonia were observed including the control group.

Key result
Dose descriptor:
NOAEL
Effect level:
125 other: mg/kg bw d
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
Remarks on result:
other: Based on reduced body weight and changes in organ weight (without histopathological correlation), clinical chemistry. The LOAEL was 250 mg/kg bw.
Critical effects observed:
not specified

Table 1: Average feed efficacy of LAS-Na for 1 month

 Sex  Doses (mg/kg)  Total intake (g)  Net gain (g)  Efficacy (%)  Total intake  Net gain  Efficacy (%)
 Male  0  346.5  108.8  31.4  747.6  199.1  25.6
   125  322.7  97.5  30.2  692.3  183.2  25.4
   250  316.4  92.8  29.4  690.2  177.5  24.6
   500  384.2  76.5  27.0  633.5  152.4  22.7
 Female  0  274.4  60.4  21.9  573.3  114.9  18.4
   125  236.6  53.7  22.6  513.1  105.4  19.5
   250  246.4  55.3  21.9  534.8  110.9  19.4
   500  231.7  49.6  21.4  486.5  97.8  18.0

Table 2: Average relative of wet-organ of LAS-Na.

Sex  Doses (mg/kg) Body weight (g)

 Liver

(wt / %)

  Kidney
 Right (wt / %)  Left (wt / %)
 Spleen (wt / %) Adrenal gland
 Right (wt / %)  Left (wt / %)
 Male  0  370 ±6.0  14.60±0.39 / 3.94±0.08  1.38 ±0.03 / 0.37 ±0.01 1.38 ±0.04 / 0.37 ±0.01  0.70 ±0.03 / 0.19 ±0.01 25.6 ±1.2 / 6.91 ±0.32  25.8 ±1.0 / 6.97 ±0.26
   125  355.1±7.5  13.72±0.33 / 3.87±0.06 1.36±0.04 / 0.39 ±0.01  1.33 ±0.04 / 0.38 ±0.01  0.68 ±0.03 / 0.19 ±0.01  24.4 ±1.8 / 6.88 ±0.48  25.3 ±1.1 / 7.16 ±0.37
   250  349.8*±7.6  13.65±0.42 / 3.90±0.09  1.31 ±0.03 / 0.38 ±0.01  1.29 ±0.03 / 0.37 ±0.01 0.62 ±0.03 / 0.18 ±0.01  24.7 ±1.4 / 7.06 ±0.34  25.4 ±1.5 / 7.24 ±0.35
   500  324.4**±9.0  13.37±0.57 / 4.11±0.11  1.21** ±0.04 / 0.37 ±0.01  1.20** ±0.04 / 0.37 ±0.01  0.53** ±0.02 / 0.16* ±0.01  26.2 ±1.0 / 8.15* ±0.40  28.5* ±0.6 / 8.88** ±0.36
 Female  0  262.6±8  10.84±0.51 / 4.11±0.08  1.09 ±0.04 / 0.41 ±0.01  1.08 ±0.05 / 0.41 ±0.02  0.55 ±0.02 / 0.21 ±0.01 34.0 ±1.2 / 13.00 ±0.38  35.8 ±1.3 / 13.63 ±0.30
   125  258.0±3.5  10.03±0.22 / 3.89* ±0.05 0.98* ±0.03 / 0.38* ±0.01 0.95* ±0.02 / 0.37* ±0.01  0.57 ±0.02 / 0.22 ±0.01  31.7 ±1.1 / 12.27 ±0.37  32.1 ±1.5 / 12.43 ±0.54
   250  261.5±4.5  10.56 ±0.30 / 4.04 ±0.08 1.00 ±0.03 / 0.38* ±0.01 0.96 ±0.03 / 0.37* ±0.01 0.56 ±0.03 / 0.22 ±0.01 31.0 ±1.8 / 11.94 ±0.84 33.4 ± 1.7 / 12.83 ± 0.76 
   500  249.7*±3.1  11.58 ±0.21 / 4.64** ±0.08  0.97** ±0.02 / 0.39* ±0.01 0.95* ±0.02 / 0.38 ±0.01 0.51 ±0.01 / 0.21 ±0.01  32.0 ±1.9 / 12.79 ±0.69 32.3 ± 2.0 / 12.89 ± 0.75 

Table 2: Average relative of wet-organ of LAS-Na (continued..)

Sex  Doses (mg/kg)

 Lung

(wt / %)

 Heart (wt / %) Testis/Ovarium
 Right (wt / %)  Left (wt / %)
 Thymus  Pituitary  Brain  
 Male  0  1.23±0.02 / 0.33±0.01 1.27 ±0.05 / 0.34 ±0.01  1.53 ±0.03 / 0.41 ±0.01 1.51±0.03 / 0.41 ±0.01  0.52 ±0.02 / 1.14 ±0.01   10.4±0.4 / 2.81 ±0.11   1.84±0.03 / 0.50 ±0.01    
   125  1.23±0.03 / 0.35±0.01  1.18 ±0.03 / 0.33±0.01  1.48 ±0.03 / 0.42 ±0.01  1.51 ±0.03 / 0.43 ±0.01 0.56 ±0.05 / 0.16 ±0.01   11.1±1.1 / 3.14 ±0.32    1.83±0.03 / 0.52 ±0.01    
   250  1.20±0.03 / 0.35±0.01  1.14 ±0.03 / 0.33* ±0.01 1.49 ±0.04 / 0.43 ±0.01  1.49 ±0.03/ 0.43 ±0.01  0.48 ±0.02 / 0.14 ±0.01   10.9±0.7 / 3.10 ±0.16    1.82±0.02 / 0.52 ±0.01    
   500  1.13*±0.03 / 0.35±0.01  0.97** ±0.03 / 0.30 ±0.01  1.49 ±0.04 / 0.46* ±0.02  1.49 ±0.04 / 0.46* ±0.02  0.41** ±0.03 / 0.13 ±0.01   9.6±0.4 / 2.99 ±0.16    1.77*±0.02 / 0.55* ±0.02    
 Female  0  1.07±0.03 / 0.41±0.01  0.96 ±0.03 / 0.37±0.01  44.8 ±2.9 / 17.04±0.95 47.8 ±3.5 / 18.13 ±1.07  0.59 ±0.02 / 0.23 ±0.01   11.8±0.4 / 4.53 ±0.18    1.80±0.02 / 0.69 ±0.02    
   125  1.10±0.05 / 0.43 ±0.02 0.88* ±0.01 / 0.34* ±0.01 46.2 ±2.5 / 17.94 ±1.04  43.7 ±1.8 / 16.93 ±0.64  0.54 ±0.02 / 0.21 ±0.01   12.3±0.8 / 4.79 ±0.30  
  1.78±0.02 / 0.69 ±0.01
   
   250  1.11 ±0.03 / 0.42 ±0.01 0.92 ±0.02 / 0.35 ±0.01 46.5±2.8 / 17.73 ±0.9 47.6 ±2.8 / 18.12 ±0.91 0.55 ± 0.03 / 0.21 ± 0.01  11.8±0.8 / 4.53 ±0.30    
  1.76±0.02 / 0.68 ±0.01
   
   500  1.01 ±0.02 / 0.41 ±0.01 0.79** ±0.03 / 0.32** ±0.01 46.3 ±3.5 / 18.51 ±1.29  46.2 ±2.7 / 18.44 ±0.97 0.44** ± 0.04 / 0.18** ± 0.02    11.8±0.9 / 4.71 ±0.34   
  1.75*±0.01 / 0.70 ±0.01
   

*p<0.05 **p<0.01

Conclusions:
Oral administration of C10-13 sodium linear alkylbenzene sulphonate (LAS-Na) to CRJ-SD rats at 0, 125, 250 and 500 mg/kg bw for 1 month resulted in a NOAEL of 125 mg/kg bw, based on reduced body weight, changes in organ weight (without histopathological correlation) and clinical chemistry at the mid and high doses. The LOAEL was 250 mg/kg bw.
Executive summary:

One month sub-acute oral toxicity study of sodium linear alkylbenzene sulphonate (LAS-Na) was performed in CRJ-SD rats. Five weeks old male and female CRJ-SD rats (obtained from Charles River Laboratories Japan, Inc.) were used in the study. Three males and 4 females were housed in the cages maintained under controlled environmental conditions (temperature: 23 ± 2°C and humidity: 55 ± 5%). Solid feed (CE-2, CLEA Japan, Inc.) and tap water were provided ad libitum. The substance was orally administered (via metallic gastric sonde) at 0 (distilled water), 125, 250 and 500 mg/kg bw (corresponding to 250, 500 and 1000 mg/mL) to male and females rats for 29 and 30 days, respectively. General symptoms of toxicity and the body weights were recorded daily while the food consumption

was measured twice a week. Urinalysis was also performed during the study. At the end of study, blood was collected for estimation of haematological and clinical chemistry parameters. After collection of blood samples, the animals were humanely euthanized, gross necropsy was performed and the organs weights determined. No mortality was observed at any dose level. Significant body weight loss was observed at 500 mg/kg bw. The relative liver weight of female animals was significantly increased at 500 mg/kg bw (p<0.01). Apart from this, increase in relative weight of adrenal glands (p<0.05 for right side; p<0.01 for left side), testes (p<0.05) and brain (p<0.05) was also observed in male animals at 500 mg/kg bw. Decrease in the relative weight of liver (only females; at 125 mg/kg bw, p<0.05), right side kidney (only females; at 250 and 500 mg/kg bw, p<0.05), spleen (only males; at 500 mg/kg bw, p<0.05), heart (both males and females at 500 mg/kg bw, p<0.01; males at 250 mg/kg bw, p<0.05; females at 125 mg/kg bw, p<0.05) and thymus (only females; at 500 mg/kg bw, p<0.01) was also observed. Although, some significant effects were observed at the low and mid dose yet they were not dose-dependent. Few significant effects were observed in the biochemical parameters mainly at the mid and high dose level. Based on the results, oral administration of C10-13 sodium linear alkylbenzene sulphonate (LAS-Na) to CRJ-SD rats at 0, 125, 250 and 500 mg/kg bw for 1 month resulted in a NOAEL of 125 mg/kg bw, based on reduced body weight, changes in organ weight (histopathology was not conducted) and clinical chemistry at the mid and high doses. The LOAEL was 250 mg/kg bw.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable study, followed scientific principles/standards, pre-dates GLP
Remarks:
This study is assigned a reliability score of 2 because the original report was not available for review. However, the study was evaluated by IPCS prior to inclusion in their criteria document.
Justification for type of information:
Read-across approach - see read-across justification in section 13
Reason / purpose:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study is to determine the sub-acute toxicity of C10-13 sodium linear alkyl benzene sulfonate (LAS-Na) in CRJ-SD rats. LAS-Na was orally administered at 0 (distilled water), 125, 250 and 500 mg/kg bw to male and females rats for 29 and 30 days, respectively. General symptoms of toxicity and the body weights were recorded daily while the food consumption was measured twice a week. Urinalysis was also performed during the study. At the end of study, blood was collected for estimation of haematological and clinical chemistry parameters. After collection of blood samples, the animals were humanely euthanized, gross necropsy was performed and organs weights were determined.
GLP compliance:
no
Remarks:
Pre-dates GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: Not reported
- Fasting period before study: Not reported
- Housing: 3 male and 4 female animals per cage.
- Diet: Solid feed (CE-2, CLEA Japan, Inc.); ad libitum
- Water: Tap water; ad libitum
- Acclimation period: After 1 week pre-housing, the animals were used in the experiment.
ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2°C
- Humidity: 55 ± 5%
- Air changes: Not reported
- Photoperiod: Not reported
Route of administration:
oral: gavage
Details on route of administration:
The test substance was administered orally through a metallic gastric sonde.
Vehicle:
not specified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in distilled water prior to dosing.
- DOSE VOLUME: 5 mL/kg bw
- Concentration in vehicle: 0, 250, 500, and 1000 mg/mL respectively for administration of doses 0, 125, 250 and 500 mg/kg bw.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
29 days for males and 30 days for females
Frequency of treatment:
Once daily
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
In distilled water (corresponding to 250 mg/mL)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
In distilled water (corresponding to 500 mg/mL)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
In distilled water (corresponding to 1000 mg/mL)
No. of animals per sex per dose:
Test group: 12 animals/sex/dose group
Control group: 15 male/group and 16 female/group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose levels for this study were selected based on the results of acute toxicity study conducted with LAS-Na in rats. 1/3 of the LD50 levels of LAS-Na, i.e. 500 mg/kg bw, was selected as the highest dose. The other two doses were set at common ratios of 2, whereby the medium and low doses were 250 (1/6 LD50) and 125 (1/12 LD50) mg/kg bw, respectively.
Positive control:
No.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption: Yes
- Time schedule for examinations: Twice/week

FOOD EFFICIENCY: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 1 month, blood was collected was collected from the carotid artery using a cannula.
- Anesthetic used for blood collection: Yes, light ether anesthesia was used.
- Animals fasted: Not reported
- How many animals: All surviving animals
- Parameters: Hemoglobin, hematocrit, red and white blood cell count were measured using an autoanalyzer SMA-4A.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 1 month, blood was collected from each animal and centrifuged (at 3000 rpm for 15 minutes) to obtain the serum samples for analysis.
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: s-GOT, s-GPT, alkaline phosphatase, total protein, albumin, glucose, cholesterol, bilirubin, creatinine, urea nitrogen, inorganic phosphates and ions (Na+, K+, Ca2+, Mg2+ and Cl-)

URINALYSIS: Yes
- Time schedule: Not reported
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: pH, proteins, sugar, ketone bodies and occult blood

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: The wet weight of liver, kidneys, spleen, adrenal glands, lungs, heart, thymus, reproductive organ, pituitary and the brain were measured. Based on this, the relative weights were calculated.

HISTOPATHOLOGY: No
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw, animals exhibited the action of wetting the area around the mouth and nose, and rubbing it against the cage. Many animals exhibited diarrhea from the start of dosing as well as a suppressed state, several hours after dosing. Soft stool was observed in some animals dosed at 125 and 250 mg/kg bw, however, the animals showed recovery at low and mid doses.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals of all dose groups and female animals of high dose group (i.e. 500 mg/kg bw) exhibit body weight suppression.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals showed a dose dependent decline in the feed consumption.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Decreased feed efficacy was observed for male animals at 500 mg/kg bw (Table 1)The oragon.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Effects at 125 mg/kg/bw: Decreased total proteins (only females), albumin (only females), Ca2+ levels (only males), and increased cholesterol (only males).
Effects at 250 mg/kg bw: Decreased s-GPT (both males and females), total proteins (only females), albumin (only females), Na+ (only males), K+ (only males), Ca2+ (both males and females), and Mg2+ (only males).
Effects at 500 mg/kg bw: Decreased s-GOT (only females), total proteins (only females), albumin (only females), glucose (only females), Ca2+ (both males and females), and increased alkalinephosphatase (only males) and urea nitrogen (only females).
Urinalysis findings:
no effects observed
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):
The organ weight findings data are presented in Table 2.
The relative liver weight of female animals was significantly increased at 500 mg/kg bw (p<0.01). Apart from this, increase in relative weight of adrenal glands (p<0.05 for right side; p<0.01 for left side), testes (p<0.05) and brain (p<0.05) was also observed in male animals at 500 mg/kg bw. Decrease in the relative weight of liver (only females; at 125 mg/kg bw, p<0.05), right side kidney (only females; at 250 and 500 mg/kg bw, p<0.05), spleen (only males; at 500 mg/kg bw, p<0.05), heart (both males and females at 500 mg/kg bw, p<0.01; males at 250 mg/kg bw, p<0.05; females at 125 mg/kg bw, p<0.05) and thymus (only females; at 500 mg/kg bw, p<0.01) was also observed. Although, some significant effects were observed at the low and mid dose yet they were not dose-dependent.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 500 mg/kg bw, the animals showed hypertrophy in the stomach wall of the proventriculus, as if the process of inflammation and recovery had repeated over and over. Therefore, formation of new capillaries was observed on the peritoneal side. There were no additional changes relative to the control group although, events of hydronephrosis, bleeding plaques on the thymus and pneumonia were observed including the control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS: At 500 mg/kg bw, animals exhibited the action of wetting the area around the mouth and nose, and rubbing it against the cage. Many animals exhibited diarrhea from the start of dosing as well as a suppressed state, several hours after dosing. Soft stool was observed in some animals dosed at 125 and 250 mg/kg bw, however, the animals showed recovery at low and mid doses.

MORTALITY: No mortality was observed at any dose level.

BODY WEIGHT AND WEIGHT CHANGES: Body weight suppression was observed in animals (both males and females) belonging to the high dose group (i.e. 500 mg/kg bw).

FOOD CONSUMPTION AND COMPOUND INTAKE: Animals showed a dose dependent decline in the feed consumption. Decreased feed efficacy was observed for male animals at 500 mg/kg bw.

CLINICAL BIOCHEMISTRY: Few significant effects were observed in the biochemical parameters mainly at the mid and high dose level.

URINALYSIS: Results of urinalysis (pH, proteins, sugar, ketone bodies and occult blood) in the test animals were comparable to the control group.

ORGAN WEIGHT: The relative liver weight of female animals was significantly increased at 500 mg/kg bw (p<0.01). Apart from this, increase in relative weight of adrenal glands (p<0.05 for right side; p<0.01 for left side), testes (p<0.05) and brain (p<0.05) was also observed in male animals at 500 mg/kg bw. Decrease in the relative weight of liver (only females; at 125 mg/kg bw, p<0.05), right side kidney (only females; at 250 and 500 mg/kg bw, p<0.05), spleen (only males; at 500 mg/kg bw, p<0.05), heart (both males and females at 500 mg/kg bw, p<0.01; males at 250 mg/kg bw, p<0.05; females at 125 mg/kg bw, p<0.05) and thymus (only females; at 500 mg/kg bw, p<0.01) was also observed. Although, some significant effects were observed at the low and mid dose yet they were not dose-dependent.

GROSS PATHOLOGY: At 500 mg/kg bw, the animals showed hypertrophy in the stomach wall of the proventriculus, as if the process of inflammation and recovery had repeated over and over. Therefore, formation of new capillaries was observed on the peritoneal side. There were no additional changes relative to the control group although, events of hydronephrosis, bleeding plaques on the thymus and pneumonia were observed including the control group.

Key result
Dose descriptor:
NOAEL
Effect level:
125 other: mg/kg bw d
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
Remarks on result:
other: Based on reduced body weight and changes in organ weight (without histopathological correlation), clinical chemistry. The LOAEL was 250 mg/kg bw.
Critical effects observed:
not specified

Table 1: Average feed efficacy of LAS-Na for 1 month

 Sex  Doses (mg/kg)  Total intake (g)  Net gain (g)  Efficacy (%)  Total intake  Net gain  Efficacy (%)
 Male  0  346.5  108.8  31.4  747.6  199.1  25.6
   125  322.7  97.5  30.2  692.3  183.2  25.4
   250  316.4  92.8  29.4  690.2  177.5  24.6
   500  384.2  76.5  27.0  633.5  152.4  22.7
 Female  0  274.4  60.4  21.9  573.3  114.9  18.4
   125  236.6  53.7  22.6  513.1  105.4  19.5
   250  246.4  55.3  21.9  534.8  110.9  19.4
   500  231.7  49.6  21.4  486.5  97.8  18.0

Table 2: Average relative of wet-organ of LAS-Na.

Sex  Doses (mg/kg) Body weight (g)

 Liver

(wt / %)

  Kidney
 Right (wt / %)  Left (wt / %)
 Spleen (wt / %) Adrenal gland
 Right (wt / %)  Left (wt / %)
 Male  0  370 ±6.0  14.60±0.39 / 3.94±0.08  1.38 ±0.03 / 0.37 ±0.01 1.38 ±0.04 / 0.37 ±0.01  0.70 ±0.03 / 0.19 ±0.01 25.6 ±1.2 / 6.91 ±0.32  25.8 ±1.0 / 6.97 ±0.26
   125  355.1±7.5  13.72±0.33 / 3.87±0.06 1.36±0.04 / 0.39 ±0.01  1.33 ±0.04 / 0.38 ±0.01  0.68 ±0.03 / 0.19 ±0.01  24.4 ±1.8 / 6.88 ±0.48  25.3 ±1.1 / 7.16 ±0.37
   250  349.8*±7.6  13.65±0.42 / 3.90±0.09  1.31 ±0.03 / 0.38 ±0.01  1.29 ±0.03 / 0.37 ±0.01 0.62 ±0.03 / 0.18 ±0.01  24.7 ±1.4 / 7.06 ±0.34  25.4 ±1.5 / 7.24 ±0.35
   500  324.4**±9.0  13.37±0.57 / 4.11±0.11  1.21** ±0.04 / 0.37 ±0.01  1.20** ±0.04 / 0.37 ±0.01  0.53** ±0.02 / 0.16* ±0.01  26.2 ±1.0 / 8.15* ±0.40  28.5* ±0.6 / 8.88** ±0.36
 Female  0  262.6±8  10.84±0.51 / 4.11±0.08  1.09 ±0.04 / 0.41 ±0.01  1.08 ±0.05 / 0.41 ±0.02  0.55 ±0.02 / 0.21 ±0.01 34.0 ±1.2 / 13.00 ±0.38  35.8 ±1.3 / 13.63 ±0.30
   125  258.0±3.5  10.03±0.22 / 3.89* ±0.05 0.98* ±0.03 / 0.38* ±0.01 0.95* ±0.02 / 0.37* ±0.01  0.57 ±0.02 / 0.22 ±0.01  31.7 ±1.1 / 12.27 ±0.37  32.1 ±1.5 / 12.43 ±0.54
   250  261.5±4.5  10.56 ±0.30 / 4.04 ±0.08 1.00 ±0.03 / 0.38* ±0.01 0.96 ±0.03 / 0.37* ±0.01 0.56 ±0.03 / 0.22 ±0.01 31.0 ±1.8 / 11.94 ±0.84 33.4 ± 1.7 / 12.83 ± 0.76 
   500  249.7*±3.1  11.58 ±0.21 / 4.64** ±0.08  0.97** ±0.02 / 0.39* ±0.01 0.95* ±0.02 / 0.38 ±0.01 0.51 ±0.01 / 0.21 ±0.01  32.0 ±1.9 / 12.79 ±0.69 32.3 ± 2.0 / 12.89 ± 0.75 

Table 2: Average relative of wet-organ of LAS-Na (continued..)

Sex  Doses (mg/kg)

 Lung

(wt / %)

 Heart (wt / %) Testis/Ovarium
 Right (wt / %)  Left (wt / %)
 Thymus  Pituitary  Brain  
 Male  0  1.23±0.02 / 0.33±0.01 1.27 ±0.05 / 0.34 ±0.01  1.53 ±0.03 / 0.41 ±0.01 1.51±0.03 / 0.41 ±0.01  0.52 ±0.02 / 1.14 ±0.01   10.4±0.4 / 2.81 ±0.11   1.84±0.03 / 0.50 ±0.01    
   125  1.23±0.03 / 0.35±0.01  1.18 ±0.03 / 0.33±0.01  1.48 ±0.03 / 0.42 ±0.01  1.51 ±0.03 / 0.43 ±0.01 0.56 ±0.05 / 0.16 ±0.01   11.1±1.1 / 3.14 ±0.32    1.83±0.03 / 0.52 ±0.01    
   250  1.20±0.03 / 0.35±0.01  1.14 ±0.03 / 0.33* ±0.01 1.49 ±0.04 / 0.43 ±0.01  1.49 ±0.03/ 0.43 ±0.01  0.48 ±0.02 / 0.14 ±0.01   10.9±0.7 / 3.10 ±0.16    1.82±0.02 / 0.52 ±0.01    
   500  1.13*±0.03 / 0.35±0.01  0.97** ±0.03 / 0.30 ±0.01  1.49 ±0.04 / 0.46* ±0.02  1.49 ±0.04 / 0.46* ±0.02  0.41** ±0.03 / 0.13 ±0.01   9.6±0.4 / 2.99 ±0.16    1.77*±0.02 / 0.55* ±0.02    
 Female  0  1.07±0.03 / 0.41±0.01  0.96 ±0.03 / 0.37±0.01  44.8 ±2.9 / 17.04±0.95 47.8 ±3.5 / 18.13 ±1.07  0.59 ±0.02 / 0.23 ±0.01   11.8±0.4 / 4.53 ±0.18    1.80±0.02 / 0.69 ±0.02    
   125  1.10±0.05 / 0.43 ±0.02 0.88* ±0.01 / 0.34* ±0.01 46.2 ±2.5 / 17.94 ±1.04  43.7 ±1.8 / 16.93 ±0.64  0.54 ±0.02 / 0.21 ±0.01   12.3±0.8 / 4.79 ±0.30  
  1.78±0.02 / 0.69 ±0.01
   
   250  1.11 ±0.03 / 0.42 ±0.01 0.92 ±0.02 / 0.35 ±0.01 46.5±2.8 / 17.73 ±0.9 47.6 ±2.8 / 18.12 ±0.91 0.55 ± 0.03 / 0.21 ± 0.01  11.8±0.8 / 4.53 ±0.30    
  1.76±0.02 / 0.68 ±0.01
   
   500  1.01 ±0.02 / 0.41 ±0.01 0.79** ±0.03 / 0.32** ±0.01 46.3 ±3.5 / 18.51 ±1.29  46.2 ±2.7 / 18.44 ±0.97 0.44** ± 0.04 / 0.18** ± 0.02    11.8±0.9 / 4.71 ±0.34   
  1.75*±0.01 / 0.70 ±0.01
   

*p<0.05 **p<0.01

Conclusions:
Oral administration of C10-13 sodium linear alkylbenzene sulphonate (LAS-Na) to CRJ-SD rats at 0, 125, 250 and 500 mg/kg bw for 1 month resulted in a NOAEL of 125 mg/kg bw, based on reduced body weight, changes in organ weight (without histopathological correlation) and clinical chemistry at the mid and high doses. The LOAEL was 250 mg/kg bw.
Executive summary:

One month sub-acute oral toxicity study of sodium linear alkylbenzene sulphonate (LAS-Na) was performed in CRJ-SD rats. Five weeks old male and female CRJ-SD rats (obtained from Charles River Laboratories Japan, Inc.) were used in the study. Three males and 4 females were housed in the cages maintained under controlled environmental conditions (temperature: 23 ± 2°C and humidity: 55 ± 5%). Solid feed (CE-2, CLEA Japan, Inc.) and tap water were provided ad libitum. The substance was orally administered (via metallic gastric sonde) at 0 (distilled water), 125, 250 and 500 mg/kg bw (corresponding to 250, 500 and 1000 mg/mL) to male and females rats for 29 and 30 days, respectively. General symptoms of toxicity and the body weights were recorded daily while the food consumption

was measured twice a week. Urinalysis was also performed during the study. At the end of study, blood was collected for estimation of haematological and clinical chemistry parameters. After collection of blood samples, the animals were humanely euthanized, gross necropsy was performed and the organs weights determined. No mortality was observed at any dose level. Significant body weight loss was observed at 500 mg/kg bw. The relative liver weight of female animals was significantly increased at 500 mg/kg bw (p<0.01). Apart from this, increase in relative weight of adrenal glands (p<0.05 for right side; p<0.01 for left side), testes (p<0.05) and brain (p<0.05) was also observed in male animals at 500 mg/kg bw. Decrease in the relative weight of liver (only females; at 125 mg/kg bw, p<0.05), right side kidney (only females; at 250 and 500 mg/kg bw, p<0.05), spleen (only males; at 500 mg/kg bw, p<0.05), heart (both males and females at 500 mg/kg bw, p<0.01; males at 250 mg/kg bw, p<0.05; females at 125 mg/kg bw, p<0.05) and thymus (only females; at 500 mg/kg bw, p<0.01) was also observed. Although, some significant effects were observed at the low and mid dose yet they were not dose-dependent. Few significant effects were observed in the biochemical parameters mainly at the mid and high dose level. Based on the results, oral administration of C10-13 sodium linear alkylbenzene sulphonate (LAS-Na) to CRJ-SD rats at 0, 125, 250 and 500 mg/kg bw for 1 month resulted in a NOAEL of 125 mg/kg bw, based on reduced body weight, changes in organ weight (histopathology was not conducted) and clinical chemistry at the mid and high doses. The LOAEL was 250 mg/kg bw.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 30th 1972 - November 27th 1972
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable study, followed scientific principles/standards, pre-dates GLP
Remarks:
This study is assigned a reliability score of 2 because the original report was not available for review. However, the study was evaluated by IPCS prior to inclusion in their criteria document.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study is to determine the sub chronic toxicity of C10 - C14 linear alkylbenzenesulfonic acid sodium salt (LAS) in Wistar SLC rats. Male and female rats were maintained on diets of 0, 0.07, 0.2, 0.6 and 1.8% LAS (equivalent to 0, 40, 115, 340, 1030 mg/kg bw/day) for 26 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. Urinalysis was also performed 2 weeks before necropsy. All the surviving animals were humanely euthanized and gross necropsy, organs weights and histopathology was performed.
GLP compliance:
no
Remarks:
Pre-dates GLP
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
SLC
Sex:
male/female
Details on test animals and environmental conditions:
Source: Male and female were obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals
- Age at study initiation: Approximately 4 weeks
- Weight at study initiation: 80 - 90 g (male rats), 65 - 80 g (female rats)
- Fasting period before study: No
- Housing: 5 animals were housed per stainless steel cage.
- Diet: FII food (from Funabashi Farm); ad libitum
- Water: Tap water; ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature: 25 ± 1°C
- Humidity: 55 - 65%
- Air changes: Not reported
- Photoperiod: 12 hours dark /12 hours light
Route of administration:
oral: feed
Details on route of administration:
C10 - C14 LAS was administered to animals by mixing 0.07, 0.2, 0.6 and 1.8% with pulverized FII feed, the powders were shaped into solid form before being freely provided for feeding along with tap water. As control, solid FII feed was provided to the animals.
Vehicle:
unchanged (no vehicle)
Remarks:
C10 - C14 LAS was administered in diet
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
26 weeks
Frequency of treatment:
Continuous in diet (ad libitum)
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
In diet (corresponding to 0.07% dose level)
Dose / conc.:
115 mg/kg bw/day (nominal)
Remarks:
In diet (corresponding to 0.2% dose level)
Dose / conc.:
340 mg/kg bw/day (nominal)
Remarks:
In diet (corresponding to 0.6% dose level)
Dose / conc.:
1 030 mg/kg bw/day (nominal)
Remarks:
In diet (corresponding to 1.8% dose level)
No. of animals per sex per dose:
10 animals/sex/dose group
Control animals:
yes, plain diet
Positive control:
No.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Weekly

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 26 weeks, blood was collected from each animal prior to autopsy (for hemoglobin, hematocrit, red blood cells, white blood fractions and platelets) and during e
uthanasia (erythrocyte membrane resistance)
- Anesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: Hemoglobin, hematocrit, red blood cells, white blood fractions and platelets, erythrocyte membrane resistance and blood coagulation time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 26 weeks, blood was collected from each animal after euthanasia.
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: Total proteins, GOT activity, ALP activity, LAP activity, urea nitrogen, A/G ratio and bilirubin

URINALYSIS: Yes
- Time schedule: 2 weeks prior to autopsy, urine isolated and collected from waste was analyzed.
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: Proteins, glucose, ketone bodies, occult blood, bilirubin and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross findings were obtained after euthanizing animals and macroscopic findings were observed.

ORGAN WEIGHTS: Brain, pituitary, thyroid, thymus, heart, lungs, liver, kidneys, spleen, adrenal gland, prostate, testes, uterus, ovaries and appendix

HISTOPATHOLOGY: Yes
Collected tissues (all tissues weighed and also stomach, large and small intestines, pancreas, epidiymis, skin and bones) were examined and subjected to microscopy through H.E., PAS and silver staining.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Severe diarrhea was observed in the high concentration group (1.8%).
Mortality:
mortality observed, treatment-related
Description (incidence):
In 24th week, 1 male animal from the 1.8% dose group died.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was significant inhibition of weight gain in both males and females of 1.8% dose group (Table 1).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was low in the 1.8% dose group relative to the control (Table 1).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was slightly low in animals of 1.8% dose group (Table 1).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In females of 1.8% dose group, a significant decrease in hematocrit and hemoglobin and significant increase in the erythrocyte membrane resistance was observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There was dose dependent significant increase in ALP levels (at mid and high dose) and significant decrease in total protein (at high dose).
Urinalysis findings:
no effects observed
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):
Organ weight findings are presented in Table 2.

1.8% dose group: Significant increase in relative weights of liver, suprarenal gland, testis, brain and caecum were observed in males and relative weights of heart, thyroid, liver, uterus, brain and caecum of females.

0.6% dose group: Significant increase in caecum relative weights in males and hypophysis in females.

0.2% dose group: Caecum relative weights were significantly increased in males and kidney relative weights were significantly decreased in females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant changes were observed in the animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The main findings were concerned with kidneys, liver and intestinal tract. Kidney damage was seen at and above 0.2% dose groups. Mild hepatocyte changes were observed in animals of 1.8% dose group and microscopic changes in intestinal tract were observed in female rats of the 1.8 and 0.6% dose groups.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS:
- Severe diarrhea was observed in animals in the high concentration groups (1.8%) immediately after starting the study, and although the diarrhea gradually became milder after about 1 week, the animals did not recover fully by the end of the study. The animals moved slowly, and there lower abdomen was dirty.

MORTALITY: During week 24, 1 male animal from 1.8% dose group died.

BODY WEIGHT AND WEIGHT CHANGES: There was significant inhibition of weight gain in both males and females of 1.8% dose group, while the 0.6% dose groups showed slight but not significant trends to suppress weight gain.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was low in the 1.8% dose group relative to the control.

WATER CONSUMPTION AND COMPOUND INTAKE: Water consumption was slightly low in the 1.8% dose group.

HAEMATOLOGY:
- A significant decrease in hematocrit and hemoglobin, a significant increase in the erythrocyte membrane resistance was observed among female animals in the 1.8% dose groups.
- Male and female rats in 1.8% dose group showed increase in white blood cell count, increase in neutrophil fraction out of the white blood cells and a decrease in lymphocytes.
CLINICAL BIOCHEMISTRY: There was dose dependent increase in ALP levels and decrease in total protein.

URINALYSIS: No effects were observed.

ORGAN WEIGHT:
- In highest concentration group (1.8%), significant increase in relative weights of liver, suprarenal gland, testis, brain and caecum were observed in males and relative weights of heart, thyroid, liver, uterus, brain and caecum of females. In 0.6% dose group, there was significant increase in caecum relative weights in males and hypophysis in females. In 0.2% dose group, caecum relative weights were significantly increased in males and kidney relative weights were significantly decreased in females.

GROSS PATHOLOGY
- The color of the liver in 2 males and 5 females from the 1.8% dose group was lighter than normal and seemed to have a yellowish white color mixed to it.
- In addition, lung tumors were seen in one female rat from the 0.2% dose group.
- Autopsy of one male rat from the LAS-1.8% dose group that died during the study had significant swelling of the abdomen. This was due to the swelling of the stomach and small intestines. The contents from the stomach to the first part of the jejunum was a blackish purple liquid. From thereon, the ileum was filled with a semi-transparent, viscous, gelatinous substance.

HISTOPATHOLOGY
The main findings were concerned with kidneys, liver and intestinal tract. The histological findings concerning the kidneys had differences in the extent and frequency of changes between the dosage and between male and female rats.
Kidneys:
- Chronic appearance of glomeruli indicating moderate level of atrophy (male/females of 0.6% treated groups)
- Glomeruli contributed to overall swelling through dilation of vessel lumen and interstitial swelling (significant in in male/female rats of 0.2 and 0.07% dose groups. They were not very significant in the 1.8 and 0.6%)
- The glomerular interstitum had thawn mildly but chronically (significant for male/female rats in 1.8 and 0.6% groups)
- Cloudy swelling/vacuolar degeneration of proximal renal tubular epithelial cells were seen (slightly significant in LAS treated groups).
- Small, yellowish brown blobs the size of nuclei were observed in the proximal renal tubular epithelial cells (significantly in male/females of 1.8% dose group)
- Blue lead color exhibited on the lumen of the Henle loop through H.E. staining, while substances giving a strong positive PAS response were forming layers in circular forms or were present in small pieces
- The renal tubule had collapsed (significantly in male/females of 1.8% dose group)
- Swelling of surrounding connective tissue was observed in the interstitium (significantly in males of LAS-0.2% dose group and both male and female rats of 0.6% dose group)
- Various changes to the kidneys as seen in 0.2% to 0.07% dose groups differ in their extent and frequency of incidence, but these changes were also seen in the control group, so they were not considered changes that lead to permanent damages like collapse of renal tubule or cyst formation.
Liver: Disappearance of basophilic substances in the hepatocytes and eosin thickening of hepatocytes were observed mildly in male and female rats of the 1.8% dose group.
Intestinal tract: Decrease in height of epithelial cells in the colonic mucosa and disappearance of vacuoles from goblet cells were observed mildly in female rats of the 1.8 and 0.6% dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Suppression of weight gain, increase in appendix weight, alteration in hematological and clin ical biochemistry parameters, and/ or tissue damages to the large intestines, liver and kidneys in dose levels at ≥ 115 mg/kg bw/day (0.2% diet).
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
115 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: body weight gain, food, water intake and feces excreation

 Sex Group (%) No. of animals  Initial BW (g)  Final BW (g)  BW gain (%) Food (g)/animal/day Water (g)/animal/day Feces (g)/animal/day 
 Male  0  10  85.00±0.87  387.00±9.50  452.10±9.8  14.17  16.90  2.31
   0.07  10  80.90±1.92   387.10±5.93   480.74±13.11  14.48  16.91  2.24
   0.2  10  84.50±0.95   383.40±6.71   453.92±7.60  13.70  18.18  2.28
   0.6  10  86.40±1.54  365.80±8.51  426.23±8.89  13.84  16.28  2.23
   1.8  9/10   85.20±0.55   290.00±5.20   341.09±5.13**  9.84  13.29  1.78
 Female  0  10  71.80±0.51  202.50±2.32  282.24±4.42  9.35  17.48  1.53
   0.07  10   76.00±2.68   203.20±7.17  269.35±10.25  9.35  19.56  1.63
   0.2  10   74.30±1.11   201.10±3.33   271.05±5.31  8.90  17.69  1.39
   0.6  10   74.10±0.67  195.80±4.15  264.25±5.2*  9.04  13.99  1.53
   1.8  10   74.30±0.94  158.80±3.41  214.02±5.29**  7.01  12.14  1.23

* p<0.05 ** p<0.01

Table 2: Organ weight observations

 Group (%)  No. of animals  Spleen (mg) / (mg/100g) Thymus  (mg) / (mg/100g)   Liver  (g) / (g/100g)    Lung  (mg) / (mg/100g) Kidney (R) (mg) / (mg/100g)    Kidney (L)  (mg) / (mg/100g) Hypophysis  (mg/100g)   Heart (mg) / (mg/100g)
 0 10 M  614±14 /  158±2  129±5.7 /  33±0.9    12.0±0.38 /  3.1±0.04   1211±44 /  314±13   1105±33 /  285±6    1126±33 /  219±6    10.0±0.73 /  2.6±0.21    930±13 /  241±3
 0.07  10 M   641±12 /  165±2   129±10.8 /  33±2.5   12.0±0.31 /  3.1±0.05    1285±47 /  332±12   1074±25 /  277±5    1083±52 /  279±12    9.3±0.50 /  2.4±0.13   932±16 /  240±2
 0.2  10 M  644±27 /  168±7    130±9.0 /  33±2.1   12.3±0.42 /  3.2±0.12   1253±63 /  326±14    1083±34 /  282±7    1071±28 /  279±4   8.9±0.48 /  2.3±0.13   918±16 /  239±2
 0.6  10 M  572±15 /  153±4    122±8.8 /  32±2.3   12.0±0.31 /  3.2±0.07   1080±21* /  289±6    1045±31 /  280±9    1049±34 /  281±9   9.4±0.30 /  2.5±0.08   878±18* /  235±4
 1.8  9 M  460±11** /  158±2    102±6.3** /  35±1.7   10.3±0.28** /  3.5±0.08**   971±35** /  324±13    836±19** /  288±3    852±22** /  294±5  8.8±0.42 /  3.0±0.15   694±11** /  239±3
                   
 0  10 F   405±5 /  200±4   134±7.7 /  66±3.9   6.5±0.12 /  3.2±0.08   822±19 /  409±11    720±22 /  359±12   738±18 /  395±11   13.9±0.76 /  6.8±0.39   596±10 /  265±6
 0.07  10 F   408±13 /  201±4   132±2.8 /  65±2.3   6.7±0.30 /  3.3±0.05   801±19 /  396±11   717±23 /  355±13     738±33 /  364±13   16.3±0.94 /  8.0±0.40   596±16 /  294±3
 0.2  10 F   405±8 /  201±4   131±4.6 /  65±2.6   6.5±0.18 /  3.2±0.07   802±26 /  398±10   654±20* /  325±7     665±28* /  330±11*   15.0±1.10 /  7.4±0.53   574±13 /  258±5
 0.9  10 F   387±7 /  198±5   125±5.8 /  63±2.2   6.5±0.22 /  3.3±0.10   793±14 /  406±8   664±16 /  340±9     715±32 /  365±14   16.2±0.94 /  8.3±0.46*   559±11* /  286±5
 1.8  10 F   294±10** /  185±6   102±6.8** /  64±4.1   6.3±0.24 /  3.9±0.12**   673±20** /  425±16   546±16** /  345±13     563±24** /  357±21   12.3±1.08 /  7.7±0.63   426±11** /  269±7*

* p<0.05 ** p<0.01

Table 2: Organ weight observations (continued..)

 Group (%)  No. of animals  Thyroid glands (mg) / (mg/100g) Suprarenal gland (R)  (mg) / (mg/100g)    Suprarenal gland (L)  (mg) / (mg/100g)  Testis/Ovary (R)  (mg) / (mg/100g) Testis/Ovary (L)  (mg) / (mg/100g) Prosate/Uterus  (mg) / (mg/100g) Brain (mg) /  (mg/100g) Caecum (mg) /  (mg/100g)
 0 10 M  14.4±0.42 /  3.7±0.16  18.0±0.61 /  5.0±0.17  19.3±0.44 /  4.6±0.18  1592±20 /  412±7  1622±21 /  420±6  252±16 /  65±4.8 1983±12 /  515±11 837±34 /  216±8
 0.07  10 M  13.6±0.54 /  3.5±0.13  18.7±0.44 /  4.8±0.11   12.0±0.31 /  3.1±0.05  1643±14 /  425±6   1676±15 /  434±7  318±21* /  82±6.1  1918±106 /  494±25 938±38 /  242±9
 0.2  10 M  13.3±0.57 /  3.4±0.11  18.6±0.84 /  4.8±0.23   12.3±0.42 /  3.2±0.12  1621±20 /  423±4    1083±34 /  282±7  298±21 /  78±5.9 2020±11 /  528±8 971±35* /  253±7**
 0.6  10 M  13.2±0.69 /  3.5±0.22  18.2±0.69 /  4.8±0.19   12.0±0.31 /  3.2±0.07  1612±36 /  432±11    1045±31 /  280±9  231±21 /  61±5.7  1969±18* /  528±12 939±41 /  251±11*
 1.8  9 M  11.5±0.76** /  3.9±0.24  17.1±0.65* /  5.9±0.27*   10.3±0.28** /  3.5±0.08**  1501±36** /  518±13**    836±19** /  288±3  231±21 /  79±7.0  1960±22 /  677±11** 1484±30** /  512±12**
                   
 0  9 F  11.2±0.32 /  5.5±0.17 21.9±0.99 /  10.8±0.64  23.2±1.24 /  11.4±0.94   23.3±1.0 /  11.5±0.5  24.4±1.1 /  12.0±0.5  638±27 /  315±13  1832±16 /  906±14   681±18 /  337±11
 0.07  10 F  12.9±0.93 /  6.4±0.55 22.6±0.95 /  11.1±0.35   24.1±0.98 /  11.8±0.33  23.0±1.9 /  11.2±0.6  24.6±1.5 /  12.0±0.5  592±18 /  292±8  1827±18 /  908±30   753±25* /  373±13
 0.2  10 F  12.2±0.96 /  6.0±0.46 21.7±1.11 /  10.7±0.49   22.2±0.92 /  11.0±0.43  23.6±1.0 /  11.7±0.4  23.3±1.2 /  11.5±0.5 594±31 /  296±17  1837±18 /  915±13   688±26 /  343±14
 0.9  10 F  13.9±1.30 /  7.1±0.70 22.9±0.56 /  11.7±0.36   24.0±0.61 /  12.3±0.44  22.8±1.2 /  11.7±0.7  24.5±1.0 /  12.6±0.6  618±32 /  315±15  1840±14 /  943±20   697±21 /  356±7
 1.8  10 F  10.9±0.73 /  6.9±0.53*  15.6±0.81** /  9.8±0.52   16.8±0.84** /  10.6±0.55  17.6±1.4** /  11.1±0.9  18.2±1.8** /  11.5±1.1  292±18** /  185±12**  1780±15* /  1126±28**  929±26** /  586±14** 

* p<0.05 ** p<0.01

Conclusions:
Administration of linear alkylbenzene sulfonic acid sodium salt (LAS) to Wistar SLC rats at dose levels of 0, 0.07, 0.2, 0.6 and 1.8% (equivalent to 0, 40, 115, 340, 1030 mg/kg bw/day) for 26 weeks revealed an NOAEL of 0.07% (40 mg/kg bw/day), based on tissue damage in caecum, kidney and liver, hematological and/or clinical chemistry parameters changes at higher dose levels (115 mg/kg bw/day and above).
Executive summary:

The 26 weeks sub-chronic oral toxicity study of linear alkylbenzene sulfonic acid sodium salt (LAS) was performed in Wistar SLC rats. Approximately 4 weeks old male and femaleWistar SLC rats(obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals) with body weight range 80 - 90 g (males), 65 - 80 g (females) were used in the study. Five animals were housed in each stainless steelcage and maintained under controlled environmental conditions (temperature: Average of 25 ± 1°C, humidity:55 - 65%, and 12 hours light /12 hours dark). FII food (from Funabashi Farm) and tap water were provided ad libitum. The animals were administered daily with the LAS in diet at dose levels of 0, 0.07, 0.2, 0.6 and 1.8%

(equivalent to 0,40, 115, 340, 1030 mg/kg bw/day) for 26 weeks. 10 animals/sex/dose group was taken in the study.

Clinical observations, water consumption, food consumption and body weights were recorded weekly. At the end of study, haematological, clinical chemistry and urine parameters were analysed. Gross findings were obtained after euthanizing animals and organs were removed for organ weight measurements.Organ weights for brain, pituitary, thyroid, thymus, heart, lungs, liver, kidneys, spleen, adrenal gland, prostate, testes, uterus, ovaries and appendixwere recorded. Liver and kidney enzymes

were also analysed. Collected tissues were then examined and subjected to microscopy through H.E., PAS and silver staining. One male animal from the 1.8% dose group died. In the 1.8% dose groups, increase in relative weight of organs associated with diarrhea and significant suppression of weight gain has been remarked. There was also anemia and an increasing trend of white blood cell count, in addition to increased serum ALP activity, decrease in serum total protein and a very high level of tissue damage in the kidneys. In the 0.6% dose groups, there were mild suppression of weight gain, increased weight of appendix as well as fluctuations in serum ALP / total protein and tissue damage to the kidneys. In the 0.2% dose groups, there were increase in weight of appendix and histological abnormalities in a part of the kidneys. In the 0.2% dose groups, there were increase in weight of appendix and histological abnormalities in a part of the kidneys. Various changes to the kidneys as seen in 0.2 to 0.07% dose groups differ in their extent and frequency of incidence, but these changes were also seen in the control group, so they were not considered changes that lead to permanent damages like collapse of renal tubule or cyst formation. Administration of linear alkylbenzene sulfonic acid sodium salt (LAS) to Wistar SLC ratsat dose levels of 0, 0.07, 0.2, 0.6 and 1.8% (equivalent to 0,40, 115, 340, 1030 mg/kg bw/day) for 26 weeks identified a NOAEL of 0.07% (40 mg/kg bw/day), based on tissue damage in caecum, kidney and liver, hematological as well as clinical chemistry parameters changes at higher dose levels (115 mg/kg bw/day and above).

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable study, followed scientific principles/standards, pre-dates GLP
Remarks:
This study is assigned a reliability score of 2 because the original report was not available for review. However, the study was evaluated by IPCS prior to inclusion in their criteria document.
Justification for type of information:
Read-across approach - see read-across justification in section 13.
Reason / purpose:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study is to determine the sub chronic toxicity of C10 - C14 linear alkylbenzenesulfonic acid sodium salt (LAS) in Wistar SLC rats. Male and female rats were maintained on diets of 0, 0.07, 0.2, 0.6 and 1.8% LAS (equivalent to 0, 40, 115, 340, 1030 mg/kg bw/day) for 26 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. Urinalysis was also performed 2 weeks before necropsy. All the surviving animals were humanely euthanized and gross necropsy, organs weights and histopathology was performed.
GLP compliance:
no
Remarks:
Pre-dates GLP
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
SLC
Sex:
male/female
Details on test animals and environmental conditions:
Source: Male and female were obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals
- Age at study initiation: Approximately 4 weeks
- Weight at study initiation: 80 - 90 g (male rats), 65 - 80 g (female rats)
- Fasting period before study: No
- Housing: 5 animals were housed per stainless steel cage.
- Diet: FII food (from Funabashi Farm); ad libitum
- Water: Tap water; ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature: 25 ± 1°C
- Humidity: 55 - 65%
- Air changes: Not reported
- Photoperiod: 12 hours dark /12 hours light
Route of administration:
oral: feed
Details on route of administration:
C10 - C14 LAS was administered to animals by mixing 0.07, 0.2, 0.6 and 1.8% with pulverized FII feed, the powders were shaped into solid form before being freely provided for feeding along with tap water. As control, solid FII feed was provided to the animals.
Vehicle:
unchanged (no vehicle)
Remarks:
C10 - C14 LAS was administered in diet
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
26 weeks
Frequency of treatment:
Continuous in diet (ad libitum)
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
In diet (corresponding to 0.07% dose level)
Dose / conc.:
115 mg/kg bw/day (nominal)
Remarks:
In diet (corresponding to 0.2% dose level)
Dose / conc.:
340 mg/kg bw/day (nominal)
Remarks:
In diet (corresponding to 0.6% dose level)
Dose / conc.:
1 030 mg/kg bw/day (nominal)
Remarks:
In diet (corresponding to 1.8% dose level)
No. of animals per sex per dose:
10 animals/sex/dose group
Control animals:
yes, plain diet
Positive control:
No.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Weekly

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 26 weeks, blood was collected from each animal prior to autopsy (for hemoglobin, hematocrit, red blood cells, white blood fractions and platelets) and during e
uthanasia (erythrocyte membrane resistance)
- Anesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: Hemoglobin, hematocrit, red blood cells, white blood fractions and platelets, erythrocyte membrane resistance and blood coagulation time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 26 weeks, blood was collected from each animal after euthanasia.
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: Total proteins, GOT activity, ALP activity, LAP activity, urea nitrogen, A/G ratio and bilirubin

URINALYSIS: Yes
- Time schedule: 2 weeks prior to autopsy, urine isolated and collected from waste was analyzed.
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: Proteins, glucose, ketone bodies, occult blood, bilirubin and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross findings were obtained after euthanizing animals and macroscopic findings were observed.

ORGAN WEIGHTS: Brain, pituitary, thyroid, thymus, heart, lungs, liver, kidneys, spleen, adrenal gland, prostate, testes, uterus, ovaries and appendix

HISTOPATHOLOGY: Yes
Collected tissues (all tissues weighed and also stomach, large and small intestines, pancreas, epidiymis, skin and bones) were examined and subjected to microscopy through H.E., PAS and silver staining.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Severe diarrhea was observed in the high concentration group (1.8%).
Mortality:
mortality observed, treatment-related
Description (incidence):
In 24th week, 1 male animal from the 1.8% dose group died.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was significant inhibition of weight gain in both males and females of 1.8% dose group (Table 1).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was low in the 1.8% dose group relative to the control (Table 1).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was slightly low in animals of 1.8% dose group (Table 1).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In females of 1.8% dose group, a significant decrease in hematocrit and hemoglobin and significant increase in the erythrocyte membrane resistance was observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There was dose dependent significant increase in ALP levels (at mid and high dose) and significant decrease in total protein (at high dose).
Urinalysis findings:
no effects observed
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):
Organ weight findings are presented in Table 2.

1.8% dose group: Significant increase in relative weights of liver, suprarenal gland, testis, brain and caecum were observed in males and relative weights of heart, thyroid, liver, uterus, brain and caecum of females.

0.6% dose group: Significant increase in caecum relative weights in males and hypophysis in females.

0.2% dose group: Caecum relative weights were significantly increased in males and kidney relative weights were significantly decreased in females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant changes were observed in the animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The main findings were concerned with kidneys, liver and intestinal tract. Kidney damage was seen at and above 0.2% dose groups. Mild hepatocyte changes were observed in animals of 1.8% dose group and microscopic changes in intestinal tract were observed in female rats of the 1.8 and 0.6% dose groups.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS:
- Severe diarrhea was observed in animals in the high concentration groups (1.8%) immediately after starting the study, and although the diarrhea gradually became milder after about 1 week, the animals did not recover fully by the end of the study. The animals moved slowly, and there lower abdomen was dirty.

MORTALITY: During week 24, 1 male animal from 1.8% dose group died.

BODY WEIGHT AND WEIGHT CHANGES: There was significant inhibition of weight gain in both males and females of 1.8% dose group, while the 0.6% dose groups showed slight but not significant trends to suppress weight gain.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was low in the 1.8% dose group relative to the control.

WATER CONSUMPTION AND COMPOUND INTAKE: Water consumption was slightly low in the 1.8% dose group.

HAEMATOLOGY:
- A significant decrease in hematocrit and hemoglobin, a significant increase in the erythrocyte membrane resistance was observed among female animals in the 1.8% dose groups.
- Male and female rats in 1.8% dose group showed increase in white blood cell count, increase in neutrophil fraction out of the white blood cells and a decrease in lymphocytes.
CLINICAL BIOCHEMISTRY: There was dose dependent increase in ALP levels and decrease in total protein.

URINALYSIS: No effects were observed.

ORGAN WEIGHT:
- In highest concentration group (1.8%), significant increase in relative weights of liver, suprarenal gland, testis, brain and caecum were observed in males and relative weights of heart, thyroid, liver, uterus, brain and caecum of females. In 0.6% dose group, there was significant increase in caecum relative weights in males and hypophysis in females. In 0.2% dose group, caecum relative weights were significantly increased in males and kidney relative weights were significantly decreased in females.

GROSS PATHOLOGY
- The color of the liver in 2 males and 5 females from the 1.8% dose group was lighter than normal and seemed to have a yellowish white color mixed to it.
- In addition, lung tumors were seen in one female rat from the 0.2% dose group.
- Autopsy of one male rat from the LAS-1.8% dose group that died during the study had significant swelling of the abdomen. This was due to the swelling of the stomach and small intestines. The contents from the stomach to the first part of the jejunum was a blackish purple liquid. From thereon, the ileum was filled with a semi-transparent, viscous, gelatinous substance.

HISTOPATHOLOGY
The main findings were concerned with kidneys, liver and intestinal tract. The histological findings concerning the kidneys had differences in the extent and frequency of changes between the dosage and between male and female rats.
Kidneys:
- Chronic appearance of glomeruli indicating moderate level of atrophy (male/females of 0.6% treated groups)
- Glomeruli contributed to overall swelling through dilation of vessel lumen and interstitial swelling (significant in in male/female rats of 0.2 and 0.07% dose groups. They were not very significant in the 1.8 and 0.6%)
- The glomerular interstitum had thawn mildly but chronically (significant for male/female rats in 1.8 and 0.6% groups)
- Cloudy swelling/vacuolar degeneration of proximal renal tubular epithelial cells were seen (slightly significant in LAS treated groups).
- Small, yellowish brown blobs the size of nuclei were observed in the proximal renal tubular epithelial cells (significantly in male/females of 1.8% dose group)
- Blue lead color exhibited on the lumen of the Henle loop through H.E. staining, while substances giving a strong positive PAS response were forming layers in circular forms or were present in small pieces
- The renal tubule had collapsed (significantly in male/females of 1.8% dose group)
- Swelling of surrounding connective tissue was observed in the interstitium (significantly in males of LAS-0.2% dose group and both male and female rats of 0.6% dose group)
- Various changes to the kidneys as seen in 0.2% to 0.07% dose groups differ in their extent and frequency of incidence, but these changes were also seen in the control group, so they were not considered changes that lead to permanent damages like collapse of renal tubule or cyst formation.
Liver: Disappearance of basophilic substances in the hepatocytes and eosin thickening of hepatocytes were observed mildly in male and female rats of the 1.8% dose group.
Intestinal tract: Decrease in height of epithelial cells in the colonic mucosa and disappearance of vacuoles from goblet cells were observed mildly in female rats of the 1.8 and 0.6% dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Suppression of weight gain, increase in appendix weight, alteration in hematological and clin ical biochemistry parameters, and/ or tissue damages to the large intestines, liver and kidneys in dose levels at ≥ 115 mg/kg bw/day (0.2% diet).
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
115 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: body weight gain, food, water intake and feces excreation

 Sex Group (%) No. of animals  Initial BW (g)  Final BW (g)  BW gain (%) Food (g)/animal/day Water (g)/animal/day Feces (g)/animal/day 
 Male  0  10  85.00±0.87  387.00±9.50  452.10±9.8  14.17  16.90  2.31
   0.07  10  80.90±1.92   387.10±5.93   480.74±13.11  14.48  16.91  2.24
   0.2  10  84.50±0.95   383.40±6.71   453.92±7.60  13.70  18.18  2.28
   0.6  10  86.40±1.54  365.80±8.51  426.23±8.89  13.84  16.28  2.23
   1.8  9/10   85.20±0.55   290.00±5.20   341.09±5.13**  9.84  13.29  1.78
 Female  0  10  71.80±0.51  202.50±2.32  282.24±4.42  9.35  17.48  1.53
   0.07  10   76.00±2.68   203.20±7.17  269.35±10.25  9.35  19.56  1.63
   0.2  10   74.30±1.11   201.10±3.33   271.05±5.31  8.90  17.69  1.39
   0.6  10   74.10±0.67  195.80±4.15  264.25±5.2*  9.04  13.99  1.53
   1.8  10   74.30±0.94  158.80±3.41  214.02±5.29**  7.01  12.14  1.23

* p<0.05 ** p<0.01

Table 2: Organ weight observations

 Group (%)  No. of animals  Spleen (mg) / (mg/100g) Thymus  (mg) / (mg/100g)   Liver  (g) / (g/100g)    Lung  (mg) / (mg/100g) Kidney (R) (mg) / (mg/100g)    Kidney (L)  (mg) / (mg/100g) Hypophysis  (mg/100g)   Heart (mg) / (mg/100g)
 0 10 M  614±14 /  158±2  129±5.7 /  33±0.9    12.0±0.38 /  3.1±0.04   1211±44 /  314±13   1105±33 /  285±6    1126±33 /  219±6    10.0±0.73 /  2.6±0.21    930±13 /  241±3
 0.07  10 M   641±12 /  165±2   129±10.8 /  33±2.5   12.0±0.31 /  3.1±0.05    1285±47 /  332±12   1074±25 /  277±5    1083±52 /  279±12    9.3±0.50 /  2.4±0.13   932±16 /  240±2
 0.2  10 M  644±27 /  168±7    130±9.0 /  33±2.1   12.3±0.42 /  3.2±0.12   1253±63 /  326±14    1083±34 /  282±7    1071±28 /  279±4   8.9±0.48 /  2.3±0.13   918±16 /  239±2
 0.6  10 M  572±15 /  153±4    122±8.8 /  32±2.3   12.0±0.31 /  3.2±0.07   1080±21* /  289±6    1045±31 /  280±9    1049±34 /  281±9   9.4±0.30 /  2.5±0.08   878±18* /  235±4
 1.8  9 M  460±11** /  158±2    102±6.3** /  35±1.7   10.3±0.28** /  3.5±0.08**   971±35** /  324±13    836±19** /  288±3    852±22** /  294±5  8.8±0.42 /  3.0±0.15   694±11** /  239±3
                   
 0  10 F   405±5 /  200±4   134±7.7 /  66±3.9   6.5±0.12 /  3.2±0.08   822±19 /  409±11    720±22 /  359±12   738±18 /  395±11   13.9±0.76 /  6.8±0.39   596±10 /  265±6
 0.07  10 F   408±13 /  201±4   132±2.8 /  65±2.3   6.7±0.30 /  3.3±0.05   801±19 /  396±11   717±23 /  355±13     738±33 /  364±13   16.3±0.94 /  8.0±0.40   596±16 /  294±3
 0.2  10 F   405±8 /  201±4   131±4.6 /  65±2.6   6.5±0.18 /  3.2±0.07   802±26 /  398±10   654±20* /  325±7     665±28* /  330±11*   15.0±1.10 /  7.4±0.53   574±13 /  258±5
 0.9  10 F   387±7 /  198±5   125±5.8 /  63±2.2   6.5±0.22 /  3.3±0.10   793±14 /  406±8   664±16 /  340±9     715±32 /  365±14   16.2±0.94 /  8.3±0.46*   559±11* /  286±5
 1.8  10 F   294±10** /  185±6   102±6.8** /  64±4.1   6.3±0.24 /  3.9±0.12**   673±20** /  425±16   546±16** /  345±13     563±24** /  357±21   12.3±1.08 /  7.7±0.63   426±11** /  269±7*

* p<0.05 ** p<0.01

Table 2: Organ weight observations (continued..)

 Group (%)  No. of animals  Thyroid glands (mg) / (mg/100g) Suprarenal gland (R)  (mg) / (mg/100g)    Suprarenal gland (L)  (mg) / (mg/100g)  Testis/Ovary (R)  (mg) / (mg/100g) Testis/Ovary (L)  (mg) / (mg/100g) Prosate/Uterus  (mg) / (mg/100g) Brain (mg) /  (mg/100g) Caecum (mg) /  (mg/100g)
 0 10 M  14.4±0.42 /  3.7±0.16  18.0±0.61 /  5.0±0.17  19.3±0.44 /  4.6±0.18  1592±20 /  412±7  1622±21 /  420±6  252±16 /  65±4.8 1983±12 /  515±11 837±34 /  216±8
 0.07  10 M  13.6±0.54 /  3.5±0.13  18.7±0.44 /  4.8±0.11   12.0±0.31 /  3.1±0.05  1643±14 /  425±6   1676±15 /  434±7  318±21* /  82±6.1  1918±106 /  494±25 938±38 /  242±9
 0.2  10 M  13.3±0.57 /  3.4±0.11  18.6±0.84 /  4.8±0.23   12.3±0.42 /  3.2±0.12  1621±20 /  423±4    1083±34 /  282±7  298±21 /  78±5.9 2020±11 /  528±8 971±35* /  253±7**
 0.6  10 M  13.2±0.69 /  3.5±0.22  18.2±0.69 /  4.8±0.19   12.0±0.31 /  3.2±0.07  1612±36 /  432±11    1045±31 /  280±9  231±21 /  61±5.7  1969±18* /  528±12 939±41 /  251±11*
 1.8  9 M  11.5±0.76** /  3.9±0.24  17.1±0.65* /  5.9±0.27*   10.3±0.28** /  3.5±0.08**  1501±36** /  518±13**    836±19** /  288±3  231±21 /  79±7.0  1960±22 /  677±11** 1484±30** /  512±12**
                   
 0  9 F  11.2±0.32 /  5.5±0.17 21.9±0.99 /  10.8±0.64  23.2±1.24 /  11.4±0.94   23.3±1.0 /  11.5±0.5  24.4±1.1 /  12.0±0.5  638±27 /  315±13  1832±16 /  906±14   681±18 /  337±11
 0.07  10 F  12.9±0.93 /  6.4±0.55 22.6±0.95 /  11.1±0.35   24.1±0.98 /  11.8±0.33  23.0±1.9 /  11.2±0.6  24.6±1.5 /  12.0±0.5  592±18 /  292±8  1827±18 /  908±30   753±25* /  373±13
 0.2  10 F  12.2±0.96 /  6.0±0.46 21.7±1.11 /  10.7±0.49   22.2±0.92 /  11.0±0.43  23.6±1.0 /  11.7±0.4  23.3±1.2 /  11.5±0.5 594±31 /  296±17  1837±18 /  915±13   688±26 /  343±14
 0.9  10 F  13.9±1.30 /  7.1±0.70 22.9±0.56 /  11.7±0.36   24.0±0.61 /  12.3±0.44  22.8±1.2 /  11.7±0.7  24.5±1.0 /  12.6±0.6  618±32 /  315±15  1840±14 /  943±20   697±21 /  356±7
 1.8  10 F  10.9±0.73 /  6.9±0.53*  15.6±0.81** /  9.8±0.52   16.8±0.84** /  10.6±0.55  17.6±1.4** /  11.1±0.9  18.2±1.8** /  11.5±1.1  292±18** /  185±12**  1780±15* /  1126±28**  929±26** /  586±14** 

* p<0.05 ** p<0.01

Conclusions:
Administration of linear alkylbenzene sulfonic acid sodium salt (LAS) to Wistar SLC rats at dose levels of 0, 0.07, 0.2, 0.6 and 1.8% (equivalent to 0, 40, 115, 340, 1030 mg/kg bw/day) for 26 weeks revealed an NOAEL of 0.07% (40 mg/kg bw/day), based on tissue damage in caecum, kidney and liver, hematological and/or clinical chemistry parameters changes at higher dose levels (115 mg/kg bw/day and above).
Executive summary:

The 26 weeks sub-chronic oral toxicity study of linear alkylbenzene sulfonic acid sodium salt (LAS) was performed in Wistar SLC rats. Approximately 4 weeks old male and femaleWistar SLC rats(obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals) with body weight range 80 - 90 g (males), 65 - 80 g (females)were used in the study. Five animals were housed in each stainless steelcage and maintained under controlled environmental conditions (temperature: Average of 25 ± 1°C, humidity:55 - 65%, and 12 hours light /12 hours dark). FII food (from Funabashi Farm)and tap water were provided ad libitum. The animals were administered daily with the LAS in diet at dose levels of0, 0.07, 0.2, 0.6 and 1.8%

(equivalent to 0,40, 115, 340, 1030 mg/kg bw/day) for 26 weeks. 10 animals/sex/dose group was taken in the study.

Clinical observations, water consumption, food consumption and body weights were recorded weekly. At the end of study, haematological, clinical chemistry and urine parameters were analysed. Gross findings were obtained after euthanizing animals and organs were removed for organ weight measurements.Organ weights for brain, pituitary, thyroid, thymus, heart, lungs, liver, kidneys, spleen, adrenal gland, prostate, testes, uterus, ovaries and appendixwere recorded. Liver and kidney enzymes

were also analysed. Collected tissues were then examined and subjected to microscopy through H.E., PAS and silver staining. One male animal from the 1.8% dose group died. In the 1.8% dose groups, increase in relative weight of organs associated with diarrhea and significant suppression of weight gain has been remarked. There was also anemia and an increasing trend of white blood cell count, in addition to increased serum ALP activity, decrease in serum total protein and a very high level of tissue damage in the kidneys. In the 0.6% dose groups, there were mild suppression of weight gain, increased weight of appendix as well as fluctuations in serum ALP / total protein and tissue damage to the kidneys. In the 0.2% dose groups, there were increase in weight of appendix and histological abnormalities in a part of the kidneys. In the 0.2% dose groups, there were increase in weight of appendix and histological abnormalities in a part of the kidneys. Various changes to the kidneys as seen in 0.2 to 0.07% dose groups differ in their extent and frequency of incidence, but these changes were also seen in the control group, so they were not considered changes that lead to permanent damages like collapse of renal tubule or cyst formation. Administration of linear alkylbenzene sulfonic acid sodium salt (LAS) to Wistar SLC ratsat dose levels of 0, 0.07, 0.2, 0.6 and 1.8% (equivalent to 0,40, 115, 340, 1030mg/kg bw/day) for 26 weeks identified a NOAEL of 0.07% (40 mg/kg bw/day), based on tissue damage in caecum, kidney and liver, hematological as well as clinical chemistry parameters changes at higher dose levels (115 mg/kg bw/day and above).

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Does not meet important criteria of today's standard methods
Justification for type of information:
Read-across approach - see read-across justificaiton in section 13
Reason / purpose:
read-across source
Qualifier:
no guideline followed
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:
Wistar
Remarks:
JCL
Sex:
male/female
Details on test animals 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
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:
unchanged (no vehicle)
Remarks:
Test substance was administered either in diet or drinking water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Nine months
Frequency of treatment:
Continuous in diet or drinking water (ad libitum)
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 (Table 1).
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 (Table 1).
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 cho
linesterase 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 (Table 2).
There was also significant decrease in absolute and relative heart weight in females of 0.6% (diet) and 0.07% (drinking water) dose groups (Table 2).
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):
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:
NOAEL
Effect level:
85 mg/kg bw/day (nominal)
Based on:
act. ingr.
Remarks:
Drinking water
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 lactat e 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
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Remarks:
Dietary study
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
water consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
other: Liver and kidney enzyme levels
Remarks on result:
other: Adverse effects were observed at all dose levels
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

Table 1: Body weight gain, Food, water and sample consumption of Rats on administration of LAS for 9 months

 Dose group  No. of Rats Initial BW (g)  Final BW (g)  BW gain (%)  Food (g/rat/day)  Water (g/rat/day)  Sample (g/kg bw/day) 
 0 8M  113.1±1.156   421.6±8.542   372.6±5.889  15  23  -
 0.6  8M   110.6±0.925   413.5±7.356   373.8±6.380  16  23 0.234
 1.8  8M   109.2±2.218   341.1±8.008   312.5±6.518**  14  33 0.747
 0.07  8M   110.6±1.572   411.0±5.255   371.9±6.908  17  29 0.051
 0.2  8M   108.3±1.900   385.1±5.453   356.1±6.346**  15  33  0.148
               
 0  8F   92.3±1.592   212.6±4.508   230.6±5.420  10  18  -
 0.6  8F   92.1±1.663   216.1±7.705    234.3±5.753  10  17  0.287
 1.8  8F   90.6±1.463   184.8±4.278    204.3±5.791**  9  18  0.969
 0.07  8F   92.7±1.770   206.0±3.555    222.4±4.478  11  20  0.082
 0.2  8F   91.6±1.893   209.3±5.383    228.4±3.857  12  18  0.173

*p<0.05 **p<0.01

Table 2: Organ weight of male and female rats on administration of LAS for 9 months

 Dose group  Brain (g) / (g/100g)  Heart (g) / (g/100g)   Lung (g) / (g/100g)   Liver (g) / (g/100g)   Spleen (g) / (g/100g)   Kidner (R) (g) / (g/100g)   Kidney (L) (g) / (g/100g) Adrenal (R) (mg) / (mg/100g)   Adrenal (L) (mg) / (mg/100g) Testis/Ovary (R) (g) / (g/100g)    Testis/Ovary (L) (g) / (g/100g)    Uterus (g) / (g/100g)  
 0 M  1.987±0.011 /  0.463±0.008   1.026±0.018 /  0.239±0.005    1.263±0.033 /  0.294±0.008  12.7±0.341 /  2.9±0.039  0.698±0.014 /  0.162±0.003    1.162±0.040 /  0.270±0.008   1.184±0.041 /  0.275±0.007  18.3±1.117 /  4.2±0.217   18.1±0.934 /  4.2±0.225   1.591±0.023 /  0.370±0.006   1.643±0.027 /  0.382±0.005  
 0.6 M   1.982±0.011 /  0.473±0.007   0.971±0.022* /  0.231±0.004    1.269±0.042 /  0.302±0.005    12.9±0.310 /  3.0±0.049  0.682±0.019 /  0.162±0.002     1.210±0.026 /  0.290±0.006   1.214±0.032 /  0.289±0.007   19.6±1.900 /  4.7±0.435  17.6±1.602 /  4.2±0.416     1.563±0.019 /  0.373±0.004   1.585±0.027 /  0.378±0.007   -  
 1.8 M   1.827±0.131 /  0.521±0.037   0.825±0.026* /  0.235±0.003    1.040±0.025* /  0.295±0.002    11.5±0.352* /  3.2±0.043*   0.501±0.163** /  0.143±0.003**  0.977±0.022** /  0.279±0.007   0.982±0.017** /  0.281±0.007    17.5±1.370 /  5.0±0.481  17.0±0.654 /  4.8±0.146    1.455±0.027** /  0.415±0.004**    1.491±0.033** /  0.425±0.004**  -  
 0.07 M   1.995±0.016 /  0.471±0.006   0.981±0.031 /  0.232±0.008    1.287±0.021 /  0.304±0.007    13.2±0.258 /  3.0±0.052   0.682±0.009 /  0.161±0.002    1.221±0.018 /  0.288±0.005   1.254±0.012 /  0.296±0.005*   18.6±0.680 /  4.4±0.156   19.2±1.024 /  4.5±0.262   1.606±0.022 /  0.379±0.006    1.566±0.056 /  0.370±0.015  -  
 0.2 M   1.881±0.108 /  0.478±0.028   0.975±0.183 /  0.247±0.003    1.154±0.037* /  0.293±0.007    11.6±0.184 /  2.9±0.042   0.595±0.013** /  0.150±0.002   1.124±0.025 /  0.286±0.007   1.154±0.031 /  0.293±0.008   18.4±0.914 /  4.6±0.232   17.5±1.309 /  4.4±0.354   1.483±0.059 /  0.377±0.016     1.589±0.013 /  0.404±0.005  -  
                           
 0 F   1.853±0.023 /  0.846±0.016   0.647±0.013 /  0.294±0.003    0.870±0.026 /  0.397±0.012    6.2±0.150 /  2.8±0.051   0.441±0.016 /  0.201±0.006    0.650±0.021 /  0.296±0.006   0.650±0.021 /  0.296±0.008   24.8±1.381 /  11.2±0.443   25.7±1.385 /  11.7±0.487   29.3±2.738 /  13.4±1.225    31.2±4.007 /  14.2±1.857    0.687±0.037 /  0.313±0.017   
 0.6 F   1.859±0.011 /  0.845±0.027   0.595±0.019* /  0.269±0.006**   0.810±0.032 /  0.367±0.015   6.6±0.355 /  2.9±0.061   0.425±0.010 /  0.193±0.009    0.648±0.023 /  0.292±0.004   0.655±0.026 /  0.296±0.008   25.8±1.563 /  11.7±0.820   24.8±1.641 /  11.3±0.863   23.3±3.504 /  10.8±1.740    32.8±3.120 /  14.9±1.380    0.660±0.033 /  0.301±0.020    
 1.8 F   1.788±0.031 /  0.954±0.015**  
 0.455±0.008 /  0.242±0.002
  0.761±0.028 /  0.405±0.009  7.1±0.195** /  3.8±0.082**    0.351±0.012** /  0.187±0.004   0.580±0.015 /  0.309±0.003   0.586±0.021* /  0.312±0.007    19.6±1.981* /  10.3±0.924  21.3±1.487* /  11.3±0.609    25.3±2.853 /  13.4±1.417     31.5±2.322 /  16.7±1.165     0.588±0.066 /  0.312±0.034   
 0.07 F   1.854±0.019 /  0.844±0.016   0.611±0.011* /  0.277±0.004**   0.801±0.027 /  0.365±0.014   6.3±0.139 /  2.8±0.063   0.424±0.007 /  0.193±0.004    0.669±0.017 /  0.304±0.006   0.677±0.010 /  0.308±0.005  22.0±1.619 /  10.0±0.720    21.0±1.763 /  9.8±0.790   30.0±1.490 /  13.6±0.740    31.7±2.300 /  12.2±1.200     0.729±0.051 /  0.331±0.022   
 0.2 F   1.691±0.112 /  0.810±0.053   0.593±0.015 /  0.287±0.004   0.817±0.040 /  0.393±0.021   6.1±0.220 /  2.9±0.071   0.428±0.014 /  0.205±0.006    0.651±0.015 /  0.312±0.006   0.670±0.020 /  0.321±0.008*   23.2±1.495 /  11.1±0.677   24.3±1.154 /  11.6±0.431   28.4±1.633 /  13.6±0.821    28.6±1.900 /  13.7±0.830    0.644±0.032 /  0.308±0.014   
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 a 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 a NOAEL of 0.07% (85 mg/kg bw/day in drinking water), based on adverse effects at all dose levels, 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.

Four week old male and female Wistar 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. Five animals were housed in each cage and maintained under controlled environmental conditions (temperature: Average of 25 ± 1°C, humidity: 50 - 60%, and 12 hours light /12 hours dark).CE-2 diet (from CLEA Japan) and water were provided ad 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) male 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, KATPase 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 on adverse effects at all dose levels, 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).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study which meets basic scientific principles
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Cox CD
Sex:
male/female
Details on test animals and environmental conditions:
20 male and 20 female per group
Diet: ad libitum
Route of administration:
oral: feed
Vehicle:
other: standard chow
Duration of treatment / exposure:
91 days
Frequency of treatment:
continuously
Remarks:
Doses / Concentrations:
0; 250; 500; 1000 mg/kg bw
Basis:

No. of animals per sex per dose:
20
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
FOOD EFFICIENCY: Yes
HISTOPATHOLOGY: Yes
HAEMATOLOGY: Yes
Sacrifice and pathology:
HISTOPATHOLOGY: Yes
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
not treatment related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
SUMMARY OF DAILY OBSERVATIONS
One rat escaped from its cage during the third week of the test and had to be eliminated because of contamination. One male from the mid-dose group and one female from the low-dose group displayed symptoms of inner ear infection.

AVERAGE BODY WEIGHT GAIN, FEED CONSUMPTION, AND EFFICIENCY
The only adverse significant differences noted were in body weight gain and feed efficiency in the female rats of the mid-dose group.

ORGAN/BODY WEIGHT RATIO
There were no significant differences notes in organ to body weight ratios.

HISTOPATHOLOGY
No lesions were found which followed a pattern of agent-induced toxicity. Tissue alterations observed were mild and were not considered significant.

PATHOLOGY
There do not appear to be any gross or histopathologic indications of a treatment-related effect.

HEMATOLOGY
The only abnormality observed was a slightly elevated WBC in one animal. This change was not considered significant. All other values are within normal limits and there were no significant differences between treatment groups.
Dose descriptor:
NOAEL
Effect level:
1 000 other: mg/kg bw
Sex:
male/female
Basis for effect level:
other: no significant effects at highest dose tested
Critical effects observed:
not specified

Bodyweight gain

 

males

females

Control

341±30

157±23

TEA-1 (250 mg/kg)

339±35

170±13

TEA-1 (500 mg/kg)

331±28

151±15

TEA-1 (1000 mg/kg)

329±32

161±22

 

Feed consumption

 

males

females

Control

2143±155

1542±105

TEA-1 (250 mg/kg)

2073±165

1592±108

TEA-1 (500 mg/kg)

2076±282

5166±115

TEA-1 (1000 mg/kg)

2091±155

1604±123

 

Feed efficiency

 

males

females

Control

15.95±0.98

10.18±1.1

TEA-1 (250 mg/kg)

16.35±0.97

10.72±0.67

TEA-1 (500 mg/kg)

15.56±0.84

8.62±2.7

TEA-1 (1000 mg/kg)

15.75±0.99

9.99±0.88

   

Organ to bodyweight ratio (LIVER)

 

males

females

Control

38.2±3.3

42.1±12.2

TEA-1 (250 mg/kg)

41.6±3.7

39.9±3.3

TEA-1 (500 mg/kg)

45.9±6.9

44.6±1.0

TEA-1 (1000 mg/kg)

49.6±2.9

45.3±2.9

 

Organ to bodyweight ratio (KIDNEY)

 

males

females

Control

7.6±0.6

8.1±0.3

TEA-1 (250 mg/kg)

9.3±1.3

10.2±1.4

TEA-1 (500 mg/kg)

9.2±0.7

11.1±0.7

TEA-1 (1000 mg/kg)

10.4±1.4

10.3±1.4

Conclusions:
The NOAEL of the test material for this study is 1000 mg/kg bw.
Executive summary:

 In a subchronic toxicity study triethanolamine (88.5%) was administered to Cox CD male and female rats in the feed at dose levels of 0, 250, 500, and 1000 mg/kg bw/day for 91 days. No treatment-related effects were observed in the animals.The NOAEL is  1000 mg/kg bw/day.This subchronic toxicity study in the rat is acceptable and it satisfies the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in the rat.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
85 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Sufficient to address the endpoint
System:
other: gastrointestinal tract and urinary
Organ:
kidney
other: Caecum

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

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study which meets basic scientific principles.
Remarks:
This study is assigned a reliability score of 2 because the original report was not available for review. However, the study was evaluated by IPCS prior to inclusion in their criteria document.
Justification for type of information:
See attached read-across justification.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 39 to 42 days
- Weight at study initiation: 59.2 - 85.4 g (males), 57.0 - 79.3 g (females)
- Fasting period before study: No
- Housing: Individual in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24
- Humidity (%): 35 - 65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Type of coverage:
not specified
Vehicle:
acetone
Details on exposure:
TEST SITE
- Area of exposure: an area extending from the animal's mid-back to dorsal interscapular region
- Time intervals for shavings or clipplings: at least 24 hours prior to initial dose, and once weekly thereafter


REMOVAL OF TEST SUBSTANCE
No data


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0, 1.0, 0.5, 0.25, 0.125 and 0 g/kg bw
- Concentration (if solution): 1120, 560, 280, 140, 70 and 0 mg/ml
- Constant volume or concentration used: yes
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days per week
Remarks:
Doses / Concentrations:
125; 250; 500; 1000; 2000 mg/kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
20; 10 special study animals (designated for periodic urinalysis, hematology, and clinical chemistry determinations), 10 base study animals (subject to the collection of clinical observations data, sperm morphology and vaginal cytology evaluations, necropsy with gross examination and tissue collection, and histopathologic examination).
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: no
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 11
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All
- Parameters examined were erythrocyte count (RBC), hemoglobin (HgB), hematocrit (HCT), leukocyte count (WBC), platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leukocyte differential count, reticulocyte count, erythrocyte and platelet morphology.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 11
- Animals fasted: No data
- How many animals: All
- Parameters examined were sorbitol dehydrogenase (SDH), glutamic-pyruvic transaminase activity (GPT), glutamic-oxaloacetic transaminase activity (GOT), urea nitrogen (BUN), creatinine, total protein, albumin, glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: week 1, week 3, week 7 and week 12
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were specific gravity, glucose, protein, microscopic examination of sediment.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
The following tissues were examined: gross lesions and tissue masses (and regional lymph nodes, if possible), blood smear (if required by the pathologist), mandibular and mesentric lymph nodes, salivary gland, sternebrae, femur, or vertebrae, including marrow, thyroids, heart, esophagus, stomach (to include forestomach and glandular stomach), uterus, brain (three sections, including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), thymus, trachea, parathyroids, small intestine (duodenum, jejunum, ileum), cecum, colon and rectum, liver, prostate, testis, epididymis, seminal vesicle, ovaries, lungs and mainstem bronchi, nasal cavity and nasal turbinates (3), preputial or clitoral glands (paired), pancreas, spleen, kidneys, adrenals, urinary bladder, pituitary, spinal cord and sciatic nerve (if neurologie signs were present), eyes (if grossly abnormal), mammary gland (including surface skin), pharynx (if grossly abnormal), skin (lesions in dosed area, unaffected skin in dosed area, and undosed control skin).

Complete histopathologic evaluation of all tissues listed was performed on all base study rats from the 2.0 and 0 g/kg dose groups. On the basis of those findings, the skin at the site of application (both males and females) and the kidney (females only)were selected as the target tissues. These tissues were examined in rats in successively lower dose groups to a no-effect level; in addition, any gross lesions detected at necropsy were examined microscopically.
Other examinations:
Sperm morphology and vaginal cytology were evaluated in the control and the three highest dose groups.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no mortality; skin effects seen at the site of the application
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
no mortality; skin effects seen at the site of the application
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
lesions on the skin (at the application site)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHTS
The 2.0 g/kg male dose group exhibited a pronounced depression in body weight gain; the differential weight gain, relative to control, for this group was -32.8 percent, whereas those for the other male dosed groups ranged from -4.8 to +8.4 percent. The mean weight gains for all female dosed groups were depressed when compared with that of the female control group; the differential weight gains (relative to control group weight gain) for the 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg female dose groups were -13.1, -10.6, -6.0, -21.4, and -35.9 percent, respectively.

MORTALITY
All animals in all dose groups survived until scheduled termination.

CLINICAL OBSERVATIONS
The only clinical abnormalities associated with treatment with triethanolamine occurred at the site of dermal dose application. Discoloration of the skin was seen in males from all dose groups (including control); it was present in 6 males in the 0 g/kg dose group, and in all 10 base study animals in each of the dosed groups. Irritation at the site of application was seen in the three highest triethanolamine dosed groups (0.5, 1.0, and 2.0 g/kg), with incidence increasing and time to onset decreasing with increasing dose level. Scaliness was observed only at the 1.0 and 2.0 g/kg levels; this was observed in 1 rat in the 1.0 g/kg group (first seen on Day 13), and in 5 rats in the 2.0 g/kg group (first seen on Day 6). Crustiness at the site of application was recorded for 10 rats in the 2.0 g/kg dose group; two males from this group were also observed to have ulceration at the application site. Other clinical abnormalities occurred sporadically.
All treatment-related abnormalities in females were seen at the site of dermal dose application. Irritation was observed at the 1.0 (7 rats) and 2.0 (10 rats) g/kg dose levels; scaliness was also seen in female rats from these groups (4 animals dosed at the 1.0 g/kg level, and 5 animals dosed at the 2.0 g/kg level). Crustiness was present in 3 female rats dosed at 2.0 g/kg. Other abnormalities were determined not to be compound induced.

NECROPSY
Gross observations
Gross lesions were common on the skin of high dose rats. The compound-related lesion seen at the application site was dermal crust. Yellow skin was seen in the lumbar region of treated and control animals. This lesion had no corresponding microscopic lesion and is attributed to application of the vehicle (acetone). Other gross lesions were considered incidental, spontaneous lesions.

Final body weight
The mean final body weights for both male and females dosed at 2.0 g/kg body weight were significantly decreased (p <0.01), compared with those of their respective control groups.

Organ weights
The weights of the brain, right kidney, spleen, thymus, liver, lung, heart, right and left testes, and right and left epididymides were recorded at the scheduled necropsy of base study animals.
The mean brain to body weight values for the 2.0 g/kg male and female dose groups were significantly increased relative to control. In the absence of statistical changes in the absolute brain weight for these groups, this increase in relative weight appears to be the result of depressed final body weights in rats dosed at 2.0 g/kg.
Statistically significant increases in mean right kidney absolute weights and weights relative to brain weight occurred in both males and females dosed at 1.0 and 2.0 g/kg. The right kidney to body weight ratios of the 0.25, 0.5, 1.0, and 2.0 g/kg male dose groups and the 1.0 and 2.0 g/kg female dose groups were also significantly increased over that of control.
The mean spleen absolute weight and spleen weight relative to brain weight of the 2.0 g/kg female dose group were significantly decreased, relative to control values. There was a significant increase in the mean spleen to body weight ratio in male rats dosed at 1.0 and 2.0 g/kg. Thymus absolute weight and weight relative to brain weight were increased in males dosed at 2.0 g/kg body weight.
Mean liver to body weight ratios for the 0.5 and 1.0 g/kg male dose groups were significantly elevated over that of control. No statistically significant changes were observed in mean liver weight values for the female dose groups.
The mean lung absolute weight and lung to brain weight ratio of the 2.0 g/kg male dose group were significantly decreased, relative to control. The mean lung to brain weight of the 0.5 g/kg male dose group was also significantly decreased. The only statistically significant change in mean lung weight values for female rats was increased lung to brain weight ratio at the 0.125 g/kg level.
No statistically significant changes were seen in mean heart weight data for male and female rats.
For both testes, there was a significant increase in weight relative to body weight in the 2.0 g/kg male dose group; this was a result of decreased body weight at necropsy.
No statistical changes were observed for mean left epididymis weight values. The 2.0 g/kg male dose group exhibited decreased right epididymis absolute weight, and increased weight relative to body weight. Right epididymis to body and to brain weight ratios were increased in males dosed at 0.25 g/kg.

CLINICAL PATHOLOGY
Hematology
Hematology was performed on blood obtained from special study rats on study Day 80. Male rats dosed at 2.0 g/kg exhibited a significantly increased leukocyte count and decreased mean corpuscular volume. Significantly decreased MCV was also exhibited by the 2.0 g/kg female dose group; the hematocrit of this group was depressed as well.
Male rats in the 2.0 g/kg dose group exhibited significant increases in both relative and absolute number of segmented neutrophils and eosinophils, and a significant reduction in the relative number of lymphocytes. Female rats treated at the same dosage level (2.0 g/kg) also exhibited a significant increase in relative and absolute numbers of segmented neutrophils, as well as a decrease in relative lymphocyte count. These hematological changes in high dose rats of both sexes can be attributed to an inflammatory response resulting from dermal irritation. The mean values for all other dosed groups of both sexes were statistically similar to control.

Clinical chemistry
The 2.0 and 0.25 g/kg male dose groups exhibited elevated SGOT levels; the mean SGPT level of the 2.0 g/kg male dose group was also significantly higher than control. No other statistically significant changes were seen when mean data from the male dosed groups were compared with that of control. The 2.0 g/kg female dose group exhibited elevated serum urea nitrogen, albumin, SGOT, and SGPT. The only other significant change in the female group mean data was a decrease in serum sorbitol dehydrogenase in rats dosed at 0.5 and 1.0 g/kg.

Urinalyses
Urine, collected from special study group rats on Days 3, 16, 44, and 86, was analyzed.
At study Day 3, mean values for all male dosed groups were statistically similar to those of the male control group. At study Day 16, the only values statistically different from control were urine protein at the 1.0 g/kg level, and the number of leukocytes at the 0.25 g/kg level. By study Day 44, there was a significant reduction in urine protein levels in males dosed at 0.5, 1.0, and 2.0 g/kg; in addition, the specific gravity of urine collected from the 2.0 g/kg male dose group was significantly increased over control. At the final urinalysis (Day 86), the specific gravity of urine from rats in the 2.0 g/kg dose group was again significantly elevated, and the urine protein levels for the 0.5, 1.0, and 2.0 g/kg male dose groups were significantly lower than control. The only other parameter statistically different from control was increased urine volume in males dosed at 0.5 g/kg.
For female rats, there was decreased urine protein concentration at the 0.25 g/kg level and above at the Day 3 urinalysis; the number of crystals found in the urine of rats in the 2.0 g/kg dose group was also significantly greater than control. Increased number of crystals was also present, in females dosed at 1.0 and 2.0 g/kg, on Day 16. Beginning on Day 16, and continuing through Days 44 and 86, the specific gravity of urine from female rats from these two highest dose groups was significantly greater than that of control females. The only other statistically significant changes observed at Day 44 were increased urine protein concentration in females dosed at 0.5 g/kg, and increased number of crystals in females dosed at 2.0 g/kg. At Day 86, in addition to the increased urine specific gravity in the two highest dose groups, the 2.0 g/kg females exhibited an increase in urine glucose concentration.

HISTOPATHOLOGY
The compound-related skin lesions were chronic-active inflammation and acanthosis, and were seen in 2.0, 1.0, 0.5, and 0.25 g/kg male rats and in 2.0, 1.0, and 0.5 g/kg female rats. The chronic-active inflammation contained epidermal and dermal components. The epidermal component was characterized by acanthotic, hyperkeratotic, focally parakeratotic epidermis that occasionally contained rete pegs; these changes were graded separately as acanthosis. Severely affected epidermis contained focal hemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers. The dermal component in severely affected rats was characterized bydermal fibrosis, neocapillarization, minimally distorted adnexal organs, and variably severe mixed inflammatory infiltrates consisting of histiocytes, lymphocytes, neutrophils and eosinophils. Although only the most severe grades of chronic-active inflammation contained all of the previously listed epidermal and dermal features, the lesion was regarded as a continuum, and, therefore, the morphology "chronic-active inflammation" was used in all dose groups. Less severe lesions of chronic-active inflammation had reduced acanthosis, and lacked one or more of the following components: epidermal hemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers. Additionally, less severe lesions contained fewer of the dermal features of the most severe lesion. In minimal inflammatory lesions the epidermal component was predominant. Where only the term acanthosis was used, the least severe lesion contained 2-3 layers of epidermal cells above the basilar layer. Occasionally, there was minimal to mild dermal fibrosis associated with the acanthosis. In males and females there was a dose dependent reduction in severity and incidence of skin lesions from high to no-effect dose.
Non-compound related microscopic lesions were seen in the kidney, liver, lung, prostate, ovary, preputial gland, clitoral gland, eye, Zymbal's gland, nose/nasal cavity; and mediastinal lymph node.
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
125 mg/kg bw/day
Sex:
male
Basis for effect level:
other: chronic-active inflammation and acanthosis at the site of application
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
250 mg/kg bw/day
Sex:
female
Basis for effect level:
other: chronic-active inflammation and acanthosis at the site of application
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
125 mg/kg bw/day
Sex:
male
Basis for effect level:
other: increased relative kidney weight
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
500 mg/kg bw/day
Sex:
female
Basis for effect level:
other: increased absolute and relative kidney weight
Critical effects observed:
not specified

The compound-induced skin lesions in males and females were chronic-active inflammation and acanthosis. The incidence, severity, and

morphology of the lesions were similar between sexes. In males, however, the compound effect extended to one dose lower than in females. Although there was increased incidence of nephropathy from low to high dose in female rats, the severity of this lesion did not vary between dose groups, and, therefore, it was regarded as incidental. Other microscopic lesions were regarded as incidental and spontaneous or associated with retrobulbar bleeding.

Conclusions:
Under the conditions of this test the NOAEL for both local and systemic effects is 125 mg/kg bw.
Executive summary:

In a repeat-dose dermal toxicity study triethanolamine in acetonewas applied to the skin of male and female Fischer 344 rats at dose levels of 125, 250, 500, 1000, and 2000 mg/kg bw/day, for 5 days/week during a 90-day period.The test material induced skin lesions in males and females (chronic-active inflammation and acanthosis), similar between sexes. In males, the compound effect extended to one dose lower than in females. Although there was increased incidence of nephropathy from low to high dose in female rats, the severity of this lesion did not vary between dose groups, and, therefore, it was regarded as incidental. Other microscopic lesions were regarded as incidental and spontaneous or associated with retrobulbar bleeding. No other treatment- related effects were observed. The NOAEL for males was set at 125 mg/kg bw both for local (chronic-active inflammation and acanthosis at the application site) and systemic effects (increased relative kidney weight). The NOAEL for female local effects local (chronic-active inflammation and acanthosis at the application site) was 250 mg/kg bw and for systemic effects 500 mg/kg bw (increased absolute and relative kidney weight).

 

This dermal toxicity study in theratisacceptableand satisfies the guideline requirement for a repeat-dose dermal toxicity study (OECD 411).

Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Rationale for reliability incl. deficiencies:
other: Study which meets basic scientific principles.
Remarks:
This study is assigned a reliability score of 2 because the original report was not available for review. However, the study was evaluated by IPCS prior to inclusion in their criteria document.
Justification for type of information:
Read-across approach - see read-across justification in section 13
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 39 to 42 days
- Weight at study initiation: 59.2 - 85.4 g (males), 57.0 - 79.3 g (females)
- Fasting period before study: No
- Housing: Individual in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24
- Humidity (%): 35 - 65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Type of coverage:
not specified
Vehicle:
acetone
Details on exposure:
TEST SITE
- Area of exposure: an area extending from the animal's mid-back to dorsal interscapular region
- Time intervals for shavings or clipplings: at least 24 hours prior to initial dose, and once weekly thereafter


REMOVAL OF TEST SUBSTANCE
No data


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0, 1.0, 0.5, 0.25, 0.125 and 0 g/kg bw
- Concentration (if solution): 1120, 560, 280, 140, 70 and 0 mg/ml
- Constant volume or concentration used: yes
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days per week
Remarks:
Doses / Concentrations:
125; 250; 500; 1000; 2000 mg/kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
20; 10 special study animals (designated for periodic urinalysis, hematology, and clinical chemistry determinations), 10 base study animals (subject to the collection of clinical observations data, sperm morphology and vaginal cytology evaluations, necropsy with gross examination and tissue collection, and histopathologic examination).
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: no
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 11
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All
- Parameters examined were erythrocyte count (RBC), hemoglobin (HgB), hematocrit (HCT), leukocyte count (WBC), platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leukocyte differential count, reticulocyte count, erythrocyte and platelet morphology.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 11
- Animals fasted: No data
- How many animals: All
- Parameters examined were sorbitol dehydrogenase (SDH), glutamic-pyruvic transaminase activity (GPT), glutamic-oxaloacetic transaminase activity (GOT), urea nitrogen (BUN), creatinine, total protein, albumin, glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: week 1, week 3, week 7 and week 12
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were specific gravity, glucose, protein, microscopic examination of sediment.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
The following tissues were examined: gross lesions and tissue masses (and regional lymph nodes, if possible), blood smear (if required by the pathologist), mandibular and mesentric lymph nodes, salivary gland, sternebrae, femur, or vertebrae, including marrow, thyroids, heart, esophagus, stomach (to include forestomach and glandular stomach), uterus, brain (three sections, including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), thymus, trachea, parathyroids, small intestine (duodenum, jejunum, ileum), cecum, colon and rectum, liver, prostate, testis, epididymis, seminal vesicle, ovaries, lungs and mainstem bronchi, nasal cavity and nasal turbinates (3), preputial or clitoral glands (paired), pancreas, spleen, kidneys, adrenals, urinary bladder, pituitary, spinal cord and sciatic nerve (if neurologie signs were present), eyes (if grossly abnormal), mammary gland (including surface skin), pharynx (if grossly abnormal), skin (lesions in dosed area, unaffected skin in dosed area, and undosed control skin).

Complete histopathologic evaluation of all tissues listed was performed on all base study rats from the 2.0 and 0 g/kg dose groups. On the basis of those findings, the skin at the site of application (both males and females) and the kidney (females only)were selected as the target tissues. These tissues were examined in rats in successively lower dose groups to a no-effect level; in addition, any gross lesions detected at necropsy were examined microscopically.
Other examinations:
Sperm morphology and vaginal cytology were evaluated in the control and the three highest dose groups.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no mortality; skin effects seen at the site of the application
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
no mortality; skin effects seen at the site of the application
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
lesions on the skin (at the application site)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHTS
The 2.0 g/kg male dose group exhibited a pronounced depression in body weight gain; the differential weight gain, relative to control, for this group was -32.8 percent, whereas those for the other male dosed groups ranged from -4.8 to +8.4 percent. The mean weight gains for all female dosed groups were depressed when compared with that of the female control group; the differential weight gains (relative to control group weight gain) for the 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg female dose groups were -13.1, -10.6, -6.0, -21.4, and -35.9 percent, respectively.

MORTALITY
All animals in all dose groups survived until scheduled termination.

CLINICAL OBSERVATIONS
The only clinical abnormalities associated with treatment with triethanolamine occurred at the site of dermal dose application. Discoloration of the skin was seen in males from all dose groups (including control); it was present in 6 males in the 0 g/kg dose group, and in all 10 base study animals in each of the dosed groups. Irritation at the site of application was seen in the three highest triethanolamine dosed groups (0.5, 1.0, and 2.0 g/kg), with incidence increasing and time to onset decreasing with increasing dose level. Scaliness was observed only at the 1.0 and 2.0 g/kg levels; this was observed in 1 rat in the 1.0 g/kg group (first seen on Day 13), and in 5 rats in the 2.0 g/kg group (first seen on Day 6). Crustiness at the site of application was recorded for 10 rats in the 2.0 g/kg dose group; two males from this group were also observed to have ulceration at the application site. Other clinical abnormalities occurred sporadically.
All treatment-related abnormalities in females were seen at the site of dermal dose application. Irritation was observed at the 1.0 (7 rats) and 2.0 (10 rats) g/kg dose levels; scaliness was also seen in female rats from these groups (4 animals dosed at the 1.0 g/kg level, and 5 animals dosed at the 2.0 g/kg level). Crustiness was present in 3 female rats dosed at 2.0 g/kg. Other abnormalities were determined not to be compound induced.

NECROPSY
Gross observations
Gross lesions were common on the skin of high dose rats. The compound-related lesion seen at the application site was dermal crust. Yellow skin was seen in the lumbar region of treated and control animals. This lesion had no corresponding microscopic lesion and is attributed to application of the vehicle (acetone). Other gross lesions were considered incidental, spontaneous lesions.

Final body weight
The mean final body weights for both male and females dosed at 2.0 g/kg body weight were significantly decreased (p <0.01), compared with those of their respective control groups.

Organ weights
The weights of the brain, right kidney, spleen, thymus, liver, lung, heart, right and left testes, and right and left epididymides were recorded at the scheduled necropsy of base study animals.
The mean brain to body weight values for the 2.0 g/kg male and female dose groups were significantly increased relative to control. In the absence of statistical changes in the absolute brain weight for these groups, this increase in relative weight appears to be the result of depressed final body weights in rats dosed at 2.0 g/kg.
Statistically significant increases in mean right kidney absolute weights and weights relative to brain weight occurred in both males and females dosed at 1.0 and 2.0 g/kg. The right kidney to body weight ratios of the 0.25, 0.5, 1.0, and 2.0 g/kg male dose groups and the 1.0 and 2.0 g/kg female dose groups were also significantly increased over that of control.
The mean spleen absolute weight and spleen weight relative to brain weight of the 2.0 g/kg female dose group were significantly decreased, relative to control values. There was a significant increase in the mean spleen to body weight ratio in male rats dosed at 1.0 and 2.0 g/kg. Thymus absolute weight and weight relative to brain weight were increased in males dosed at 2.0 g/kg body weight.
Mean liver to body weight ratios for the 0.5 and 1.0 g/kg male dose groups were significantly elevated over that of control. No statistically significant changes were observed in mean liver weight values for the female dose groups.
The mean lung absolute weight and lung to brain weight ratio of the 2.0 g/kg male dose group were significantly decreased, relative to control. The mean lung to brain weight of the 0.5 g/kg male dose group was also significantly decreased. The only statistically significant change in mean lung weight values for female rats was increased lung to brain weight ratio at the 0.125 g/kg level.
No statistically significant changes were seen in mean heart weight data for male and female rats.
For both testes, there was a significant increase in weight relative to body weight in the 2.0 g/kg male dose group; this was a result of decreased body weight at necropsy.
No statistical changes were observed for mean left epididymis weight values. The 2.0 g/kg male dose group exhibited decreased right epididymis absolute weight, and increased weight relative to body weight. Right epididymis to body and to brain weight ratios were increased in males dosed at 0.25 g/kg.

CLINICAL PATHOLOGY
Hematology
Hematology was performed on blood obtained from special study rats on study Day 80. Male rats dosed at 2.0 g/kg exhibited a significantly increased leukocyte count and decreased mean corpuscular volume. Significantly decreased MCV was also exhibited by the 2.0 g/kg female dose group; the hematocrit of this group was depressed as well.
Male rats in the 2.0 g/kg dose group exhibited significant increases in both relative and absolute number of segmented neutrophils and eosinophils, and a significant reduction in the relative number of lymphocytes. Female rats treated at the same dosage level (2.0 g/kg) also exhibited a significant increase in relative and absolute numbers of segmented neutrophils, as well as a decrease in relative lymphocyte count. These hematological changes in high dose rats of both sexes can be attributed to an inflammatory response resulting from dermal irritation. The mean values for all other dosed groups of both sexes were statistically similar to control.

Clinical chemistry
The 2.0 and 0.25 g/kg male dose groups exhibited elevated SGOT levels; the mean SGPT level of the 2.0 g/kg male dose group was also significantly higher than control. No other statistically significant changes were seen when mean data from the male dosed groups were compared with that of control. The 2.0 g/kg female dose group exhibited elevated serum urea nitrogen, albumin, SGOT, and SGPT. The only other significant change in the female group mean data was a decrease in serum sorbitol dehydrogenase in rats dosed at 0.5 and 1.0 g/kg.

Urinalyses
Urine, collected from special study group rats on Days 3, 16, 44, and 86, was analyzed.
At study Day 3, mean values for all male dosed groups were statistically similar to those of the male control group. At study Day 16, the only values statistically different from control were urine protein at the 1.0 g/kg level, and the number of leukocytes at the 0.25 g/kg level. By study Day 44, there was a significant reduction in urine protein levels in males dosed at 0.5, 1.0, and 2.0 g/kg; in addition, the specific gravity of urine collected from the 2.0 g/kg male dose group was significantly increased over control. At the final urinalysis (Day 86), the specific gravity of urine from rats in the 2.0 g/kg dose group was again significantly elevated, and the urine protein levels for the 0.5, 1.0, and 2.0 g/kg male dose groups were significantly lower than control. The only other parameter statistically different from control was increased urine volume in males dosed at 0.5 g/kg.
For female rats, there was decreased urine protein concentration at the 0.25 g/kg level and above at the Day 3 urinalysis; the number of crystals found in the urine of rats in the 2.0 g/kg dose group was also significantly greater than control. Increased number of crystals was also present, in females dosed at 1.0 and 2.0 g/kg, on Day 16. Beginning on Day 16, and continuing through Days 44 and 86, the specific gravity of urine from female rats from these two highest dose groups was significantly greater than that of control females. The only other statistically significant changes observed at Day 44 were increased urine protein concentration in females dosed at 0.5 g/kg, and increased number of crystals in females dosed at 2.0 g/kg. At Day 86, in addition to the increased urine specific gravity in the two highest dose groups, the 2.0 g/kg females exhibited an increase in urine glucose concentration.

HISTOPATHOLOGY
The compound-related skin lesions were chronic-active inflammation and acanthosis, and were seen in 2.0, 1.0, 0.5, and 0.25 g/kg male rats and in 2.0, 1.0, and 0.5 g/kg female rats. The chronic-active inflammation contained epidermal and dermal components. The epidermal component was characterized by acanthotic, hyperkeratotic, focally parakeratotic epidermis that occasionally contained rete pegs; these changes were graded separately as acanthosis. Severely affected epidermis contained focal hemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers. The dermal component in severely affected rats was characterized bydermal fibrosis, neocapillarization, minimally distorted adnexal organs, and variably severe mixed inflammatory infiltrates consisting of histiocytes, lymphocytes, neutrophils and eosinophils. Although only the most severe grades of chronic-active inflammation contained all of the previously listed epidermal and dermal features, the lesion was regarded as a continuum, and, therefore, the morphology "chronic-active inflammation" was used in all dose groups. Less severe lesions of chronic-active inflammation had reduced acanthosis, and lacked one or more of the following components: epidermal hemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers. Additionally, less severe lesions contained fewer of the dermal features of the most severe lesion. In minimal inflammatory lesions the epidermal component was predominant. Where only the term acanthosis was used, the least severe lesion contained 2-3 layers of epidermal cells above the basilar layer. Occasionally, there was minimal to mild dermal fibrosis associated with the acanthosis. In males and females there was a dose dependent reduction in severity and incidence of skin lesions from high to no-effect dose.
Non-compound related microscopic lesions were seen in the kidney, liver, lung, prostate, ovary, preputial gland, clitoral gland, eye, Zymbal's gland, nose/nasal cavity; and mediastinal lymph node.
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
125 mg/kg bw/day
Sex:
male
Basis for effect level:
other: chronic-active inflammation and acanthosis at the site of application
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
250 mg/kg bw/day
Sex:
female
Basis for effect level:
other: chronic-active inflammation and acanthosis at the site of application
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
125 mg/kg bw/day
Sex:
male
Basis for effect level:
other: increased relative kidney weight
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
500 mg/kg bw/day
Sex:
female
Basis for effect level:
other: increased absolute and relative kidney weight
Critical effects observed:
not specified

The compound-induced skin lesions in males and females were chronic-active inflammation and acanthosis. The incidence, severity, and

morphology of the lesions were similar between sexes. In males, however, the compound effect extended to one dose lower than in females. Although there was increased incidence of nephropathy from low to high dose in female rats, the severity of this lesion did not vary between dose groups, and, therefore, it was regarded as incidental. Other microscopic lesions were regarded as incidental and spontaneous or associated with retrobulbar bleeding.

Conclusions:
Under the conditions of this test the NOAEL for both local and systemic effects is 125 mg/kg bw.
Executive summary:

In a repeat-dose dermal toxicity study triethanolamine in acetonewas applied to the skin of male and female Fischer 344 rats at dose levels of 125, 250, 500, 1000, and 2000 mg/kg bw/day, for 5 days/week during a 90-day period.The test material induced skin lesions in males and females (chronic-active inflammation and acanthosis), similar between sexes. In males, the compound effect extended to one dose lower than in females. Although there was increased incidence of nephropathy from low to high dose in female rats, the severity of this lesion did not vary between dose groups, and, therefore, it was regarded as incidental. Other microscopic lesions were regarded as incidental and spontaneous or associated with retrobulbar bleeding. No other treatment- related effects were observed. The NOAEL for males was set at 125 mg/kg bw both for local (chronic-active inflammation and acanthosis at the application site) and systemic effects (increased relative kidney weight). The NOAEL for female local effects local (chronic-active inflammation and acanthosis at the application site) was 250 mg/kg bw and for systemic effects 500 mg/kg bw (increased absolute and relative kidney weight).

 

This dermal toxicity study in theratisacceptableand satisfies the guideline requirement for a repeat-dose dermal toxicity study (OECD 411).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Information available only for TEA
System:
urinary
Organ:
kidney

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study which meets basic scientific principles.
Remarks:
This study is assigned a reliability score of 2 because the original report was not available for review. However, the study was evaluated by IPCS prior to inclusion in their criteria document.
Justification for type of information:
See attached read-across justification.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 39 to 42 days
- Weight at study initiation: 59.2 - 85.4 g (males), 57.0 - 79.3 g (females)
- Fasting period before study: No
- Housing: Individual in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24
- Humidity (%): 35 - 65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Type of coverage:
not specified
Vehicle:
acetone
Details on exposure:
TEST SITE
- Area of exposure: an area extending from the animal's mid-back to dorsal interscapular region
- Time intervals for shavings or clipplings: at least 24 hours prior to initial dose, and once weekly thereafter


REMOVAL OF TEST SUBSTANCE
No data


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0, 1.0, 0.5, 0.25, 0.125 and 0 g/kg bw
- Concentration (if solution): 1120, 560, 280, 140, 70 and 0 mg/ml
- Constant volume or concentration used: yes
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days per week
Remarks:
Doses / Concentrations:
125; 250; 500; 1000; 2000 mg/kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
20; 10 special study animals (designated for periodic urinalysis, hematology, and clinical chemistry determinations), 10 base study animals (subject to the collection of clinical observations data, sperm morphology and vaginal cytology evaluations, necropsy with gross examination and tissue collection, and histopathologic examination).
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: no
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 11
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All
- Parameters examined were erythrocyte count (RBC), hemoglobin (HgB), hematocrit (HCT), leukocyte count (WBC), platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leukocyte differential count, reticulocyte count, erythrocyte and platelet morphology.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 11
- Animals fasted: No data
- How many animals: All
- Parameters examined were sorbitol dehydrogenase (SDH), glutamic-pyruvic transaminase activity (GPT), glutamic-oxaloacetic transaminase activity (GOT), urea nitrogen (BUN), creatinine, total protein, albumin, glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: week 1, week 3, week 7 and week 12
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were specific gravity, glucose, protein, microscopic examination of sediment.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
The following tissues were examined: gross lesions and tissue masses (and regional lymph nodes, if possible), blood smear (if required by the pathologist), mandibular and mesentric lymph nodes, salivary gland, sternebrae, femur, or vertebrae, including marrow, thyroids, heart, esophagus, stomach (to include forestomach and glandular stomach), uterus, brain (three sections, including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons), thymus, trachea, parathyroids, small intestine (duodenum, jejunum, ileum), cecum, colon and rectum, liver, prostate, testis, epididymis, seminal vesicle, ovaries, lungs and mainstem bronchi, nasal cavity and nasal turbinates (3), preputial or clitoral glands (paired), pancreas, spleen, kidneys, adrenals, urinary bladder, pituitary, spinal cord and sciatic nerve (if neurologie signs were present), eyes (if grossly abnormal), mammary gland (including surface skin), pharynx (if grossly abnormal), skin (lesions in dosed area, unaffected skin in dosed area, and undosed control skin).

Complete histopathologic evaluation of all tissues listed was performed on all base study rats from the 2.0 and 0 g/kg dose groups. On the basis of those findings, the skin at the site of application (both males and females) and the kidney (females only)were selected as the target tissues. These tissues were examined in rats in successively lower dose groups to a no-effect level; in addition, any gross lesions detected at necropsy were examined microscopically.
Other examinations:
Sperm morphology and vaginal cytology were evaluated in the control and the three highest dose groups.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no mortality; skin effects seen at the site of the application
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
no mortality; skin effects seen at the site of the application
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
lesions on the skin (at the application site)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHTS
The 2.0 g/kg male dose group exhibited a pronounced depression in body weight gain; the differential weight gain, relative to control, for this group was -32.8 percent, whereas those for the other male dosed groups ranged from -4.8 to +8.4 percent. The mean weight gains for all female dosed groups were depressed when compared with that of the female control group; the differential weight gains (relative to control group weight gain) for the 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg female dose groups were -13.1, -10.6, -6.0, -21.4, and -35.9 percent, respectively.

MORTALITY
All animals in all dose groups survived until scheduled termination.

CLINICAL OBSERVATIONS
The only clinical abnormalities associated with treatment with triethanolamine occurred at the site of dermal dose application. Discoloration of the skin was seen in males from all dose groups (including control); it was present in 6 males in the 0 g/kg dose group, and in all 10 base study animals in each of the dosed groups. Irritation at the site of application was seen in the three highest triethanolamine dosed groups (0.5, 1.0, and 2.0 g/kg), with incidence increasing and time to onset decreasing with increasing dose level. Scaliness was observed only at the 1.0 and 2.0 g/kg levels; this was observed in 1 rat in the 1.0 g/kg group (first seen on Day 13), and in 5 rats in the 2.0 g/kg group (first seen on Day 6). Crustiness at the site of application was recorded for 10 rats in the 2.0 g/kg dose group; two males from this group were also observed to have ulceration at the application site. Other clinical abnormalities occurred sporadically.
All treatment-related abnormalities in females were seen at the site of dermal dose application. Irritation was observed at the 1.0 (7 rats) and 2.0 (10 rats) g/kg dose levels; scaliness was also seen in female rats from these groups (4 animals dosed at the 1.0 g/kg level, and 5 animals dosed at the 2.0 g/kg level). Crustiness was present in 3 female rats dosed at 2.0 g/kg. Other abnormalities were determined not to be compound induced.

NECROPSY
Gross observations
Gross lesions were common on the skin of high dose rats. The compound-related lesion seen at the application site was dermal crust. Yellow skin was seen in the lumbar region of treated and control animals. This lesion had no corresponding microscopic lesion and is attributed to application of the vehicle (acetone). Other gross lesions were considered incidental, spontaneous lesions.

Final body weight
The mean final body weights for both male and females dosed at 2.0 g/kg body weight were significantly decreased (p <0.01), compared with those of their respective control groups.

Organ weights
The weights of the brain, right kidney, spleen, thymus, liver, lung, heart, right and left testes, and right and left epididymides were recorded at the scheduled necropsy of base study animals.
The mean brain to body weight values for the 2.0 g/kg male and female dose groups were significantly increased relative to control. In the absence of statistical changes in the absolute brain weight for these groups, this increase in relative weight appears to be the result of depressed final body weights in rats dosed at 2.0 g/kg.
Statistically significant increases in mean right kidney absolute weights and weights relative to brain weight occurred in both males and females dosed at 1.0 and 2.0 g/kg. The right kidney to body weight ratios of the 0.25, 0.5, 1.0, and 2.0 g/kg male dose groups and the 1.0 and 2.0 g/kg female dose groups were also significantly increased over that of control.
The mean spleen absolute weight and spleen weight relative to brain weight of the 2.0 g/kg female dose group were significantly decreased, relative to control values. There was a significant increase in the mean spleen to body weight ratio in male rats dosed at 1.0 and 2.0 g/kg. Thymus absolute weight and weight relative to brain weight were increased in males dosed at 2.0 g/kg body weight.
Mean liver to body weight ratios for the 0.5 and 1.0 g/kg male dose groups were significantly elevated over that of control. No statistically significant changes were observed in mean liver weight values for the female dose groups.
The mean lung absolute weight and lung to brain weight ratio of the 2.0 g/kg male dose group were significantly decreased, relative to control. The mean lung to brain weight of the 0.5 g/kg male dose group was also significantly decreased. The only statistically significant change in mean lung weight values for female rats was increased lung to brain weight ratio at the 0.125 g/kg level.
No statistically significant changes were seen in mean heart weight data for male and female rats.
For both testes, there was a significant increase in weight relative to body weight in the 2.0 g/kg male dose group; this was a result of decreased body weight at necropsy.
No statistical changes were observed for mean left epididymis weight values. The 2.0 g/kg male dose group exhibited decreased right epididymis absolute weight, and increased weight relative to body weight. Right epididymis to body and to brain weight ratios were increased in males dosed at 0.25 g/kg.

CLINICAL PATHOLOGY
Hematology
Hematology was performed on blood obtained from special study rats on study Day 80. Male rats dosed at 2.0 g/kg exhibited a significantly increased leukocyte count and decreased mean corpuscular volume. Significantly decreased MCV was also exhibited by the 2.0 g/kg female dose group; the hematocrit of this group was depressed as well.
Male rats in the 2.0 g/kg dose group exhibited significant increases in both relative and absolute number of segmented neutrophils and eosinophils, and a significant reduction in the relative number of lymphocytes. Female rats treated at the same dosage level (2.0 g/kg) also exhibited a significant increase in relative and absolute numbers of segmented neutrophils, as well as a decrease in relative lymphocyte count. These hematological changes in high dose rats of both sexes can be attributed to an inflammatory response resulting from dermal irritation. The mean values for all other dosed groups of both sexes were statistically similar to control.

Clinical chemistry
The 2.0 and 0.25 g/kg male dose groups exhibited elevated SGOT levels; the mean SGPT level of the 2.0 g/kg male dose group was also significantly higher than control. No other statistically significant changes were seen when mean data from the male dosed groups were compared with that of control. The 2.0 g/kg female dose group exhibited elevated serum urea nitrogen, albumin, SGOT, and SGPT. The only other significant change in the female group mean data was a decrease in serum sorbitol dehydrogenase in rats dosed at 0.5 and 1.0 g/kg.

Urinalyses
Urine, collected from special study group rats on Days 3, 16, 44, and 86, was analyzed.
At study Day 3, mean values for all male dosed groups were statistically similar to those of the male control group. At study Day 16, the only values statistically different from control were urine protein at the 1.0 g/kg level, and the number of leukocytes at the 0.25 g/kg level. By study Day 44, there was a significant reduction in urine protein levels in males dosed at 0.5, 1.0, and 2.0 g/kg; in addition, the specific gravity of urine collected from the 2.0 g/kg male dose group was significantly increased over control. At the final urinalysis (Day 86), the specific gravity of urine from rats in the 2.0 g/kg dose group was again significantly elevated, and the urine protein levels for the 0.5, 1.0, and 2.0 g/kg male dose groups were significantly lower than control. The only other parameter statistically different from control was increased urine volume in males dosed at 0.5 g/kg.
For female rats, there was decreased urine protein concentration at the 0.25 g/kg level and above at the Day 3 urinalysis; the number of crystals found in the urine of rats in the 2.0 g/kg dose group was also significantly greater than control. Increased number of crystals was also present, in females dosed at 1.0 and 2.0 g/kg, on Day 16. Beginning on Day 16, and continuing through Days 44 and 86, the specific gravity of urine from female rats from these two highest dose groups was significantly greater than that of control females. The only other statistically significant changes observed at Day 44 were increased urine protein concentration in females dosed at 0.5 g/kg, and increased number of crystals in females dosed at 2.0 g/kg. At Day 86, in addition to the increased urine specific gravity in the two highest dose groups, the 2.0 g/kg females exhibited an increase in urine glucose concentration.

HISTOPATHOLOGY
The compound-related skin lesions were chronic-active inflammation and acanthosis, and were seen in 2.0, 1.0, 0.5, and 0.25 g/kg male rats and in 2.0, 1.0, and 0.5 g/kg female rats. The chronic-active inflammation contained epidermal and dermal components. The epidermal component was characterized by acanthotic, hyperkeratotic, focally parakeratotic epidermis that occasionally contained rete pegs; these changes were graded separately as acanthosis. Severely affected epidermis contained focal hemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers. The dermal component in severely affected rats was characterized bydermal fibrosis, neocapillarization, minimally distorted adnexal organs, and variably severe mixed inflammatory infiltrates consisting of histiocytes, lymphocytes, neutrophils and eosinophils. Although only the most severe grades of chronic-active inflammation contained all of the previously listed epidermal and dermal features, the lesion was regarded as a continuum, and, therefore, the morphology "chronic-active inflammation" was used in all dose groups. Less severe lesions of chronic-active inflammation had reduced acanthosis, and lacked one or more of the following components: epidermal hemorrhage, fibrin and/or mineral deposits, bacterial colonies, serum pockets, pustules, erosions and/or ulcers. Additionally, less severe lesions contained fewer of the dermal features of the most severe lesion. In minimal inflammatory lesions the epidermal component was predominant. Where only the term acanthosis was used, the least severe lesion contained 2-3 layers of epidermal cells above the basilar layer. Occasionally, there was minimal to mild dermal fibrosis associated with the acanthosis. In males and females there was a dose dependent reduction in severity and incidence of skin lesions from high to no-effect dose.
Non-compound related microscopic lesions were seen in the kidney, liver, lung, prostate, ovary, preputial gland, clitoral gland, eye, Zymbal's gland, nose/nasal cavity; and mediastinal lymph node.
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
125 mg/kg bw/day
Sex:
male
Basis for effect level:
other: chronic-active inflammation and acanthosis at the site of application
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
250 mg/kg bw/day
Sex:
female
Basis for effect level:
other: chronic-active inflammation and acanthosis at the site of application
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
125 mg/kg bw/day
Sex:
male
Basis for effect level:
other: increased relative kidney weight
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
500 mg/kg bw/day
Sex:
female
Basis for effect level:
other: increased absolute and relative kidney weight
Critical effects observed:
not specified

The compound-induced skin lesions in males and females were chronic-active inflammation and acanthosis. The incidence, severity, and

morphology of the lesions were similar between sexes. In males, however, the compound effect extended to one dose lower than in females. Although there was increased incidence of nephropathy from low to high dose in female rats, the severity of this lesion did not vary between dose groups, and, therefore, it was regarded as incidental. Other microscopic lesions were regarded as incidental and spontaneous or associated with retrobulbar bleeding.

Conclusions:
Under the conditions of this test the NOAEL for both local and systemic effects is 125 mg/kg bw.
Executive summary:

In a repeat-dose dermal toxicity study triethanolamine in acetonewas applied to the skin of male and female Fischer 344 rats at dose levels of 125, 250, 500, 1000, and 2000 mg/kg bw/day, for 5 days/week during a 90-day period.The test material induced skin lesions in males and females (chronic-active inflammation and acanthosis), similar between sexes. In males, the compound effect extended to one dose lower than in females. Although there was increased incidence of nephropathy from low to high dose in female rats, the severity of this lesion did not vary between dose groups, and, therefore, it was regarded as incidental. Other microscopic lesions were regarded as incidental and spontaneous or associated with retrobulbar bleeding. No other treatment- related effects were observed. The NOAEL for males was set at 125 mg/kg bw both for local (chronic-active inflammation and acanthosis at the application site) and systemic effects (increased relative kidney weight). The NOAEL for female local effects local (chronic-active inflammation and acanthosis at the application site) was 250 mg/kg bw and for systemic effects 500 mg/kg bw (increased absolute and relative kidney weight).

 

This dermal toxicity study in theratisacceptableand satisfies the guideline requirement for a repeat-dose dermal toxicity study (OECD 411).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125
Study duration:
subchronic
Species:
rat
Quality of whole database:
Information available only regarding TEA

Additional information

Justification for classification or non-classification

Based on the results of repeated dose toxicity studies on two analogues, the substance does not need to be classified. Although adverse effects have been seen after repeated exposures of animals to the analogue LAS Na, these were seen at dose levels well above the classification criteria limit.