Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.

Endpoint summary

Description of key information

Repeated dose toxicity studies were conducted through oral route in rats (Yoneyama 1976 a, Yoneyama 1972; Ito 1978 a, b, c; Oser 1965; study 1 - 6 in CSR) and mice (Yoneyama 1976 b, study 7 CSR) and through dermal route in rats (Ito 1978 b) and through subcutaneous route in monkeys (Heywood 1978).

One oral study was performed with C10-13 LAS Na (Ito 1978a), two oral studies were performed with C10-13 LAS Mg (Ito 1978b and c) and four oral studies were performed with C10-14 LAS Na (Yoneyama 1976 a and b, Yoneyama 1972 and Oser 1965). The dermal study was performed with C10-13 LAS Mg (Ito 1978 d).

C10-13 LAS Mg and C10-13 LAS Na are considered to be a similar substance as C10-13 LAS H as they all disassociate in vivo to form the identical LAS anion.

 

C10-14 LAS Na (source substance) has very similar composition to C10-13 LAS (target substance). Therefore read-across is considered to be acceptable.

The target and source substance only differ slightly in the C10 and C14 content. The substances present a similar potency of toxicological behaviour with respect to acute oral toxicity, with LD50 being 1080 mg/kg bw for the target substance and 900 mg/kg bw for the source substance. With respect to repeated dose endpoint, for the studies available on the source and target substances, the doses at which effects were observed were similar (after consideration of exposure route and dose spacing) as were the observed effects. 

For dietary exposures, LOAELs ranged from 300 to 500 for source and target substances, respectively. In both substances, the significant secondary effects observed at higher doses were similar. For the key studies available for the source substance, no significant effects in liver and kidneys were observed at the NOAELs of 40 mg/kg bw/day and 85 mg/kg bw/day. The consistency in the adverse effects seen in liver and kidneys in both studies supports the selection of 85 mg/kg bw/day as the appropriate NOAEL.

 

For the target substance, the supporting (limited) study by the same route resulted in a NOAEL of 150 mg/kg/day. Therefore, there is no evidence in the available data set that there are significant differences in potency between the source and target substances. In addition, use of data from the source substance represents an overall conservative approach in terms of the quantitative aspects of the repeated dose toxicity of these substances.

In conclusion, considering the entire dataset, the highest NOAEL for systemic toxicity below the lowest LOAEL (115 mg/kg bw/day from the 26 weeks dietary study with C10-14 LAS Na in rats, Yoneyama 1972, based on histopathological changes in liver and kidney) is 85 mg/kw bw/day from the 9 months drinking water study in rats with C10-14 LAS Na (Yoneyama 1976 a). In this drinking water study, the LOAEL was slightly higher at 145 mg/kg bw/day and based on liver and kidney enzyme changes. The consistency in the adverse effects seen in liver and kidneys in these studies (i.e. Yoneyama 1976 and Yoneyama 1972) supports the selection of 85 mg/kg bw/day as the appropriate NOAEL. Further, the selection of 85 mg/ kg bw/day as the appropriate NOAEL is also supported by the higher NOAEL of 150 mg/kg bw/day observed in the 26 weeks oral gavage study in rats with C10-13 LAS Mg (Ito 1978 b). Therefore, based on the entire data set, the point of departure taken forward for risk assessment purposes is the NOAEL of 85 mg/kg bw/day.

 

For dermal exposure, the systemic toxicity NOAEL was established at 5.0% (equivalent to 2500 mg/kg bw/day), based on no toxicologically relevant changes at any dose level in a sub-chronic 26 -weeks dermal repeated dose toxicity study with C10-13 LAS Mg in rats (Ito 1978 d).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic 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: read-across from a study predating current guidelines and GLP, but adequate for assessment.

Justification for type of information:

The repeated dose toxicity study of C10-13 LAS is based on read-across with C10-14 LAS. The read-across justification is presented in the assessment reports section. A cross-reference is made to that record.

Reason / purpose for cross-reference:

read-across source

Remarks:

study 1

Reason / purpose for cross-reference:

read-across source

Remarks:

study 2

Reason / purpose for cross-reference:

read-across source

Remarks:

study 3

Reason / purpose for cross-reference:

read-across source

Remarks:

study 4

Reason / purpose for cross-reference:

read-across source

Remarks:

study 5

Reason / purpose for cross-reference:

read-across source

Remarks:

study 6

Reason / purpose for cross-reference:

read-across source

Remarks:

study 7

Reason / purpose for cross-reference:

read-across: supporting information

Remarks:

read-across justification

Key result

Dose descriptor:

NOAEL

Remarks:

study 1

Effect level:

85 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical biochemistry

other: Liver and kidney enzymes

Remarks on result:

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

Key result

Dose descriptor:

LOAEL

Remarks:

study 1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

water consumption and compound intake

other: Liver and kidney enzyme levels

Remarks on result:

other: Adverse effects were observed at all dose levels

Key result

Dose descriptor:

NOAEL

Remarks:

study 2

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Suppression of weight gain, increase in appendix weight, alternations in haematological and clinical 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).

Dose descriptor:

NOAEL

Remarks:

study 6

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: There was a significant increase in relative liver weights in females fed with 250 mg/kg bw/day.

Dose descriptor:

NOAEL

Remarks:

study 7 (drinking water study)

Effect level:

133 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

other: Liver and kidney enzyme levels

Remarks on result:

other: This is based on alteration in liver and kidney weights and enzyme levels at higher tested dose level. As no other parameters (histopathology, clinical chemistry and haematological parameters) were evaluated in the study.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

145 other: mg/kg bw day; study 1

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 other: mg/kg bw/day; study 1

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

115 other: mg/kg bw/day; study 2

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

250 other: mg/kg bw/day; study 6

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

380 other: mg/kg bw/day; study 7

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

380 other: mg/kw bw/day; study 7

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

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

GLP compliance:

no

Remarks:

(pre-dates GLP)

Limit test:

no

Species:

rat

Strain:

other: Wistar JCL

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

IN-LIFE DATES: Not reported

Route of administration:

other: oral: drinking water and feed

Details on route of administration:

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

Vehicle:

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

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

9 months

Frequency of treatment:

Continuous in diet or drinking water (ad libitum)

Dose / conc.:

0.6 other: %

Remarks:

in diet (corresponding to 300 mg/kg bw/day)

Dose / conc.:

1.8 other: %

Remarks:

in diet (corresponding to 900 mg/kg bw/day)

Dose / conc.:

0.07 other: %

Remarks:

in drinking water (corresponding to 85 mg/kg bw/day)

Dose / conc.:

0.2 other: %

Remarks:

in drinking water (corresponding to 145 mg/kg bw/day)

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), Haemoglobin (Hgb), Haematocrit (Hct), mean corpuscular volume (MCV) and mean corpuscular haemoglobin 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 consuming LAS-containing water exhibited a slight redness at the tips of their facial fur, and coarse fur all over their bodies.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A significant reduction in WBC was observed in 0.6% (diet) male rats compared to controls, and a significant reduction 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):

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.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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

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

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

(dietery study)

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

haematology

organ weights and organ / body weight ratios

water consumption and compound intake

other: Liver and kidney enzyme levels

Remarks on result:

other: Adverse effects were observed at all dose levels

Dose descriptor:

NOAEL

Effect level:

85 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

(drinking water study)

Sex:

male/female

Basis for effect level:

clinical biochemistry

other: Liver and kidney enzyme levels

Remarks on result:

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

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

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 (Yoneyama et. al., 1976)

Sex	Groups	No. of rats	Initial body weight (g)	Final Body weight (g)	Body weight gain (%)	Food (g/rat/day)	Water (g/rat/day)	Sample (g/kg BW/day)
Male	Control	8	113.1± 1.156	421.6 ± 8.542	372.6 ± 5.889	15	23	-
	0.6 F	8	110.6 ± 0.925	413.5 ± 7.356	373.8 ± 6.380	16	23	0.234
	1.8 F	8	109.2 ± 2.218	341.1 ± 8.008	312.5 ± 6.518**	14	33	0.747
	0.07 W	9	110.6 ± 1.572	411.0 ± 5.255	371.9 ± 6.908	17	29	0.051
	0.2 W	9	108.3 ± 1.900	385.1 ± 5.453	356.1 ± 6.346 **	15	33	0.148
Female	Control	8	92.3 ± 1.592	212.6 ± 4.508	230.5 ± 5.420	10	18	-
	0.6 F	8	92.1 ± 1.663	216.1 ± 7.705	234.3 ± 5.753	10	17	0.287
	1.8 F	8	90.6 ± 1.463	184.8 ± 4.278	204.3 ± 5.791 **	9	18	0.969
	0.07 W	9	92.7 ± 1.770	206.0 ± 3.555	222.4 ± 4.478	11	20	0.082
	0.2 W	9	91.6 ± 1.893	209.3 ± 5.383	228.4 ± 3.857	12	18	0.173

Values: mean ± S.E.

*p<0.05

**p<0.01

Table 2: Blood components of rats on administration of LAS for 9 months (Yoneyama et. al., 1976)

Sex	Groups	WBC ( x 10^3)	RBC ( x 10^6)	HGB (g)	HCT (%)	MCV (µ^3)	MCH (µµg)	MCHC (%)
Male	Control	5.5±0.414	5.7 ± 0.142	10.9 ± 0.195	28.0 ± 0.663	49.0 ± 0.189	19.5 ± 0.247	39.5 ± 0.504
	0.6 F	4.2 ± 0.221 *	5.9 ± 0.159	11.2 ± 0.291	29.2 ± 1.059	49.6 ± 0.778	19.4 ± 0.436	38.9 ± 0.851
	1.8 F	4.9 ± 0.461	5.8 ± 0.076	10.7 ± 0.118	27.8 ± 0.561	49.2 ± 0.250	19.1 ± 0.268	38.5 ± 0.499
	0.07 W	4.7 ± 0.209	5.9 ± 0.072	11.3 ± 0.094	29.1 ± 0.317	49.2 ± 0.147	19.1 ± 0.210	38.6 ± 0.447
	0.2 W	4.9 ± 0.304	6.0 ± 0.119	11.3 ± 0.141	29.3 ± 0.566	48.8 ± 0.200	19.1 ± 0.238	38.7 ± 0.493
Female	Control	2.6 ± 0.297	5.6 ± 0.163	11.0 ± 0.243	28.8 ± 0.859	51.7 ± 0.164	20.0 ± 0.364	38.4 ± 0.689
	0.6 F	2.5 ±0.150	5.7 ± 0.265	11.2 ± 0.485	29.3 ± 1.400	51.2 ± 0.313	19.8 ± 0.356	38.5 ± 0.699
	1.8 F	3.7 ± 0.556	5.6 ± 0.294	10.5 ± 0.599	28.2 ± 1.546	50.0 ± 0.378 **	18.9 ± 0.193*	37.5 ± 0.294
	0.07 W	2.4 ± 0.213	6.0 ± 0.302	11.5 ± 0.556	30.5 ± 1.551	51.5 ± 0.176	19.5 ± 0.159	37.7 ± 0.299
	0.2 W	2.7 ± 0.328	5.6 ± 0.100	11.0 ± 0.176	28.9 ± 0.624	52.0 ± 0.463	20.0 ± 0.349	38.3 ± 0.601

Values: mean ± S.E.

*p<0.05

**p<0.01

Table 3: Biochemical values in serum of rats on administration of LAS for 9 months (Yoneyama et. al., 1976)

Sex	Groups	GOT (K.U/mL)	GPT (K.U/mL)	GLU (mg/dL)	UN (mg/dL)	CHO (mg/dL)	ALB (g/dL)	ALP ((KA-U/dL)	ChE (ΔpH)
Male	Control	151.3 ± 7.285	107.5 ± 8.177	163.0 ± 11.488	21.0 ± 0.333	77.5 ± 3.417	4.87 ± 0.055	41.3 ± 1.308	0.166 ± 0.010
	0.6 F	133.2 ± 5.573	102.3 ± 8.485	132.2 ± 4.139*	21.0 ± 0.684	71.6 ± 2.314	47.2 ± 0.075	44.7 ± 2.782	0.187 ± 0.013
	1.8 F	126.7 ± 6.439*	93.0 ± 3.665	144.7 ± 10.099	23.1 ± 0.490**	53.0 ± 1.282**	44.7 ± 0.068**	82.3 ± 3.731**	0.281 ± 0.007 **
	0.07 W	142.8 ± 10.111	108.5 ± 12.381	131.6 ± 3.088*	22.8 ± 0.647*	68.2 ± 2.527*	47.5 ± 0.059	44.0 ± 1.239	0.131 ± 0.015
	0.2 W	143.8 ± 9.041	103.3 ± 9.513	165.1 ± 14.043	21.0 ± 1.462	62.5 ± 2.534**	43.2 ± 0.251	34.4 ± 3.193	0.171 ± 0.008
Female	Control	142.3 ± 14.359	104.2 ± 12.474	135.5 ± 5.246	23.2 ± 0.793	95.8 ± 4.778	47.1 ± 0.265	45.0 ± 5.728	0.963 ± 0.073
	0.6 F	116.1 ± 5.244	68.7 ± 2.670*	126.4 ± 3.657	21.5 ± 0.602	86.7 ± 4.230	4.85 ± 0.095	40.7 ± 1.539	1.056 ± 0.023
	1.8 F	133.6 ± 4.007	88.1 ± 4.362	130.8 ± 3.945	26.7 ± 1.935	67.8 ± 7.472**	42.8 ± 0.097	73.8 ± 11.808*	0.861 ± 0.053
	0.07 W	121.2 ± 7.003	74.2 ± 13.529	127.2 ± 2.035	21.9 ± 0.872	95.0 ± 3.755	49.0 ± 0.187	45.6 ± 12.960	0.881 ± 0.171
	0.2 W	128.5 ± 12.209	85.7 ± 9.362	136.1 ± 10.070	21.7 ± 0.950	77.2 ± 8.826	46.8 ± 0.155	44.0 ± 5.756	1.053 ± 0.042

Values: mean ± S.E.

*p<0.05

**p<0.01

Table 4:  Organ weight of male and female rats on administration of LAS for 9 months (Yoneyama et. al., 1976)

Sex

Groups

Brain

Heart

Lung

Liver

Spleen

Kidney (R)

Kidney (L)

Adrenal (R)

Adrenal (L)

Testis(R)

Testis(L)

Ovary (R)

Ovary (L)

Caecum

Uterus

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

g

g/100g

Male

Control

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 ± 0.40

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

-

-

-

-

1.238 ± 0.068

0.281 ± 0.015

-

-

0.6 F

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

-

-

-

-

1.303 ± 0.057

0.305 ± 0.010

-

-

1.8 F

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**

-

-

-

-

2.205 ± 0.141 **

0.647 ± 0.032**

-

-

0.07 W

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

-

-

-

-

1.338 ± 0.040

0.314 ± 0.009

-

-

0.2 W

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

-

-

-

-

1.210 ± 0.035

0.309 ± 0.008

-

-

Female

Control

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 ± 1.226

31.2 ± 4.007

14.2 ± 1.857

0.828 ± 0.010

0.377 ± 0.005

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

0.859 ± 0.064

0.381 ± 0.015

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

1.436 ± 0.080**

0.775 ± 0.039**

0.588 ± 0.066

0.312 ± 0.034

0.07 W

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 ± 0.780

31.7 ± 2.300

12.2 ± 1.200

0.785 ± 0.051

0.358 ± 0.024

0.729 ± 0.051

0.331 ± 0.022

0.2 W

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

0.800 ± 0.045

0.379 ± 0.026

0.644 ± 0.032

0.308 ± 0.014

Values: mean ± S.E.

*p<0.05

**p<0.01

Table 5: Liver enzyme activities of rats on administration of LAS for 9 months (Yoneyama et. al., 1976)

Sex	Groups	G6 Pase (µmoles Pi/mg protein /hr	G6P-DH (x 10^-3)ΔE/mg protein / min	LDH ΔE/mg protein / min	GPT (x 10^2) KU/mg protein	GOT (x 10^2) KU/mg protein
Male	Control	3.77 ± 0.22	60.3 ± 9.9	11.33 ± 0.53	4.06 ± 0.345	5.80 ± 0.525
	0.6 F	3.82 ± 0.50	40.9 ± 9.1 **	10.28 ± 0.84*	3.94 ± 0.484	5.49 ± 0.810
	1.8 F	2.60 ± 0.015**	36.0 ± 7.7**	9.26 ± 0.99**	3.09 ± 0.225**	4.95 ± 0.510**
	0.07 W	3.57 ± 0.36	68.0 ± 15.1	10.92 ± 0.91	3.89 ± 0.360	5.61 ± 0.410
	0.2 W	3.71 ± 0.35	56.8 ± 14.9	9.28 ± 0.71**	4.10 ± 0.420	6.16 ± 0.545
Female	Control	2.73 ± 0.57	143.6 ± 30.0	70.6 ± 11.6	2.71 ± 0.405	4.91 ± 0.635
	0.6 F	2.67 ± 0.51	100.7 ± 39.9*	63.4 ± 14.2	2.60 ± 0.505	4.30 ± 0.560
	1.8 F	2.17 ± 0.15*	58.5 ± 12.2*	62.2 ± 4.7	2.37 ± 0.225	4.05 ± 0.450 **
	0.07 W	2.61 ± 0.20	138.3 ± 41.0	65.8 ± 8.5	2.57 ± 2.290	5.61 ± 0.410*
	0.2 W	2.76 ± 0.28	120.9 ± 27.6	66.5 ± 11.3	2.89 ± 0.195	6.16 ± 0.545**

Values: mean ± S.D.

*p<0.05

**p<0.01

Table 6: Kidney enzyme activities of rats on administration of LAS for 9 months  (Yoneyama et. al., 1976)

Sex	Groups	ALP (µmoles/mg/hr)	ACP (µmoles/mg/hr)	G6Pase (µmoles/mg/hr)	Na, K-A Tpase (µmoles/mg/hr)	GPT (KU/mL)	GOT (KU/mL)	LDH (ΔE/mg/min)	MDH (ΔE/mg/min)
Male	Control	16.37 ± 1.984	1.80 ± 0.188	3.97 ± 0.590	3.94 ± 0.613	23.99 ± 5.300	192.11 ± 35.741	0.339 ± 0.043	2.308 ± 0.0447
	0.6 F	14.44 ± 2.046	1.72 ± 0.141	3.92 ± 0.618	3.63 ± 0.640	23.16 ± 5.885	199.95 ± 22.869	0.334 ± 0.050	2.325 ± 0.575
	1.8 F	15.74 ± 1.933	1.78 ± 0.179	3.47 ± 0.732	3.32 ± 1.311	21.11 ± 4.690	194.18 ± 13.668	0.358 ± 0.070	2.354 ± 0.554
	0.07 W	14.73 ± 2.387	1.85 ± 0.194	4.26 ± 0.547	3.52 ± 1.170	23.85 ± 4.640	207.39 ± 21.308	0.347 ± 0.098	2.617 ± 0.628
	0.2 W	15.03 ± 1.169	1.66 ± 0.109	4.03 ± 0.428	3.09 ± 0.224*	24.44 ± 3.535	202.94 ± 15.708	0.362 ± 0.058	2.679 ± 0.740
Female	Control	15.94 ± 3.369	2.02 ± 0.182	3.96 ± 0.545	4.29 ± 1.027	28.27 ± 4.407	188.33 ± 21.189	0.350 ± 0.035	2.387 ± 0.601
	0.6 F	17.11 ± 2.919	2.03 ± 0.179	3.63 ± 0.521	4.29 ± 0.656	30.26 ± 5.736	196.48 ± 21.273	0.338 ± 0.044	2.231 ± 0.433
	1.8 F	16.86 ± 2.369	1.85 ± 0.195	3.13 ± 0.426**	3.19 ± 0.613*	32.60 ± 3.737	184.89 ± 11.276	0.291 ± 0.025**	2.181 ± 0.404
	0.07 W	16.24 ± 2.381	1.98 ± 0.218	3.72 ± 0.443	4.14 ± 1.452	27.19 ± 3.319	198.09 ± 29.803	0.322 ± 0.035	2.052 ± 0.306
	0.2 W	15.02 ± 3.244	1.90 ± 0.136	3.45 ± 0.651	4.59 ± 1.154	27.03 ± 4.430	187.36 ± 20.235	0.316 ± 0.052	2.070 ± 0.362

Values: mean ± S.D.

*p<0.05

**p<0.01

Conclusions:

Administration of LAS to Wistar JCL rats by test diets at dose levels of 0, 0.6 and 1.8% for 9 months (focusing on the liver and kidneys) revealed an LOAEL of 0.6% (300 mg/kg bw/day in diet), based on adverse effects at all dose levels.
Administration of LAS to Wistar JCL rats in drinking water at dose levels of 0, 0.07 and 0.2% for 9 months (focusing on the liver and kidneys) revealed an NOAEL of 0.07% (85 mg/kg bw/day in drinking water), based on 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:

A sub-chronic (9 months) repeated dose toxicity study was conducted with C10-14 LAS, sodium salt in male and female Wistar JCL rats. The test substance was administered at the following dose levels: through diet at 0, 0.6 and 1.8% (equivalent to 0, 300 and 900 mg/kg bw/day) to 8 animals/sex/dose and via drinking water at 0, 0.07 and 0.2% (equivalent to 0, 85 and 145 mg/kg bw/day to 9 animals/sex/dose. Rats at the 0.6 and 1.8% dose group exhibited severe weight loss so LAS administration via diet was stopped after 2 weeks. Clinical observations, water consumption, food consumption and body weights were recorded weekly. Terminal blood collection was done for estimation of haematological and clinical chemistry parameters. Gross findings were observed after euthanizing animals and organs were removed for organ weight measurements. No histopathology was performed. Body weight gain was reduced at the highest dose group only. Significant decreases in transaminase activity and renal Na,K-ATPase were seen at 145 mg/kg bw/day. No significant haematological or organ weight changes were noted. Based on significant decrease in the liver and renal enzyme levels at 145 mg/kg bw/day, the systemic toxicity NOAEL was established at 85 mg/kg bw/day (Yoneyama, 1976).  

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1972

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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 alkylbenzene sulfonic 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 were performed.

GLP compliance:

no

Remarks:

(pre-dates GLP)

Limit test:

no

Species:

rat

Strain:

other: Wistar SLC

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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

IN-LIFE DATES: From: May 30, 1972 To: Nov. 27, 1972

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:

other: 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 haemoglobin, hematocrit, red blood cells, white blood fractions and platelets) and during euthanasia (erythrocyte membrane resistance)
- Anesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All surviving animals
- Parameters: Haemoglobin, haematocrit, 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 along with stomach, large and small intestines, pancreas, epididymis, skin and bones) were weighed, examined and subjected to microscopy through H.E., PAS, lipid and silver staining.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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 their lower abdomen was dirty.

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, while the 0.6% dose groups showed slight but not significant trends to suppress weight gain.

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.

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 relative to the control

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- A significant decrease in haematocrit and haemoglobin, 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 findings:

effects observed, treatment-related

Description (incidence and severity):

There was dose dependent significant increase in ALP levels (at 0.6 and 1.8% dose groups and significant decrease in total protein (at 1.8% dose group).

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):

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:

effects observed, non-treatment-related

Description (incidence and severity):

- 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 s welling 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 there on, the ileum was filled with a semi-transparent, viscous, gelatinous substance.

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.

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% and 1.8% treated groups)
- Glomeruli contributed to overall swelling through dilation of vessel lumen and interstitial swelling (significant in male/female rats of 0.2 and 0.07% dose groups. They were not very significant in the 1.8 and 0.6% dose groups)
- The glomerular interstitum had thawn mildly but chronically (present in control group but was significant for male/female rats in 1.8 and 0.6% groups)
- Cloudy swelling/vacuolar degeneration of proximal renal tubular epithelial cells were seen (mildly present in control group and slightly significant in LAS treated groups).
- Small, yellowish brown blobs the size of nuclei was 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
-Lumen dilation of the paroximal renal tube and flattening of epithelial cells (significantly in males of 0.2% dose group, no effect observed in females and control groups)
- The renal tubule had collapsed (significantly in male/females of 1.8% dose group)
- Swelling of surrounding connective tissue was observed in the interstitium (observed in control group and 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.

Further details on are provided in "Any other information on results inc.tables"

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Suppression of weight gain, increase in appendix weight, alteration in hematological and clinical 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).

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, detergent, water intake and faeces excretion in males (Yoneyama et al., 1972)

Group	No. of Rats	Initial body wt (g) (mean±S.E)	Final body wt (g) (mean±S.E)	Body weight gain (1) (%) (mean±S.E)	Food (g/rat/day)	Detergent		water (g/rat/day)	Feces (g/rat/day)
						mg/rat/day	mg/kg/day
Control	10	85±0.871	387±9.509	452.1±9.841	14.17	-	-	16.9	2.31
1.80%	10-9	85.2±0.553	290.00±5.206	341.09±5.136**	9.84	177.12	814.8	13.29	1.78
0.60%	10	86.40±1.543	365.80±8.517	426.23±8.893	13.84	83.04	322.3	16.28	2.23
0.20%	10	84.50±0.957	383.40±6.715	453.92±7.603	13.7	27.4	101.5	18.18	2.28
0.07%	10	80.9±1.923	387.10±5.930	480.74±13.113	14.48	10.14	37.2	16.91	2.24

Table 2:  Body weight gain, food, detergent, water intake and feces excretion in females (Yoneyama et al., 1972)

Group	No. of Rats	Initial body wt (g) (mean±S.E)	Final body wt (g) (mean±S.E)	Body weight gain (%) (1) (mean±S.E)	Food (g/rat/day)	Detergent		water (g/rat/day)	Feces (g/rat/day)
						mg/rat/day	mg/kg/day
Control	10	71.80±0.512	202.50±2.325	282.24±4.422	9.35	-	-	17.48	1.53
1.80%	10	74.30±0.943	158.80±3.411	214.02±5.288**	7.01	126.18	913	12.14	1.23
0.60%	10	74.10±0.673	195.80±4.149	264.25±5.207*	9.04	54.24	332.2	13.99	1.53
0.20%	10	74.30±1.116	201.10±3.334	271.05±5.314	8.9	17.8	108.7	17.69	1.39
0.07%	10	76.00±2.683	203.20±7.172	269.35±10.255	9.35	6.55	39.4	19.56	1.63

where (1) refers to Final BW/Initial BW-1) into100

*P<0.05

**P<0.01

Table 3 : Haematology-I in males and females rats (Yoneyama et al., 1972)

Group	No. of Rats	RBC*10^4/mm^3	Ht (%)	Hb (g/dl)	MCH (rr)	MCV (µ8)	MCC%
Male
Control	10	910±13	53.1±0.4	19.8±0.3	21.8±0.4	58.4±1.0	37.4±0.6
1.80%	9	924±27^1)	54.6±0.4	20.6±0.5	22.4±0.8	59.3±1.3	37.7±0.8
0.60%	10	890±14	54.0±0.4	19.8±0.1	22.3±0.3	60.7±0.9	36.7±0.1
0.20%	10	914±25	53.3±0.3	19.5±0.1	21.5±0.6	58.6±1.6	36.7±0.2
0.07%	10	925±20	53.3±0.4	19.6±0.1	21.2±0.5	57.8±1.2	36.7±0.2
Female
Control	10	838±22	52.1±0.5	19.9±0.5	23.9±0.9	62.5±1.6	38.2±1.0
1.80%	10	785±14	49.5±0.3**	18.0±0.1**	23.0±0.4	63.2±0.9	36.4±0.1
0.60%	10	795±17	50.9±0.3	19.0±0.1	24.0±0.5	64.2±1.1	37.4±0.2
0.20%	10	778±16	51.1±0.2	19.1±0.1	24.7±0.4	65.9±1.3	37.5±0.2
0.07%	10	806±18	50.6±0.4	18.9±0.1	23.6±0.6	63.0±1.5	37.4±0.3

Table 4 : Haematology-II in males and females rats (Yoneyama et al., 1972)

Group	No. of Rats	Thrombocyte*10^4(A)	Osmotic fragility (A)	Whole blood clotting time (min)
				Beginning	Ending
Male
Control	10	70.5±9 (8)	213.6±9.4 (9)	1.06±0.13	2.12±0.26
1.80%	9	70.2±4.9 (8)	199.9±6.6 (9)	1.14±0.11	2.32±0.27
0.60%	10	71.6±8.4	211.4±7.9	1.42±0.23	2.71±0.29
0.20%	10	64.4±5.5(9)	228.1±6.5(9)	1.34±0.19	2.50±0.35
0.07%	10	80.1±13.2	225.1±10.5	1.40±0.19	2.52±0.33
Female
Control	10	52.0±3.8 (9)	240.4±7.5 (8)	1.29±0.29	2.95±0.30
1.80%	10	58.4±4.5 (9)	181.8±6.5(8)*	1.37±0.14	2.57±0.19
0.60%	10	54.8±3.3 (9)	226.0±5.9 (9)	1.24±0.17	2.81±0.22
0.20%	10	59.8±5.9 (9)	245.4±9.3(9)	1.26±0.19	2.92±0.22
0.07%	10	63.3±5.3 (8)	248.4±10.2	1.22±0.21	3.30±0.41

*P<0.05

**P<0.01

(A): No of rats

¹⁾ (mean±SE)

Table 5: Percent differential of Leucocytes (Yoneyama et al., 1972)

Group	No. of Rats	Neutro	Lympho	Mono	Eosino
Male
Control	10	9.8±0. 88 (1)	89.08±0.954	0.28±0.102	0.83±0.203
1.80%	9	12.09±0.698*	87.22±0.624	0.09±0.061	0.58±0.164
0.60%	10	10.77±1.017	87.82±0.860	0.26±0.094	0.60±0.158
0.20%	10	10.34±0.521	88.22±0.625	0.40±0.102	0.62±0.128
0.07%	10	9.41±1.074	89.35 ± 0.998	0.50± 0.088	0.73±0.125
Female
Control	10	8.24±0.56	90.85±0.685	0.18±0.073	0.72±0.150
1.80%	10	8.67±0.747	90.61 ± 0.762	0.28±0.076	0.42±0.108
0.60%	10	10.21±1 .054	88.49±1.065	0.32±0.071	0.96±0.143
0.20%	10	8.75±0.754	90.15±0.781	0.38±0.096	0.70±0.146
0.07%	10	8.65±0.752	90.39 ± 0.771	0.40±0.099	0.54±0.108

Table 6: Counts differential of leucocytes.10²/mm³ (Yoneyama et al., 1972)

Group	No. of Rats	WBC	Neutro	Lympho	Mono	Eosino
Male
Control	10	88.12±3.686 (1)	8.49±0.678	78.65±3.779	0.24±0.087	0.67±0.148
1.80%	9	96.16±5.726	11.78±1.113	83.76±4.805	0.08 ±0.057	0.54±0.142
0.60%	10	91.47±5.430	9.96±1.154	80.27±4.686	0.25±0.088	0.58±0.142
0.20%	10	90.55±6.435	9.61±1.166	79.64±5.196	0.36 ±0.089	0.60±0.155
0.07%	10	96.15±3.329	9.08±1.077	85.85±2.913	0.50±0.093	0.71±0.135
Female
Control	10	92.00±3.802	7.59±0.579	83.57±3.517	0.16±0.066	0.67±0.139
1.80%	10	109.30±3.831**	9.34±0.664	95.03 ± 7.208	0.31 ± 0.085	0.47±0.120
0.60%	10	87.90±3.631	8.87±0.847	77.89±3.679	0.26±0.058	0.85±0.133
0.20%	10	83.15±4.350	7.38±0.637	76.67 ± 5.063	0.32±0.078	0.61 ± 0.138
0.07%	10	94.40±5.110	8.36±1.176	85.13±4.180	0.37±0.091	0.51 ± 0.101

*P<0.05

**P<0.01

(A): No of rats

¹⁾(mean±SE)

Table 7: Serum Chemistry (Yoneyama et al., 1972)

Group	No. of Rats	ALP K-A Unit/dl	LAP G-R Unit	GOT karmen unit/ml	UN mg/dl	A/G ratio	Total protein g/dl	Bililubin
								(Indirect) mg/dl	(Direct) mg/dl	Total mg/dl
Male
Control	10	24.07±1.109^1)	185.81± 9.586	132.75± 6.357	16.92±1.102	1.42±0.082	7.36±0.071	0.20±0.050	0.11±0.036	0.29±0.044
1.80%	9	44.96±4.01**	179.80± 9.74	139.12± 9.397	16.30±0.634	1.59±0.129	6.55±0.191**	0.40±0.250	0.12±0.027	0.48±0.268
0.60%	10	29.22±1.093**	180.54± 7.897	133.50± 5.559	15.76±0.611	1.46±0.096	7.20±0.084	0.30±0.201	0.32±0.211	0.56±0.415
0.20%	10	24.34±1.173	181.23± 9.637	140.25±14.732	13.04±1.224*	1.47±0.091	7.20±0.059	0.41±0.231	0.31±0.188	0.68±0.422
0.07%	10	25.48±0.816	178.99± 9.4271	144.60±11.501	15.36±1.221	1.38±0.082	7.26±0.118	0.35±0.201	0.34±0.197	0.64±0.401
Female
Control	10	24.41±1.380	198.54± 8.899	156.30±12.716	17.52±1.733	1.53±0.062	7.80±0.122	0.12±0.050	0.14±0.054	0.23±0.054
1.80%	10	61.41±6.124**	208.72± 9.097	154.60±8.297	19.00±1.043	1.76±0.121	6.42±0.127**	0.12±0.025 (9)	0.12±0.033 (9)	0.25±0.054 (9)
0.60%	10	31.80±1.599**	187.58± 10.642	139.50±3.024	17.15±2.020	1.70±0.097**	7.22±0.186*	0.14±0.036	0.12±0.038	0.27±0.062
0.20%	10	27.23±1.049	193.65± 7.686	146.55±9.144	14.14±1.404	1.48±0.164	7.38±0.184	0.20±0.044 (9)	0.11±0.023 (9)	0.31±0.052 (9)
0.07%	10	25.77±1.237	192.23± 8.056	128.25±5.432	15.09±1.245	1.72±0.067	7.68±0.163	0.15±0.059 (9)	0.10±0.018 (9)	0.24±0.06 (9)

where:

*P<0.05

**P<0.01

(A): No of rats

¹⁾(mean±SE)

Table 8 : Urinalysis in female and male rats (Yoneyama et al., 1972)

Group	No. of Rats	pH				Protein					Glucose		Keton			Blood			Bilirubin		Urobilin
		8	7	6	5	-	±	+	++	+++	-	+	-	±	+	-	±	+	-	+	0.1*	1
Male
Control	10	1	4	5	-	-	-	7	2	1	10	-	7	3	-	10	-	-	10	-	10	-
1.80%	9	-	-	9	-	-	-	2	3	4	9	-	2	7	-	9	-	-	9	-	9	-
0.60%	10	-	5	5	-	-	2	5	2	1	10	-	5	5	-	10	-	-	10	-	10	-
0.20%	10	-	2	8	-	-	4	3	3		10	-	6	4	-	10	-	-	10	-	10	-
0.07%	10	-	4	6	-	1	-	6	1	2	10	-	8	2	-	10	-	-	10	-	10	-
Female
Control	10	2	2	6	-	2	2	5	1	-	10	-	5	5	-	10	-	-	10	-	10	-
1.80%	10	1	-	9	-	3	3	4	-	-	10	-	4	5	-	10	-	-	10	-	9	-
0.60%	10	-	5	5	-	2	3	2	3	-	10	-	7	3	-	10	-	-	10	-	10	-
0.20%	10	-	3	7	-	2	2	5	1	-	10	-	4	6	-	10	-	-	10	-	10	-
0.07%	10	-	3	7	-	3	3	4	-	-	10	-	7	3	-	10	-	-	10	-	9	-

*Ehrlich units

Table 9: Organ Weights (Male-1) (Yoneyama et al., 1972)

Group	No. of rats	Heart [Actual weight (mg)/weight (mg)/100 g]	Spleen [Actual weight (mg)/weight (mg)/100 g]	Thymus [Actual weight (mg)/weight (mg)/100 g]	Liver [Actual weight (g)/weight (g)/100 g]	Lung [Actual weight (mg)/weight (mg)/100 g]	Kidneys [Actual weight (mg)/weight (mg)/100 g]		Hypophysis [Actual weight (mg)/weight (mg)/100 g]
							R	L
Control	10	930±13/241± 3^1)	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
1.80%	9	694±11**/239±3	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
0.60%	10	878±18*/235± 4	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
0.20%	10	918±16/239± 2	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
0.07%	10	932±16/240± 2	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

Table 10: Organ Weights (Male-2) (Yoneyama et al., 1972)

Group	No. of rats	Thyroid glands [Actual weight (mg)/weight (mg)/100 g]	Supranal gland [Actual weight (mg)/weight (mg)/100 g]		Testis [Actual weight (mg)/weight (mg)/100 g]		Prostate glands [Actual weight (mg)/weight (mg)/100 g]	Brain [Actual weight (mg)/weight (mg)/100 g]	Caecum [Actual weight (mg)/weight (mg)/100 g]
			R	L	R	L
Control	10	14.4±0.421^1)/3.7±0.16	18.0±0.61/4.6±0.18	19.3±0.44/5.0±0.17	1592±20/412± 7	1622±21/420± 6	252±16/65±4.8	1983±12/515±11	837±34/216± 8
1.80%	9	11.5±0.76**/3.9±0.24	15.5±0.44**/5.3±0.17*	17.1±0.65*/5.9±0.27*	1513±23*/522±7**	1501±36**/518±13**	231±21/79±7.0	1960±22/677±11 **	1484±30**/512±12**
0.60%	10	13.2±0.69/3.5±0.22	17.5±0.76/4.6±0.20	18.2±0.69/4.8±0.19	1576±27/422± 11	1612±36/432±11	231±21/61±5.7	1969±18*/528±12	939±41/251±11*
0.20%	10	13.3±0.57/3.4±0.11	17.3±0.61/4.5±0.17	18.6±0.84/4.8±0.23	1564±22/408± 5	1621±20/423± 4	298±21/78±5.9	2020±11/528±8	971±35*/253 ± 7**
0.07%	10	13.6±0.54/3.5±0.13	16.8±0.85/4.3± 0.22	18.7±0.44/4.8± 0.11	1643±14/425± 6	1676±15/434± 7	318±21*/82±6.1	1918±106/494±25	938±38/242± 9

where,

*P<0.05

**P<0.01

(A): No of rats

¹⁾(mean±SE)

Table 11: Organ Weights (Female-1) (Yoneyama et al., 1972)

Group	No. of rats	Heart [Actual weight (mg)/weight (mg)/100 g]	Spleen [Actual weight (mg)/weight (mg)/100 g]	Thymus [Actual weight (mg)/weight (mg)/100 g]	Liver [Actual weight (g)/weight (g)/100 g]	Lung [Actual weight (mg)/weight (mg)/100 g]	Kidneys [Actual weight (mg)/weight (mg)/100 g]		Hypophysis [Actual weight (mg)/weight (mg)/100 g]
							R	L
Control	10	596±100^1)/265± 6	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
1.80%	10	426± 11**/269±7*	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±.63
0.60%	10	559± 11*/286± 5	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*
0.20%	10	574±13/258± 5	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
0.07%	10	596±16/294± 3	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

Table 12: Organ Weights (Female-2) (Yoneyama et al., 1972)

Group	No. of rats	Thyroid glands [Actual weight (mg)/weight (mg)/100 g]	Supranal gland [Actual weight (mg)/weight (mg)/100 g]		Ovary [Actual weight (mg)/weight (mg)/100 g]		Uterus [Actual weight (mg)/weight (mg)/100 g]	Brain [Actual weight (mg)/weight (mg)/100 g]	Caecum [Actual weight (mg)/weight (mg)/100 g]
			R	L	R	L
Control	10	11.2±0.321^1)/ 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
1.80%	10	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**
0.60%	10	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
0.20%	10	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
0.07%	10	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

where,

*P<0.05

**P<0.01

(A): No of rats

¹⁾(mean±SE)

Table 13: Summary of microscopic  changes in kindey (Yoneyama et al., 1972)

Microscopic findings	#	Male					Female
		C	LAS				C	LAS
			0.07%	0.2%	0.6%	1.8%		0.07%	0.2%	0.6%	1.8%
Swelling in glomerulus	S E M O S L	0 2 (10) 2	0 5 (10) 4	1 6 (10) 3	0 7 (10) 2	0 5 (9) 4	0 2 (10) 3	1 5 (10) 4	1 5 (10) 4	0 7 (10) 3	0 5 (10) 5
Histolysis in glomerulus	S E M O S L	0 2 2	0 7 2	1 5 4	1 5 4	1 5 3	0 3 1	0 5 5	0 9 4	1 6 3	1 5 4
Atrophy in glomerulus	S E M O S L	0 1 1	0 5 5	1 6 2	2 7 1	3 6 0	0 1 1	0 4 5	0 6 3	1 7 2	4 5 1
Cloudy swelling in epithelium of proximal tubules	S E M O S L	0 0 4	0 1 5	0 6 2	0 8 2	0 7 2	0 0 3	0 0 4	0 3 3	0 3 3	0 4 5
Vacuolation in epithelium of proximal tubules	S E M O S L	0 0 4	0 6 4	0 3 7	0 4 6	0 5 4	0 1 5	0 7 3	0 6 4	0 7 3	0 7 3
Lipofuscin in epithelium of proximal tubules	S E M O S L	0 0 0	0 0 0	0 0 5	0 0 5	2 7 0	0 0 0	0 0 0	0 0 1	0 0 4	5 5 0
PAS-positive materies in loop of Henle	S E M O S L						0 0 4	0 3 7	0 3 6	0 3 6	0 0 3
Dilatation in lumina of distal tubules	S E M O S L	0 0 2	0 4 6	3 3 4	2 4 4	0 5 4	0 0 0	0 0 5	0 3 3	0 5 5	0 3 5
Decrease in cell height of epithelium in distal tubules	S E M O S L	0 0 1	0 0 6	2 3 5	2 3 4	0 4 5	0 0 0	0 0 3	0 0 5	0 3 5	0 3 5
Destruction in urinary tubules	S E M O S L	0 0 0	0 0 0	0 0 2	0 2 3	0 3 4	0 0 0	0 0 2	0 0 3	0 0 5	0 4 5
edema in stroma	S E M O S L	0 1 2	1 5 4	1 6 3	1 3 5	0 4 5	0 1 1	1 4 5	1 5 4	1 6 3	0 2 7
Swelling in perivascular connective tissues	S E M O S L	0 1 1	1 4 4	1 3 6	0 5 5	0 3 4	0 1 1	0 4 5	0 5 5	0 7 3	0 1 6
Proliferation in interstitial connective tissues	S E M O S L	0 0 1	0 0 5	0 0 5	0 1 4	0 2 6	0 0 2	0 0 2	0 0 5	0 0 6	0 0 6

where, SE: Severe

SL: Slight

MO: Moderate

(A) No. of rats

Table 14:  Summary of microscopic changes in liver and large intestine (Yoneyama et al., 1972)

Microscopic findings	#	Male					Female
		C	LAS				C	LAS
			0.07%	0.2%	0.6%	1.8%		0.07%	0.2%	0.6%	1.8%
Decrease in basoplam in liver cells	S E M O S L	0 0 (10) 0	0 0 (10) 0	0 0 (10) 1	0 0 (10) 1	0 0 (9) 6	0 0 (10) 0	0 0 (10) 0	0 0 (10) 0	0 0 (10) 3	0 1 (10) 6
Increase in eosinophilia in liver cells	S E M O S L	0 0 1	0 0 0	0 0 0	0 0 0	0 0 6	0 0 1	0 0 0	0 0 0	0 0 2	0 2 7
Swelling in liver cells	S E M O S L	0 0 0	0 0 0	0 0 0	0 3 3	0 3 3	0 0 0	0 0 0	0 1 3	0 1 4	0 2 6
Rarefaction in cytoplasm of liver cells	S E M O S L	0 0 3	0 0 8	0 1 6	0 1 6	0 1 6	0 0 2	0 0 6	0 1 7	0 1 7	0 1 7
Decrease in cell height of epithelium in large instestine	S E M O S L	0 0 0	0 0 0	0 0 2	0 0 4	0 0 5	0 0 0	0 0 0	0 0 3	0 1 4	0 1 4

where, SE: Severe

SL: Slight

MO: Moderate

(A) No. of rats

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, haematological and/or clinical chemistry parameters changes at higher dose levels (115 mg/kg bw/day and above).

Executive summary:

A sub-chronic repeated dose toxicity study was conducted with C10-14 LAS, sodium salt in Wistar SLC rats. The test substance was administered to groups of 10 male and female rats/dose in the diet for 26 weeks at doses of 0, 40, 115, 340 and 1030 mg/kg/day. 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 pathological examinations were observed after necropsy and the major organs were subjected to histopathological examinations. Significant diarrhoea, suppressed growth, increased caecal weight and degeneration of renal tubes were observed in the highest dose group. Similar but less severe signs were seen in other doses with the exception of the lowest dose of 40 mg/kg/day, which showed no adverse effects related to exposure to the LAS. Under the study conditions, systemic toxicity NOAEL was determined to be 40 mg/kg/day, based on tissue damage in caecum, kidney and liver, haematological as well as clinical chemistry parameters changes at higher dose levels (Yoneyama, 1972). 

Reference 4

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

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:

other: CRJ-SD rats

Sex:

male/female

Details on test animals or test system 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

EXPERIMENTAL START DATE: Not reported

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered orally through a metallic gastric sonde.

Vehicle:

other: Distilled water

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 of 0, 125, 250 and 500 mg/kg bw.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

29 days for males and 30 days for females

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: Haemoglobin, haematocrit, red and white blood cell count were measured using an auto-analyzer 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.

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 alkaline phosphatase (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 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

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

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/day.

Critical effects observed:

not specified

Table 1: Effect of LAS on average feed efficiency of rats after 1 month (po) exposure (Ito et al., 1978)

Sex	Doses	2 weeks			4 weeks
		Total intake (g)	Net gain (g)	Efficacy (%)	Total intake (g)	Net gain (g)	Efficacy (%)
Males	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	284.2	76.5	27	633.5	152.4	22.7
Females	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

Table 2: Effect of LAS on haematological parameters of rats after 1 month (po) exposure (Ito et al., 1978)

Sex	Doses	RBC (10^6/mm^3)	Hb (g/dL)	Ht (%)	WBC (10^3/mm^3)
Males	0	7.5 ± 0.11	16.5 ± 0.17	48.8 ± 0.63	16.5 ± 1.04
	125	7.4 ± 0.08	16.0 ± 0.13	47.4 ± 0.49	17.4 ± 1.12
	250	7.4 ± 0.17	15.9 ± 0.36	47.4 ± 1.13	17.8 ± 1.30
	500	7.5 ± 0.07	16.0 ± 0.12	48.1 ± 0.34	18.8 ± 1.41
Females	0	6.6 ± 0.15	15.2 ± 0.18	44.0 ± 0.65	12.1 ± 0.49
	125	6.6 ± 0.15	15.2 ± 0.15	44.4 ± 0.56	12.0 ± 0.87
	250	6.6 ± 0.11	15.2 ± 0.13	44.5 ± 0.51	13.5 ± 0.91
	500	6.8 ± 0.06	15.2 ± 0.10	44.2 ± 0.41	14.5 ± 1.31

Mean ± SE

* Significantly different from control (p<0.05)

** Significantly different from control (p<0.01)

Table 3a: Effect of LAS on biochemical parameters of rats after 1 month (po) exposure (Ito et al., 1978)

Sex	Dose (mg/kg)	sGOT	sGPT	ALP	TP	Alb	Glu	Chol
		mu/mL	mu/mL	mu/mL	mg%	mg%	mg%	mg%
Males	0	137.0± 5.4	44.5 ± 1.6	294.8 ± 11.6	5.4 ± 0.08	3.3 ± 0.05	228.7 ± 5.8	55.4 ± 1.27
	125	134.3 ± 8.4	39.5 ± 1.9	280.2 ± 17.5	5.1 ± 0.14	3.2 ± 0.08	228.9 ± 9.0	60.8* ± 2.38
	250	133.3 ± 7.3	39.3 ± 1.5*	311.8 ± 26.6	5.1 ± 0.27	3.1 ± 0.16	225.3 ± 11.5	54.5 ± 2.48
	500	145.4 ± 7.3	45.0 ± 2.1	374.0* ± 30.0	5.2 ± 0.07	3.3 ± 0.04	218.3 ± 3.5	53.4 ± 1.25
Females	0	130.3 ± 6.5	48.5 ± 1.1	184.9 ± 8.2	6.1 ± 0.08	3.8 ± 0.05	224.1 ± 5.7	67.7 ± 2.24
	125	141.0 ± 7.3	44.4 ± 1.1	205.2 ± 10.0	5.8** ± 0.7	3.7* ± 0.05	231.7 ± 8.4	63.9 ± 1.89
	250	134.5 ± 7.1	39.9 ± 0.8*	182.5 ± 11.5	5.7* ± 0.16	3.5** ± 0.09	216.2 ± 7.1	64.9 ± 2.81
	500	162.5* ± 10.7	44.1 ± 2.0	222.7 ± 17.9	5.7** ± 0.08	3.6** ± 0.05	207.8* ± 5.5	62.3 ± 2.30

Mean ± SE

* Significantly different from control (p<0.05)

** Significantly different from control (p<0.01)

Table 3b: Effect of LAS on biochemical parameters of rats after 1 month (po) exposure (Ito et al., 1978)

Sex	Dose (mg/kg)	T-Bili	Creat	BUN	InP	Na	K	Ca	Mg	Cl
		mg%	mg%	mg%	mg%	meg/L	meg/L	mg/dL	mg/dL	meg/L
Males	0	0.9 ± 0.03	0.5 ± 0.01	17.4 ± 0.99	6.8 ± 0.14	147.6 ± 1.7	5.0 ± 0.10	5.1 ± 0.07	2.5 ± 0.04	102.5 ± 0.62
	125	0.8 ± 0.04	0.5 ± 0.02	17.1 ± 1.32	6.8 ± 0.16	142.3 ± 2.0	4.8 ± 0.14	4.6* ± 0.14	2.5 ± 0.10	103.7 ± 0.65
	250	0.8 ± 0.04	0.4 ±0.03	17.0 ± 0.09	6.4 ± 0.26	135.8* ± 3.1	4.5** ± 0.11	4.6** ± 0.15	2.2** ± 0.05	104.6 ± 0.77
	500	0.9 ± 0.02	0.5 ± 0.02	19.3 ± 1.17	6.8 ± 0.10	141.5 ± 2.9	5.0 ± 0.17	4.8* ± 0.12	2.4 ± 0.06	104.4 ± 1.22
Females	0	0.9 ± 0.09	0.5 ± 0.02	17.9 ± 0.82	6.2 ± 0.14	143 ± 1.3	4.5 ± 0.09	5.2 ± 0.08	2.5 ± 0.07	105.4 ± 0.43
	125	0.8 ± 0.02	0.5 ± 0.01	17.9 ± 1.51	6.3 ± 0.23	143.4 ± 1.9	4.5 ± 0.08	5.0 ± 0.11	2.5 ± 0.07	106.8 ± 0.66
	250	0.8 ± 0.05	0.5 ± 0.01	18.7 ± 0.99	6.3 ± 0.22	145.3 ± 1.5	4.4 ± 0.15	4.9* ± 0.06	2.5 ± 0.06	106.8 ± 0.54
	500	0.9 ± 0.06	0.6 ± 0.02	24.0* ± 1.41	6.1 ± 0.21	142.6 ± 1.2	4.5 ± 0.08	4.9** ± 0.08	2.4 ± 50.0	106.6 ± 0.42

Mean ± SE

* Significantly different from control (p<0.05)

** Significantly different from control (p<0.01)

Table 4a: Effect of LAS on organ weight of rats after 1 month (po) exposure (Ito et al., 1978)

Sex

Dose (mg/kg)

Body weight (g)

Liver

Kidney

Spleen

Adrenal gland

Right

Left

Right

Left

wt (g)

%

wt (g)

%

wt (g)

%

wt (g)

%

wt (mg)

%

wt (mg)

%

Males

0

370.7± 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

Females

0

262.6± 8.0

10.84 ± 0.51

4.11 ± 0.08

1.09 ± 0.04

0.41 ± 0.01

1.08 ± 0.05

0.41 ± 0.2

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

Mean ± SE

* Significantly different from control (p<0.05)

** Significantly different from control (p<0.01)

%: X10^-3

Table 4b: Effect of LAS on organ weights of rats after 1 month (po) exposure (Ito et al., 1978)

Sex

Dose (mg/kg)

Lung

Heart

Thymus

Testis/Ovarium

Pituitary

Brain

Right

Left

wt (g)

%

wt (g)

%

wt (g)

%

wt (mg)

%

wt (mg)

%

wt (mg)

%

wt (g)

%

Males

0

1.23± 0.02

0.33± 0.01

1.27 ± 0.05

0.34 ± 0.01

0.52 ± 0.02

0.14 ± 0.01

1.53 ± 0.03

0.41 ± 0.01

1.51 ± 0.03

0.41 ± 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

0.56 ± 0.05

0.16 ± 0.01

1.48 ± 0.03

0.42 ± 0.01

1.51 ± 0.03

0.43 ± 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

1.41 ± 0.03

0.33* ± 0.01

0.48 ± 0.02

0.14 ± 0.01

1.49 ± 0.04

0.43 ± 0.01

1.49 ± 0.03

0.43 ± 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

0.41** ± 0.03

0.13 ± 0.01

1.49 ± 0.04

0.46* ± 0.02

1.49 ± 0.04

0.46* ± 0.02

9.6 ± 0.4

2.99 ± 0.16

1.77* ± 0.02

0.55* ± 0.02

Females

0

1.07 ± 0.03

0.41 ± 0.01

0.96 ± 0.03

0.37 ± 0.01

0.59 ± 0.02

0.23 ± 0.01

44.8 ± 2.9

17.04 ± 0.95

47.8 ± 3.5

18.13 ± 1.07

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

0.54 ± 0.02

0.21 ± 0.01

46.2 ± 2.5

17.94 ± 1.04

43.7 ± 1.8

16.93 ± 0.64

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

0.55 ± 0.03

0.21 ± 0.01

46.5 ± 2.8

17.73 ± 0.94

47.6 ± 2.8

18.12 ± 0.91

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

0.44** ± 0.04

0.18** ± 0.02

46.3 ± 3.5

18.51 ± 1.29

46.2 ± 2.7

18.44 ± 0.97

11.8 ± 0.9

4.71 ± 0.34

1.75* ± 0.01

0.70 ± 0.01

Mean ± SE

* Significantly different from control (p<0.05)

** Significantly different from control (p<0.01)

%: X10^-3

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 250 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 500 mg/kg bw.

Executive summary:

A short-term repeated dose toxicity study was conducted with C10-13 LAS, sodium salt in CRJ-SD rats. The test substance was administered via oral gavage to groups of male and female rats for 28 days at dose levels of 0, 125, 250 and 500 mg/kg/day. Clinical signs 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, following which the animals were humanely euthanized, gross necropsy was performed, the organs weights were determined and histopathological examination of the major organs were performed. Body weight gain was suppressed, some serum biochemical measures were different from the controls, and some organ weights were either decreased (spleen, heart, thymus) or increased (liver) at the high dose. No mortalities or histopathological abnormalities were observed. No adverse effects were up to 125 mg/kg bw/day mg/kg/day. Under the study conditions, the systemic toxicity NOAEL was determined to be 125 mg/kg/day based on reduced body weight, changes in organ weight (without histopathological correlation) and clinical chemistry at the mid and high doses (Ito, 1978a). 

Reference 5

Endpoint:

sub-chronic 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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of this study is to determine the chronic toxicity (oral) of C10 - C13 linear alkylbenzene sulfonic acid magnesium salt (LAS-Mg) in CRJ-SD rats. Male and female rats were administered with 0, 75, 150 and 300 mg/kg bw/day of test substance for 26 weeks. General symptoms and body weight of animals were recorded on a daily-basis and food intake were recorded weekly. Urinalysis and haematological examinations were performed in week 5 and 13 of the study. At the end of study, urinalysis, haematological and serum biochemistry were also done. All the surviving animals were humanely euthanized after 26 weeks and organs (liver, kidneys, spleen, adrenal glands, lungs, heart, thymus, reproductive organ, pituitary, brain and thyroid) were collected for recording absolute and relative organ weights. All the collected organs (including stomach, duodenum, ileum, cecum, pancreas, bone marrow) were examined microscopically.

GLP compliance:

no

Remarks:

(pre-dates GLP)

Limit test:

no

Species:

rat

Strain:

other: CRJ-SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

IN LIFE DATES: Not reported

Route of administration:

oral: gavage

Details on route of administration:

Test substance was forcibly administered using a metallic gastric sonde, once daily

Vehicle:

water

Remarks:

(distilled)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance solutions were prepared in distilled water to prepare required concentrations of 75, 150 and 300 mg/kg bw/day
- Storage conditions: Not reported
- DOSE VOLUME: 3 mL/kg bw for test substance as well as control group
- Preparation (%): 0, 2.5, 5 and 10% respectively for administration of doses of 0, 75, 150 and 300 mg/kg bw/day

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

Once daily for 26 weeks (excluding Sundays)

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 animals/sex/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the subacute toxicity study of LAS-Mg, in which there was slight weight suppression of animals at 620 mg/kg bw/day (1/3 LD50). Hence, the high dose for this study was selected as 300 mg/kg (1/6 LD50). The lower doses were set at a common ratio of 2, with the medium dose being 150 mg/kg (1/12 LD50) and the low dose being 75 mg/kg (1/24 LD50).
- Rationale for animal assignment: Not reported
- Rationale for selecting satellite groups: No satellite groups were used in the study

Positive control:

Not included

Observations and examinations performed and frequency:

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

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

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

FOOD EFFICIENCY: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During weeks 5, 11 and 26
- Anaesthetic used for blood collection: Yes. The animals were anesthetized with sodium pentobarbital
- Animals fasted: Not reported
- How many animals: During weeks 5 and 11, blood was collected from 10 animals/sex from the control group and 300 mg/kg bw/day group and in week 26, blood was collected from all animals.
- Parameters checked: Red blood cell count, white blood cell count, haemoglobin level and haematocrit level were measured, and white blood cell fractions were calculated by Giemsa-staining the blood smear samples.

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

URINALYSIS: Yes
- Time schedule for collection of urine: During weeks 5, 11 and 26
- Metabolism cages used for collection of urine: No
- Animals fasted: Not reported
- How many animals: During weeks 5 and 11, urine was collected from 10 animals/sex from the control group and 300 mg/kg bw/day group and in week 26, urine was collected from all animals.
- Method: Lab sticks, Urobilistix and iktstix method (Semi-quantitative investigation)
- Parameters: pH, protein, glucose, ketone bodies, occult blood, urobilinogen and bilirubin

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

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

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

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were wetting in the area surrounding animals’ mouths after dosing, and a few animals had abnormal airway sounds. Soft stools were seen in animals after one month, then recovered in few days.

Mortality:

mortality observed, treatment-related

Description (incidence):

One of the male animals in the 300 mg/kg bw/day dose group died (in week 21) from weakness due to significant weight loss involving diarrhea and loss of appetite.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was slight body weight suppression in the male animals of 300 mg/kg bw/day dose group.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

5 weeks interim analysis: There were decrease in segmented neutrophils and increase in lymphocytes in male animals in the 300 mg/kg bw/day dose group
13 weeks interim analysis: Significant decrease in basophils and haematocrit levels was observed for females and significant increase in segmented neutrophils and white blood cell count and significant decrease in lymphocytes were observed for males the 300 mg/kg bw/day dose group.
6 month analysis: Significant reduction in haematocrit levels for female animals in 300 mg/kg bw/day dose group was the only effect seen in haematological parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In 300 mg/kg bw/day dose groups: There was significant reduction in bilirubin, protein, albumin and calcium levels in male animals and in s-GPT, glucose, calcium and magnesium levels in female animals
In 150 mg/kg bw/day dose groups: There was significant reduction in s-GOT, s-GPT, alkaline phosphatase, protein, albumin, sodium and calcium levels in male animals and in chloride levels in female animals.
No effects were observed in 75 mg/kg bw/day treated animals.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

5 weeks interim analysis: No significant effects (on protein, occult blood, Urobilinogen and pH) were observed in treated animals as compare to control animals
13 weeks interim analysis: Male animals in the 300 mg/kg bw/day dose group showed + protein results. Others parameters (occult blood, Urobilinogen and pH) were comparable in control and treated animals.
26 weeks analysis: There were changes in protein levels in treated male animals as compare to control groups.

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 300 mg/kg bw/day dose groups: Significant increased liver weights (absolute weight in females and relative weight in males), actual and relative thymus weights (males) and relative pituitary (males) weights were observed in treated animals as compare to control. Reduction in actual and relative heart weights (females), relative kidneys weights (males), and relative adrenal gland weights (females) were also observed in highest dose treated group.

In 150 mg/kg bw/day dose groups: Significant increased actual and relative thymus weights and actual pituitary weights were observed in male animals as compare to control. There was decreased relative heart weights in females and relative kidneys weights in males of this dose group.

In 75 mg/kg bw/day dose group: Significant dip in absolute heart weights and relative adrenal glands weights of females was seen in low dose group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Yellow nodules was observed on liver for one male animal in the 75 mg/kg group.
- White spots were observed on adrenal glands for one female animal in the 75 mg/kg group.
- Bleeding spots in stomach were observed for two female animals in the 300 mg/kg group and one male animal in the 75 mg/kg group. Erosion was observed for one male animal in the 150 mg/kg group. Thickening of the proventriculus and yellow crust formation on the mucosal surface were observed for 11 male animals in the 300 mg/kg group and one male animal in the 150 mg/kg group.
- Stasis and diffuse emphysema in lungs were observed for one male animal in the control group.
- Oedema was observed in pituitary for one male animal in the 150 mg/kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Control group:
- Hyperpyrexia of the bile duct was observed for one female, while steatosis was observed for another female animal.
- Renal tubular hyaline casts were observed for two each of male and female animals.
- Fibrosis of islets of Langerhans observed for two male animals.
- Stasis was observed for one male animal.
- Interstitial myocarditis was observed for two male animals, while epicarditis was observed for another animal.
300 mg/kg bw/day dose group:
- Interstitial small round cell infiltration was observed in kidney for one male animal and renal tubular calcification was observed for one female animal.
- Small round cell infiltration in the submucosal layer of stomach was observed for one male animal.

150 mg/kg bw/day dose group:
- Kupffer cell migration was observed for one male animal.
- Renal tubular hyaline casts were observed for one male animal, interstitial small round cell infiltration was seen for two male animals, renal tubular epithelial cell swelling was observed for one male animal and renal tubular calcification was observed for one female animal.
- Interstitial myocarditis was observed for two male animals.

75 mg/kg bw/day dose group:
- Focal necrosis was observed for one male animal, while Kupffer cell migration and sinusoidal dilation were observed for one female animal.
- Renal tubular hyaline casts were observed for three male animals, while interstitial small round cell infiltration was observed for two other male animals.
- Fibrosis of islets of Langerhans was observed for one male animal, and mild bleeding in the peripancreatic lymph glands were observed for another male animal.
-Interstitial pneumonia was observed for one female animal.
- Although interstitial myocarditis was observed for five male animals, one of them was a moderate change. Another animal experienced epicarditis.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other:

Remarks:

Based on body weight gain suppression, mortality, fluctuations in hematological and serum biochemistry, altered organ weights and histopathological alterations in kidney at 300 mg/kg bw/day.

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Oral administration of C10-13 magnesium linear alkylbenzene sulphonate (LAS-Mg) to CRJ-SD rats at dose levels of 0, 75, 150 and 300 kg/kg bw/day for 26 weeks revealed no observed adverse effect level (NOAEL) of 150 mg/kg bw/day, based on weight suppression, mortality, fluctuations in haematological and serum biochemistry, altered organ weights and histopathological alterations in kidney at 300 mg/kg bw/day.

Executive summary:

A sub-chronic repeated dose toxicity study was conducted with C10-13 LAS, magnesium salt in CRJ-SD rats. The test substance was administered daily at dose levels of 0, 75, 150 and 300 mg/kg bw/day via oral gavage to groups of 20 animals/sex for 26 weeks. General symptoms and body weight of animals were recorded on a daily basis and food intake was recorded weekly. Urinalysis, haematological and clinical biochemistry examinations were performed during the in-life phase as well as at termination. Following necropsy of the surviving animals, gross pathological and histopathological examinations were performed. There was slight body weight suppression in the males at 300 mg/kg bw/day. Haematological findings revealed significant reduction in haematocrit levels for females at 300 mg/kg bw/day. There was significant reduction in bilirubin, protein, albumin and calcium levels in male animals and significant reduction in SGPT, glucose, calcium and magnesium levels in female animals at 300 mg/kg bw/day. Reduction in relative heart weight (females), kidney weight (males) and adrenal gland weight (females) were also observed in the highest dose group. In the kidneys, renal tubular epithelial cell swelling, hyaline casts and interstitial small round cell infiltration were observed at 300 mg/kg bw/day. There were no toxicologically significant effects at the lower doses. Under the study conditions, the systemic toxicity NOAEL was established at 150 mg/kg bw/day, based on reduction in body weight, fluctuations in haematological and serum biochemistry, altered organ weights and histopathological alterations in kidney at 300 mg/kg bw/day (Ito, 1978b).

Reference 6

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

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 Magnesium Linear Alkyl Benzene Sulfonate Salts (LAS-Mg) in CRJ-SD rats. LAS-Mg was orally administered at 0 (distilled water), 155, 310 and 620 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, and gross necropsy, organs weights and histopathology was performed.

GLP compliance:

no

Remarks:

(pre-dates GLP)

Limit test:

no

Species:

rat

Strain:

other: CRJ-SD rats

Sex:

male/female

Details on test animals or test system 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

EXPERIMENTAL START DATE: Not reported

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered orally through a metallic gastric sonde.

Vehicle:

other: Distilled water

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, 310, 620, and 1240 mg/mL respectively for administration of doses of 0, 155, 310 and 620 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.:

155 mg/kg bw/day (actual dose received)

Remarks:

In distilled water (corresponding to 310 mg/mL)

Dose / conc.:

310 mg/kg bw/day (actual dose received)

Remarks:

In distilled water (corresponding to 620 mg/mL)

Dose / conc.:

620 mg/kg bw/day (actual dose received)

Remarks:

In distilled water (corresponding to 1240 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-Mg in rats. 1/3 of the LD50 levels of LAS-Mg, i.e. 620 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 310 (1/6 LD50) and 155 (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: Haemoglobin, haematocrit, red and white blood cell count were measured using an auto-analyzer 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: Yes
Based on the results of the organ weight measurements, histopathology was performed only for the livers of male animals from the test and control groups. After Bouin fixing, paraffin embedding and H&E staining, the samples were examined under the microscope.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 620 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 155 and 310 mg/kg bw, however, the animals showed recovery at low and mid doses.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male animal (Day 6) and two female animals (Day 7 and 12, respectively) died from the high dose group (i.e. 620 mg/kg bw). In each case, the animal died from weakness after significant loss of body weight involving diarrhea and loss of appetite, and the necropsy revealed congestion of the gastric mucosa, intestinal relaxation, ascites retention, and it also involved lung congestion.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 620 mg/kg bw, males exhibited a body weight suppression on Day 2. Thereafter, the growth curve showed the same rate of increase as the control group, but the suppression was observed again after 3 weeks. In the end, body weight suppression was observed in animals (both males and females) belonging to the high dose group (i.e. 620 mg/kg bw).

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 620 mg/kg bw.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased haemoglobin and haematocrit level was observed in the male animals of high dose group (i.e. 620 mg/kg bw). No changes were observed in the RBC or WBC count.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Decreased s-GOT (only males), K+ (only males) and Ca2+ levels (only females) was observed in the low dose group (i.e. 155 mg/kg bw). Increased s-GOT (both males and females) and alkaline phosphatase (only males), and decreased total proteins (only males), creatinine (only males) and Ca2+ levels (both males and females) was observed in the mid dose group (i.e. 310 mg/kg bw). Increased s-GOT, s-GPT and alkaline phosphatase (both males and females), and decreased total proteins (only females), albumin (only females), glucose (only females), cholesterol (only males) K+ (only males) and Ca2+ levels (both males and females) was observed in the high dose group (i.e. 620 mg/kg bw). The effects were not dose-dependent.

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 relative liver weight of male and female animals was significantly increased at 620 mg/kg bw (p<0.01). Apart from this, increase in relative weight of testes (p<0.01) and brain (p<0.01) was also observed at 620 mg/kg bw. Decrease in the relative weight of kidney (both side at 310 mg/kg bw; right side at 155 mg/kg bw, p<0.05), adrenal gland (both sides at 155 and 310 mg/kg bw, p<0.05), heart (at 310 and 620 mg/kg bw, p<0.01) and thymus (at 620 mg/kg bw, p<0.05) was observed in the female animals. Although, some significant effects were observed at low and mid dose yet they were not dose-dependent.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 620 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:

effects observed, non-treatment-related

Description (incidence and severity):

Focal necrosis and increased glycogen levels were observed in the liver of few animals belonging to mid and low dose groups, but these findings were common with the control group and also showed no dose-dependency.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

310 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: Based on mortality, reduced body weight and changes in organ weight (without biochemical or histopathological correlation). The LOAEL was 620 mg/kg bw.

Critical effects observed:

not specified

Conclusions:

Oral administration of C10-13 magnesium linear alkylbenzene sulphonate (LAS-Mg) to CRJ-SD rats at 0, 155, 310 and 620 mg/kg bw for 1 month resulted in a NOAEL of 310 mg/kg bw, based on mortality, reduced body weight and changes in organ weight (without biochemical or histopathological correlation) at higher dose level. The LOAEL was 620 mg/kg bw.

Executive summary:

A sub-acute repeated dose toxicity study was conducted with C10-13 LAS, magnesium salt in CRJ-SD rats. The test substance was administered to groups of male and female rats via oral gavage at doses of 0, 155, 310 and 620 mg/kg bw/day for 29/30 days. Clinical signs 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, following which the animals were humanely euthanized, gross necropsy was performed and the organs weights were determined for the major organs. Based on the results of organ weight measurements, histopathology was performed only for the livers of male animals from the test and control groups. 1 male and 2 female animals died at the highest dose. The relative liver weight of male and female animals was significantly increased at 620 mg/kg bw/day. Apart from this, increase in relative testes weight and brain weight were also observed at this dose. Decrease in the relative heart weight (at 310 and 620 mg/kg bw/day) and thymus (at 620 mg/kg bw/day) was observed in the female animals. Although, some significant effects were observed at low and mid doses yet they were not dose-dependent. Histopathology revealed focal necrosis and increased glycogen levels in the liver of few animals belonging to the 155 and 310 mg/kg bw dose groups but these findings were comparable to the control group and were not dose-dependent. Under the study conditions, the systemic toxicity NOAEL was established at 310 mg/kg bw/day, based on mortality, reduced body weight and changes in organ weight (without biochemical or histopathological correlation) at the higher dose level (Ito, 1978c). 

Reference 7

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1964

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

(two dose levels are used in the study against the guideline which recommends at least three dose levels should be used.)

GLP compliance:

no

Remarks:

(pre-GLP)

Limit test:

no

Species:

rat

Strain:

other: FDRL strain (Wistar-derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Not specified
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: All animals were at its weaning stage
- Weight at study initiation: Not specified
- Fasting period before study: Not specified
- Housing: Animals were housed individually in raised-bottom, wire-mesh cages
- Diet: Purina laboratory chow, ralston purina co., St. Louis, Missouri; ad libitum
- Water: ad libitum
- Acclimation period: Not specified

No information was provided for food and water quality and environmental conditions.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
No details were provided regarding the preparation of diet. Since rats consume approximately 2.4 times as much diet in proportion to body weight when they are first weaned as when they are adult, lower concentrations were incorporated in their diets initially, and increments in concentration made at biweekly intervals so that the intake of the test materials was relatively constant throughout. By the 11th week on test, when the animals had attained maturity, the levels in the diet were equivalent to 1000 and 5000 ppm of LAS.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

12 weeks

Frequency of treatment:

daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Not specified
- Rationale for selecting satellite groups: No satellite group was included in the study.
- Post-exposure recovery period in satellite groups: not specified

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- Body weights were recorded for approximately half the rats of each sex.

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
- Food consumption was recorded for approximately half the rats of each sex.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Not examined

OPHTHALMOSCOPIC EXAMINATION: Not examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During 6th and 12th week of test
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: Half animals from each group (not subjected to body weights and food consumption analysis)
- Parameters: Haemoglobin, haematocrit, total and differential leukocyte counts were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During 6th and 12th week on test
- Animals fasted: Not specified
- How many animals: Half animals from each group (not subjected to body weights and food consumption analysis)
- Parameters: Blood glucose and urea nitrogen were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During 6th and 12th week on test
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Half animals from each group (not subjected to body weights and food consumption analysis)
- Parameters: Albumin, pH, glucose, and microscopic examination of the sediment were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Following the 12-week clinical examinations, all surviving rats were euthanized by over dosage with sodium pentobarbital, and examined at necropsy for evidence of gross pathologic changes. The liver, kidneys, spleen, heart, adrenals, pituitary, and cecum of each rat were weighed. Representative specimens of all organs and tissues from each animal were preserved in formalin.

Microscopic examinations were made of haematoxylin-eosin stained sections of the liver, spleen, stomach, small intestine, large intestine, pancreas, kidney, bladder, adrenal, gonads, thyroid, pituitary, thymus, salivary gland, lymph node, heart, lung, femoral marrow, aorta, muscle, spinal cord, and brain of 5 rats of each sex in the control and 250 mg/kg bw/day groups. Microscopic examinations were also made of the liver, spleen, stomach, kidneys, and cecum of all animals in the 50 mg/kg bw/day groups.

Clinical signs:

no effects observed

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Occasional signs of wetting over the posterior ventral region of were noted during the first 14 days for females fed with 0.25 g/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The t values for relative liver weight in the females in the 0.25 g/kg bw/day LAS group indicated a statistical significant increase in weight of this organ, compared to the controls. The weights of other organs did not show significant differences from the controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The frequent finding at gross necropsy was hemorrhagic areas in the lungs, characteristic of the common type of pneumonitis seen in rats. The scattered incidence of fluid-containing kidneys of rats in some groups was also noticed. However, both of these findings were not associated with the test materials or dosage levels and was unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: There was significant increase in relative liver weights in females fed with 250 mg/kg bw/day

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 1: Weight gain, efficiency of food utilization and sample intake of rats fed alkyl benzene for 12 weeksa. (Oser and Morgareidge, 1965)

Dosage^b	sex	Mean weight gain (g)	Efficiency of food utilization (g/100 g)^c	sample intake (g/kg/day)
Controls	M	313	18.8	None
	F	169	13
0.05 g/kg/day	M	315	18.6	0.05
	F	164	12.6
0.25 g/kg/day	M	314	18.8	0.256
	F	163	12.6

a: The values given are the means of 7 or 8 animals of each sex.

b: These are nominal dosages. The actual dosages, calculated from food consumption, are given

as “sample intake” in the last column.

c: As grams of weight gained per 100 g of food eaten.

Table 2: Organ weights of rats fed alkyl benzene sulfonates in the diet for 12 weeks (Oser and Morgareidge, 1965)

Organ	Sex	Organ weight^a(%)
		Controls	0.05 g LAS/kg/day	0.25 g LAS/kg/day
Liver	M	3.58	3.81	3.79
	F	3.86	3.77	4.34^b
Kidneys	M	0.7	0.75	0.73
	F	0.79	0.8	0.78
Spleen	M	0.21	0.25	0.22
	F	0.31	0.28	0.3
Heart	M	0.36	0.33	0.34
	F	0.39	0.4	0.37
Adrenals	M	0.013	0.012	0.012
	F	0.027	0.029	0.027
Pituitary	M	0.004	0.004	0.004
	F	0.007	0.006	0.007
Cecum	M	0.47	0.49	0.5
	F	0.58	0.57	0.58

a: as percentage of body weight.

b: Different from corresponding controls of same sex, P < 0.01.

Table3: Gross pathologic findings in rat fed alkyl benzene sulfonates in the diet for 12 weeksa (Oser and Morgareidge, 1965)

Organ and findings	Control		0.05 g LAS/kg/day		0.25 g LAS/kg/day
	M	F	M	F	M	F
Cecum: Slight hyperemia	-	-	-	5112	-	-
Kidneys: Soft and hollow	-	-	5098	5106; 5111	5124	5146; 5148
Hollow and filled with fluid	-	5022	-	5113	-	5147
Congenital absence of right kidney left; hypertrophied	5009	-	-	-	-	-
Adrenals: Hyperemic and/or slightly hemorrhagic	5003;5012	-		5114; 5119	5124; 5125	5145
Thymus: Petecbial hemorrhage	-	-	-	5113	5130	-
Lungs: Hemorrhagic areas	5002	5029	5092	5114; 5115; 5119	5124; 5130	5144; 5150
Purulent leisions	-	-	5102	-	-	-
Edema	-	-	-	-	-	5136
Areas of emphysema	-	5030	5091	5110	-	-

a: The numbers given are the identifying numbers of the individual animals that exhibited the lesion.

Conclusions:

Administration of sodium linear alkylbenzene sulphonate with nominal chain length of C12 (range C9-15) to albino rats at dose levels of 0.05 and 0.25 g/kg bw/day resulted in a NOAEL of 0.05 g/kg bw/day for female rats, based on increased relative liver weights and 0.25 g/kg bw/day for male rats, based on no abnormal variations in growth, food intake, haematology, clinical biochemistry, urinalysis, gross pathology, organ weights and histopathology in any of the groups observed

Executive summary:

A sub-chronic repeated dose dietary toxicity study was conducted with C10-14 LAS, sodium salt in Wistar FDRL rats. The test substance was administered to groups of 5 male and 5 female rats at dietary dose levels of 0, 50 and 250 mg/kg bw/day. Appearance, behaviour and overt signs of toxicity were observed daily. Food consumption and body weights of the rats of were recorded once weekly. During 6th and 12th week on test haematological, clinical chemistry and urine parameters were analysed. Following the 12-week clinical examinations, all surviving rats were sacrificed and gross pathological examination was performed. The major organs were weighed and histopathology was performed. No adverse effects on body weight gain, food consumption and behaviour were observed. The clinical data showed no abnormal variations in any of the dose groups. The relative organ weights and the histopathological evaluation did not show significant differences among the dose groups except for a significant liver weight increase in females of the highest dose group. Under the study conditions, the systemic toxicity NOAEL was established at 50 mg/kg bw/day based on increased relative liver weights in the females at 250 mg/kg bw/day (Oser, 1965).

Reference 8

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

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 SLC-ICR mice, focusing on the liver and kidneys. Male and female mice were maintained on either test diets (0 and 0.6%) or drinking water (0, 0.07, 0.2 and 0.6%) for 9 months. Mice in 1.8% dose group of both feeding and 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, enzyme tests on the liver and kidneys were performed and organs weights were measured. No other parameters (histopathology, clinical chemistry and haematological parameters) were evaluated in the study.

GLP compliance:

no

Remarks:

predates GLP

Limit test:

no

Species:

mouse

Strain:

other: SLC-ICR mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Male and female were obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals
- Age at study initiation: 4-weeks old
- Weight at study initiation: 24 - 31 g (male mice), 20 - 25 g (female mice)
- Fasting period before study: No
- Housing: 5 animals were housed per cage.
- Diet: CE-2 food (from CLEA Japan); ad libitum
- Water: ad libitum
- Acclimation period: 1 week before the start of treatment

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

IN-LIFE DATES: Not reported

Route of administration:

other: oral: drinking water and feed

Details on route of administration:

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

Vehicle:

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

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

9 months

Frequency of treatment:

Continuous in diet or drinking water (ad libitum)

Dose / conc.:

780 mg/kg bw/day (nominal)

Remarks:

in diet (corresponding to 0.6% dose level)

Dose / conc.:

2 340 mg/kg bw/day (nominal)

Remarks:

in diet (corresponding to 1.8% dose level) was also included, however due to severe weight loss so LAS administration was stopped after 2 weeks.

Dose / conc.:

133 mg/kg bw/day (nominal)

Remarks:

in drinking water (corresponding to 0.07% dose level)

Dose / conc.:

380 mg/kg bw/day (nominal)

Remarks:

in drinking water (corresponding to 0.2% dose level)

Dose / conc.:

1 140 mg/kg bw/day (nominal)

Remarks:

in drinking water (corresponding to 0.6% dose level)

Dose / conc.:

3 420 mg/kg bw/day (nominal)

Remarks:

in drinking water (corresponding to 1.8% dose level) was 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): 9 males in 0.6% fed group and 8 males in control group; 8 females in control as well as 0.6% fed group
Drinking water study: 9 animals each in test substance treatment groups and 8 animals in control group

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: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross findings were obtained after euthanizing animals and organs were removed for organ weight measurements.

ORGAN WEIGHTS: Heart, lungs, liver, spleen, kidneys, testes and uterus

HISTOPATHOLOGY: No

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The number of males in 0.2% dose group (drinking water study) decrease from 9 to 5 animals due to an accident with housing management during the experiment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dietary study: There was a significant increase in body weight compared to controls in males of 0.6% dose group.

Drinking water study: There was significant inhibition of weight increase in both males and females of 0.6% dose group consuming LAS water compared to controls, while there was a significant increase in body weight compared to controls in males of 0.07% dose group consuming LAS water.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Individual daily food consumption was higher in all treatment groups compared to controls.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Dietary study: Water consumption was reduced in females and increased in males of 0.6% dose group (dietary study)

Drinking water study: Water consumption was reduced in males of 0.6% dose group and in females of 0.2% dose group and increased in males of 0.07 and 0.2% dose groups and females of 0.07% dose groups.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

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):

Dietary study: In males of 0.6% dose group, the absolute liver weight was significantly increased, in males of 0.6% dose group (dietary study), the relative liver weight was significantly increased. In females, the relative liver weight was significantly increased in all treatment groups.

Drinking water study: In males of 0.07% dose group, the absolute liver weight was significantly increased, in males of 0.2 and 0.6% dose groups, the relative liver weight was significantly increased, and in 0.07% males the relative liver weight was increased. Relative lung, heart and kidney weights were also increased in male mice consuming 0.07% LAS water. In females, the relative liver weight was significantly increased in all treatment groups. Relative spleen, heart and kidney weights were also increased in female mice consuming 0.6% LAS water.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Liver enzyme tests:
Dietary study: Only there was reductions in LDH activity in male mice of 0.6% group
Drinking water study: GOT activity in male mice of 0.6% group consuming LAS water were significant. GOT activity in female mice of 0.2% group was also significantly reduced.

Renal enzyme tests: G6Pase was significantly reduced in males and female mice of 0.6% group consuming LAS water compared to controls, and also reduced in other treatment groups.

Dose descriptor:

other: no NOAEL can be determined for dietary study

Remarks:

Dietary study

Remarks on result:

other: Only one dose level for LAS was fed to animals throughout the study.

Dose descriptor:

NOAEL

Remarks:

Drinking water study

Effect level:

133 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

other: Liver and kidney enzyme levels

Remarks on result:

other: This is based on alteration in liver and kidney weights and enzymes levels at higher tested dose levle. As no other parameters (histopathology, clinical chemistry and hematological parameters) were evaluated in the study.

Critical effects observed:

yes

Lowest effective dose / conc.:

380 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

380 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Administration of LAS to SLC-ICR mice by either test diets (0 and 0.6) or drinking water (0, 0.07, 0.2 and 0.6) for 9 months (focusing on the liver and kidneys) revealed an NOAEL of 0.07% (133 mg/kg bw/day in drinking water), based on observed adverse effects on organ weight and liver and kidney enzymes. Repeated administration of LAS impair renal and liver function.

Executive summary:

The 9 months sub-chronic oral toxicity study of LAS was performed in SLC-ICR mice, focusing on the liver and kidneys.

4 weeks old male and female SLC-ICR mice(obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals) with body weight range 24 - 31 g (males), 20 - 25 g (females) were used in the study. 5 animals were housed in each cage and maintained under controlled environmental conditions (temperature: Average of25 ± 1°C, humidity:50 - 60%, and 12 hours light /12 hours dark). CE-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 and 0.6% (equivalent to 0 and 780 mg/kg bw/day); 9 males in 0.6% fed group and 8 males in control group; 8 females in control as well as 0.6% fed group

Dissolved in drinking water: 0, 0.07, 0.2 and 0.6 (equivalent to 0,133, 380 and 1140 mg/kg bw/day); 9 animals each in test substance treatment groups and 8 animals in control group

Mice in 1.8% dose group of both feeding and drinking water study exhibited severe weight loss so 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 obtained 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 appendix were recorded. Liver and kidney enzymes were also analysed.

No mortality was observed throughout the study. There was significant inhibition of weight increase in both males and females of 0.6% dose group consuming LAS water compared to controls, while there was a significant increase in body weight compared to controls in males of 0.6% dose group fed with LAS in diet and 0.07% dose group consuming LAS water. Individual daily food consumption was higher in all treatment groups compared to controls. Water consumption was reduced in males of 0.6% dose group (drinking water study) and in females of 0.6% dose group (dietary study) and 0.2% dose group (drinking water study) and increased in males of 0.6% dose group (dietary study), 0.07 and 0.2% dose groups (drinking water study) and female of 0.07% dose groups (drinking water study).

In males of 0.6% dose group (dietary study) and 0.07% dose group (drinking water study), the absolute liver weight was significantly increased, in males of 0.6% dose group (dietary study), 0.2 and 0.6 dose groups (drinking water study), the relative liver weight was significantly increased, and in 0.07% (drinking water) males the relative liver weight was increased. Relative lung, heart and kidney weights were also increased in male mice consuming 0.07% LAS water. In females, the relative liver weight was significantly increased in all treatment groups. Relative spleen, heart and kidney weights were also increased in female mice consuming 0.6% LAS water.

There was reductions in LDH activity in male mice of 0.6% group fed with LAS diet and GOT activity in male mice of 0.6% group consuming LAS water were significant. GOT activity in female mice of 0.2% group (drinking water study) was also significantly reduced. G6Pase was significantly reduced in males and female mice of 0.6% group consuming LAS water compared to controls, and also reduced in other treatment groups.

No NOAEL can be identified for dietary study in mice, as only one dose level for LAS was fed to animals throughout the study.

Administration of LAS to SLC-ICR mice by either test diets (0 and 0.6) or drinking water (0, 0.07, 0.2 and 0.6) for 9 months (focusing on the liver and kidneys) revealed an NOAEL of 0.07% (133 mg/kg bw/day in drinking water), based on observed adverse effects on organ weight and liver and kidney enzymes. Repeated administration of LAS impair renal and liver function.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

85 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

other: Hepatobiliary and urinary

Organ:

kidney

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

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

GLP compliance:

no

Remarks:

(pre-dates GLP)

Limit test:

no

Species:

rat

Strain:

other: CRJ-SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

IN LIFE DATES: Not reported

Type of coverage:

not specified

Vehicle:

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

Details on exposure:

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

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

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

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

USE OF RESTRAINERS FOR PREVENTING INGESTION: No

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

Once daily for 26 weeks (excluding Sundays)

Dose / conc.:

0.5 other: %

Dose / conc.:

1 other: %

Dose / conc.:

5 other: %

No. of animals per sex per dose:

20 animals/sex/dose group

Control animals:

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

Details on study design:

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

Positive control:

Not included

Observations and examinations performed and frequency:

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

DERMAL IRRITAION: Yes

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

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

FOOD EFFICIENCY: Yes

OPHTHALMOSCOPIC EXAMINATION: No

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

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

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

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

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

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

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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Urinalysis findings:

no effects observed

Description (incidence and severity):

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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

Histopathological findings: neoplastic:

not examined

Details on results:

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

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

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

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

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

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

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

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

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

Key result

Dose descriptor:

NOAEL

Effect level:

5 other: %

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

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

Remarks on result:

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

Conclusions:

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

Executive summary:

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

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

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

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

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

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

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

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

GLP compliance:

no

Remarks:

(pre-dates GLP)

Limit test:

no

Species:

rat

Strain:

other: CRJ-SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

IN LIFE DATES: Not reported

Type of coverage:

not specified

Vehicle:

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

Details on exposure:

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

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

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

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

USE OF RESTRAINERS FOR PREVENTING INGESTION: No

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

Once daily for 26 weeks (excluding Sundays)

Dose / conc.:

0.5 other: %

Dose / conc.:

1 other: %

Dose / conc.:

5 other: %

No. of animals per sex per dose:

20 animals/sex/dose group

Control animals:

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

Details on study design:

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

Positive control:

Not included

Observations and examinations performed and frequency:

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

DERMAL IRRITAION: Yes

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

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

FOOD EFFICIENCY: Yes

OPHTHALMOSCOPIC EXAMINATION: No

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

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

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

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

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

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

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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Urinalysis findings:

no effects observed

Description (incidence and severity):

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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

Histopathological findings: neoplastic:

not examined

Details on results:

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

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

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

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

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

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

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

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

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

Key result

Dose descriptor:

NOAEL

Effect level:

5 other: %

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

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

Remarks on result:

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

Conclusions:

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

Executive summary:

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

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

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

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

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

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

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

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

Additional information

Oral 

Study 1 (C10-14LAS): 

A sub-chronic (9 months) repeated dose toxicity study was conducted with C10-14 LAS, sodium salt in male and female Wistar JCL rats. The test substance was administered at the following dose levels: through diet at 0, 0.6 and 1.8% (equivalent to 0, 300 and 900 mg/kg bw/day) to 8 animals/sex/dose and via drinking water at 0, 0.07 and 0.2% (equivalent to 0, 85 and 145 mg/kg bw/day to 9 animals/sex/dose. Rats at the 0.6 and 1.8% dose group exhibited severe weight loss so LAS administration via diet was stopped after 2 weeks. Clinical observations, water consumption, food consumption and body weights were recorded weekly. Terminal blood collection was done for estimation of haematological and clinical chemistry parameters. Gross findings were observed after euthanizing animals and organs were removed for organ weight measurements. No histopathology was performed. Body weight gain was reduced at the highest dose group only. Significant decreases in transaminase activity and renal Na,K-ATPase were seen at 145 mg/kg bw/day. No significant haematological or organ weight changes were noted. Based on significant decrease in the liver and renal enzyme levels at 145 mg/kg bw/day, the systemic toxicity NOAEL was established at 85 mg/kg bw/day (Yoneyama, 1976a).

 

Study 2 (C10-14LAS): 

A sub-chronic repeated dose toxicity study was conducted with C10-14 LAS, sodium salt in Wistar SLC rats. The test substance was administered to groups of 10 male and female rats/dose in the diet for 26 weeks at doses of 0, 40, 115, 340 and 1030 mg/kg/day. 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 pathological examinations were observed after necropsy and the major organs were subjected to histopathological examinations. Significant diarrhoea, suppressed growth, increased caecal weight and degeneration of renal tubes were observed in the highest dose group. Similar but less severe signs were seen in other doses with the exception of the lowest dose of 40 mg/kg/day, which showed no adverse effects related to exposure to the LAS. Under the study conditions, systemic toxicity NOAEL was determined to be 40 mg/kg/day, based on tissue damage in caecum, kidney and liver, haematological as well as clinical chemistry parameters changes at higher dose levels (Yoneyama, 1972)

  

Study 3 (C10-13LAS): 

A short-term repeated dose toxicity study was conducted with C10-13 LAS, sodium salt in CRJ-SD rats. The test substance was administered via oral gavage to groups of male and female rats for 28 days at dose levels of 0, 125, 250 and 500 mg/kg/day. Clinical signs 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, following which the animals were humanely euthanized, gross necropsy was performed, the organs weights were determined and histopathological examination of the major organs were performed. Body weight gain was suppressed, some serum biochemical measures were different from the controls, and some organ weights were either decreased (spleen, heart, thymus) or increased (liver) at the high dose. No mortalities or histopathological abnormalities were observed. No adverse effects were up to 125 mg/kg bw/day mg/kg/day. Under the study conditions, the systemic toxicity NOAEL was determined to be 125 mg/kg/day based on reduced body weight, changes in organ weight (without histopathological correlation) and clinical chemistry at the mid and high doses (Ito, 1978a). 

 

Study 4 (C10-13LAS): 

A sub-chronic repeated dose toxicity study was conducted with C10-13LAS, magnesium salt in CRJ-SD rats. The test substance was administered daily at dose levels of 0, 75, 150 and 300 mg/kg bw/day via oral gavage to groups of 20 animals/sex for 26 weeks. General symptoms and body weight of animals were recorded on a daily basis and food intake was recorded weekly. Urinalysis, haematological and clinical biochemistry examinations were performed during the in-life phase as well as at termination. Following necropsy of the surviving animals, gross pathological and histopathological examinations were performed. There was slight body weight suppression in the males at 300 mg/kg bw/day. Haematological findings revealed significant reduction in haematocrit levels for females at 300 mg/kg bw/day. There was significant reduction in bilirubin, protein, albumin and calcium levels in male animals and significant reduction in SGPT, glucose, calcium and magnesium levels in female animals at 300 mg/kg bw/day. Reduction in relative heart weight (females), kidney weight (males) and adrenal gland weight (females) were also observed in the highest dose group. In the kidneys, renal tubular epithelial cell swelling, hyaline casts and interstitial small round cell infiltration were observed at 300 mg/kg bw/day. There were no toxicologically significant effects at the lower doses. Under the study conditions, the systemic toxicity NOAEL was established at 150 mg/kg bw/day, based on reduction in body weight, fluctuations in haematological and serum biochemistry, altered organ weights and histopathological alterations in kidney at 300 mg/kg bw/day (Ito, 1978b).  

  

Study 5 (C10-13LAS): 

A sub-acute repeated dose toxicity study was conducted with C10-13LAS, magnesium salt in CRJ-SD rats. The test substance was administered to groups of male and female rats via oral gavage at doses of 0, 155, 310 and 620 mg/kg bw/day for 29/30 days. Clinical signs 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, following which the animals were humanely euthanized, gross necropsy was performed and the organs weights were determined for the major organs. Based on the results of organ weight measurements, histopathology was performed only for the livers of male animals from the test and control groups. 1 male and 2 female animals died at the highest dose. The relative liver weight of male and female animals was significantly increased at 620 mg/kg bw/day. Apart from this, increase in relative testes weight and brain weight were also observed at this dose. Decrease in the relative heart weight (at 310 and 620 mg/kg bw/day) and thymus (at 620 mg/kg bw/day) was observed in the female animals. Although, some significant effects were observed at low and mid doses yet they were not dose-dependent. Histopathology revealed focal necrosis and increased glycogen levels in the liver of few animals belonging to the 155 and 310 mg/kg bw dose groups but these findings were comparable to the control group and were not dose-dependent. Under the study conditions, the systemic toxicity NOAEL was established at 310 mg/kg bw/day, based on mortality, reduced body weight and changes in organ weight (without biochemical or histopathological correlation) at the higher dose level (Ito, 1978c).

  

Study 6 (C10-14LAS): 

A sub-chronic repeated dose dietary toxicity study was conducted with C10-14 LAS, sodium salt in Wistar FDRL rats. The test substance was administered to groups of 5 male and 5 female rats at dietary dose levels of 0, 50 and 250 mg/kg bw/day. Appearance, behaviour and overt signs of toxicity were observed daily. Food consumption and body weights of the rats of were recorded once weekly. During 6th and 12th week on test haematological, clinical chemistry and urine parameters were analysed. Following the 12-week clinical examinations, all surviving rats were sacrificed and gross pathological examination was performed. The major organs were weighed and histopathology was performed. No adverse effects on body weight gain, food consumption and behaviour were observed. The clinical data showed no abnormal variations in any of the dose groups. The relative organ weights and the histopathological evaluation did not show significant differences among the dose groups except for a significant liver weight increase in females of the highest dose group. Under the study conditions, the systemic toxicity NOAEL was established at 50 mg/kg bw/day based on increased relative liver weights in the females at 250 mg/kg bw/day (Oser, 1965).

 

Study 7 (C10-14 LAS)

The 9 months sub-chronic oral toxicity study of LAS was performed in SLC-ICR mice, focusing on the liver and kidneys.

4 weeks old male and female SLC-ICR mice(obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals) with body weight range 24 - 31 g (males), 20 - 25 g (females) were used in the study. 5 animals were housed in each cage and maintained under controlled environmental conditions (temperature: Average of25 ± 1°C, humidity:50 - 60%, and 12 hours light /12 hours dark). CE-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 and 0.6% (equivalent to 0 and 780 mg/kg bw/day); 9 males in 0.6% fed group and 8 males in control group; 8 females in control as well as 0.6% fed group Dissolved in drinking water: 0, 0.07, 0.2 and 0.6 (equivalent to 0,133, 380 and 1140 mg/kg bw/day); 9 animals each in test substance treatment groups and 8 animals in control group Mice in 1.8% dose group of both feeding and drinking water study exhibited severe weight loss so 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 obtained 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 appendix were recorded. Liver and kidney enzymes were also analysed.

No mortality was observed throughout the study. There was significant inhibition of weight increase in both males and females of 0.6% dose group consuming LAS water compared to controls, while there was a significant increase in body weight compared to controls in males of 0.6% dose group fed with LAS in diet and 0.07% dose group consuming LAS water. Individual daily food consumption was higher in all treatment groups compared to controls. Water consumption was reduced in males of 0.6% dose group (drinking water study) and in females of 0.6% dose group (dietary study) and 0.2% dose group (drinking water study) and increased in males of 0.6% dose group (dietary study), 0.07 and 0.2% dose groups (drinking water study) and female of 0.07% dose groups (drinking water study). In males of 0.6% dose group (dietary study) and 0.07% dose group (drinking water study), the absolute liver weight was significantly increased, in males of 0.6% dose group (dietary study), 0.2 and 0.6 dose groups (drinking water study), the relative liver weight was significantly increased, and in 0.07% (drinking water) males the relative liver weight was increased. Relative lung, heart and kidney weights were also increased in male mice consuming 0.07% LAS water. In females, the relative liver weight was significantly increased in all treatment groups. Relative spleen, heart and kidney weights were also increased in female mice consuming 0.6% LAS water.

There was reductions in LDH activity in male mice of 0.6% group fed with LAS diet and GOT activity in male mice of 0.6% group consuming LAS water were significant. GOT activity in female mice of 0.2% group (drinking water study) was also significantly reduced. G6Pase was significantly reduced in males and female mice of 0.6% group consuming LAS water compared to controls, and also reduced in other treatment groups. No NOAEL can be identified for dietary study in mice, as only one dose level for LAS was fed to animals throughout the study. Administration of LAS to SLC-ICR mice by either test diets (0 and 0.6) or drinking water (0, 0.07, 0.2 and 0.6) for 9 months (focusing on the liver and kidneys) revealed an NOAEL of 0.07% (133 mg/kg bw/day in drinking water), based on observed adverse effects on organ weight and liver and kidney enzymes. Repeated administration of LAS impair renal and liver function. (Yoneyama, 1976b)

  

Dermal 

Study 1 (C10 -13LAS): 

A sub-chronic 26 -weeks repeated dose toxicity study was conducted in male and female CRJ-SD rats to evaluate the effects of C10-13LAS, magnesium salt. The test substance was applied daily on the shaved backs of animals at dose levels of 0, 0.5, 1.0 and 5.0% dissolved in 3% polyethylene glycol (PEG) for 26 weeks with 20 animals/sex/dose group. General symptoms and body weight of animals were recorded on a daily-basis and food intake were recorded weekly. Urinalysis and haematological examinations were performed on Weeks 5 and 13 and also at termination, along with clinical biochemical examination. All the surviving animals were humanely euthanized after 26 weeks and organs were collected for recording absolute and relative weights and histopathological observation. There was slight reduction of body weight in males at 0.5% at the end of the study. No toxicologically significant changes were observed in food consumption, urinalysis, haematological and serum biochemical findings. No changes were seen in the organ weights and gross pathology of the collected organs. Histopathological findings were non-specific and had no dose-dependency. Under the study conditions, the systemic toxicity NOAEL was established at 5.0% (equivalent to 2500 mg/kg bw/day), based no toxicologically relevant changes at any dose level (Ito, 1978d).

Other routes 

Study 1 (C10-13 LAS):

A 28-day repeated dose toxicity was conducted with C10-13LAS, sodium salt in Rhesus monkeys via oral and subcutaneous routes. The test substance was administered to the monkeys at doses of 0, 30, 150 and 300 mg/kg bw/day and at doses of 0, 0.1, 0.5 and 1.0 mg/kg bw/day for 28 days by simultaneous oral gavage and subcutaneous injection (into the dorsal aspect of the upper thorax) respectively. Clinical observations for any signs of toxicity, water consumption and food consumption were observed daily, and body weights was recorded once weekly. Ophthalmological, haematological, clinical chemistry and urine parameters were analysed before the start of the study and during the final week of dosing. Organ weights, gross pathological and histopathological examinations were performed on the major organs. No significant adverse effect was observed at food and water consumption of animals. All animals at the highest dose vomited on numerous occasions, usually within 3 h of being dosed. Vomiting was often associated with salivation. These effects are probably related to the inherent irritative effects of LAS rather than to its systemic toxicity. There was no further evidence of treatment-related effects among the ophthalmological, laboratory and other pathological investigations performed during this study. There was also a dose-related increase in the occurrence of chronic inflammatory cell infiltration (mainly fibroblasts) at the subcutaneous injection sites. Fibrosis of the injection sites was found among the entire test group, the incidence and severity being dose-related. Under the study conditions, a systemic toxicity NOAEL of 150 mg/kg bw/day (oral) and 0.5 mg/kg bw/day (sc) (Heywood, 1978). 

Justification for classification or non-classification

Repeated dose toxicity studies were conducted through oral route in rats, mice and monkeys, through dermal route in rats and through subcutaneous route in monkeys. These studies were conducted with the C10-13 LAS Na as well as with the read-across substance C10-14 LAS. No significant effects were observed at the sub-chronic NOAEL of 85 mg/kg bw/day in the 9 months oral gavage study (Yoneyama, 1976) with LAS. Slight to moderate histopathological lesions were observed at the LOAEL of 115 mg/kg bw/day (Yoneyama, 1972) which is above the guidance value recommended in the CLP guidance. Higher systemic NOAEL of 150 mg/kg bw/day were established in the other sub-chronic study (Ito, 1978b). Therefore, based on the results of the repeated dose studies, no classification is warranted for this endpoint according to EU CLP (1272/2008/EC) criteria.