Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1971

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to evaluate linear alkylbenzene sulphonate sodium salt (LAS) having C10 - 14 chain length distribution for reproductive toxicity potential in rats. Rats were divided into 4 groups and administered 0 (control), 0.02, 0.1 and 0.5% LAS added to their diets. Each group consisted of 50 males and 50 females randomized according to their litter and weight. After administration of LAS for 84 days when the parent animals (Po) were 107 – 112 days old, 20 female rats from each group were mated with 20 male rats from the same group. All females were separated from males on completion of seventeen days and placed in the opaque plastic litter boxes containing clean, dry saw dust and sufficient paper for nesting. Litters were examined for deformities and number of pups were counted. The first litters of F1a and F2a generation were sacrificed at 21 days of age and 10 days after sacrifice of litters, all females were remated with different males from same group to obtain the F1b generation. Twenty males and females of each group were selected at weaning to continue their respective diets and to be used for further reproduction studies and remaining weanling rats were examined and sacrificed. Reproduction studies on the F1b and F2b generations were started when the rats were 80 to 85 days old and were continued until the F3b generation was weaned. Average body weight, feed consumption and feed efficiency were recorded on weekly basis for the first 8 weeks for F1a and F2b generations. Once the reproductive studies were completed, five male and five female rats from each parenteral groups (F1a and F2b) were selected for necropsy and body weight, organ to body weight ratios were recorded. Routine hematology and histology studies were also performed. Weanling animals from F3a generation were treated similarly.
GLP compliance:
no
Remarks:
(predates GLP)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Details on test material
- Name of test material (as cited in study report): Sodium salt of LAS (C10-14)
- Activity: 98.1% on an anhydrous basis (41.9% active)
- Impurities (identity and concentrations): Unreacted alkylbenzene and Na2SO4
- Synthesis: The sodium salt of LAS was prepared according to the factory specifications by sulphonation of linear alkylbenzene having a hydrocarbon chain length distribution of C10 through C14.

Test animals

Species:
rat
Strain:
other: Charles River
Sex:
male/female
Details on test animals and environmental conditions:
Details on test animals and environmental conditions
TEST ANIMALS
- Age at study initiation:
a) Parent animals (Po): 107 – 112 days
b) F1b and F2b generation: 80 – 85 days
- Parent animals body weight at study initiation: 59.4 - 59.9 g (males) and 57.0 - 57.3 g (females)
- Housing:
a) Females after mating were placed in opaque plastic boxes containing clean, dry sawdust and paper for nesting
b) Weanling rats: Individual wire bottom cages
- Diet: Purina rat chow; ad libitum
- Drinking water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 76 ± 3 °F
- Humidity: 50 ± 5 %
- Photoperiod (hrs dark / hrs light): 12/12 hrs

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
VEHICLE
- Concentration in vehicle: LAS was added to their diets at levels of 0.02, 0.1 and 0.5%

Details on mating procedure
- Premating exposure period: 84 days (Po animals) and 80 – 85 days (F1b and F2b animals)
- Length of cohabitation: 17 days
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
2 years (3 generations)
Frequency of treatment:
Continuous in feed
Details on study schedule:
- F1 parental animals were not mated until 80-85 days old
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the Po animals in the study: 107-112 days old

Doses / concentrationsopen allclose all
Dose / conc.:
14 mg/kg bw/day
Remarks:
0.02% in diet
Dose / conc.:
70 mg/kg bw/day
Remarks:
0.1% in diet
Dose / conc.:
350 mg/kg bw/day
Remarks:
0.5% in diet
No. of animals per sex per dose:
20 males and 20 females per group
Control animals:
yes, concurrent no treatment
Details on study design:
Rats were divided into 4 groups and administered 0 (control), 0.02, 0.1 and 0.5% LAS added to their diets. Each group consisted of 50 males and 50 females randomized according to their litter and weight. After administration of LAS for 84 days when the parent animals (Po) were 107 – 112 days old, 20 female rats from each group were mated with 20 male rats from the same group. All females were separated from males on completion of seventeen days and placed in the opaque plastic litter boxes containing clean, dry saw dust and sufficient paper for nesting. Litters were examined for deformities and number of pups were counted. The first litters of F1a and F2a generation were sacrificed at 21 days of age and 10 days after sacrifice of litters, all females were remated with different males from same group to obtain the F1b generation. Twenty males and females of each group were selected at weaning to continue their respective diets and to be used for further reproduction studies and remaining weanling rats were examined and sacrificed. Reproduction studies on the F1b and F2b generations were started when the rats were 80 to 85 days old and were continued until the F3b generation was weaned. Average body weight, feed consumption and feed efficiency were recorded on weekly basis for the first 8 weeks for F1a and F2b generations. Once the reproductive studies were completed, five male and five female rats from each parenteral groups (F1a and F2b) were selected for necropsy and body weight, organ to body weight ratios were recorded. Routine hematology and histology studies were also performed. Weanling animals from F3a generation were treated similarly.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Gross examination of all animals along with routine hematology was performed after completion of reproductive study.

BODY WEIGHT: Body weight was recorded on weekly basis for first 8 weeks (F1b and F2b generation)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Feed consumption and feed efficiency were recorded on weekly basis for 12 weeks.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
Deformities and number of pups, average body weights, feed consumption, feed efficiency.

Postmortem examinations (parental animals):
Necropsy, body weight, organ to body weight ratios, routine hematology and histology
Postmortem examinations (offspring):
Necropsy, body weight, organ to body weight ratios, routine hematology and histology.
Reproductive indices:
Fertility, gestation, parturition, neonatal viability, lactation, and post-weaning growth
Offspring viability indices:
Body weight gain

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

Mortality: There was no significant difference between mortality in control and treated group of rats as overall survival in this study exceeded 56% with the highest occurring in 350 mg/kg bw/day dose group (0.5% LAS in diet) as compared to 53% in control.

Body weight, organ weight and organ to body weight ratio: Body weight and organ to body weight ratios of selected animals from high level group and controls at 8, 15 and 24 months were within normal limits. A statistically significant decrease in liver weights was noted in male rats at the low and mid dose levels at the 8-month sacrifice. As the decreased liver weight was within normal range, was not seen at the highest dose level, nor was seen at the 15- and 24-month sacrifices, it was not considered biologically significant.

Food consumption and food efficiency: All groups were comparable in terms of food consumption and food efficiency.

Hematological findings: During routine hematological evaluations, statistically significant borderline values were scattered among various treatment groups and were not considered as treatment related.

Gross pathological and histopathological findings: No unusual lesions/abnormalities were noted upon gross examination of the visceral organs and there were no microscopic differences test and control animals as no test related response were observed in any of the tissues. Although higher incidences of incurrent degenerative diseases like chronic interstitial nephritis and adrenal telangiectasis and fibroadenomas were observed in final autopsy but these were not related to test material related exposure.

Reproductive performance: General reproduction including fertility, gestation, parturition, neonatal viability, lactation, and post-weaning growth were normal for all test groups and did not vary from controls.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food efficiency
haematology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive performance
Remarks on result:
other: No effects observed at the highest dose of 350 mg/kg bw/day(0.5% LAS in diet)

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

Mortality, body weight, organ to weight ratios, gross pathological and histopathological findings: These parameters were comparable to the control group for all three treatment groups.

Hematological parameters: In F1b female group receiving 70 mg/kg bw/day (0.1% LAS in diet) dose, red blood cell count was high but as it was within the normal range as determined by previous experiments conducted in the same laboratory.

Reproductive parameters: General reproductive parameters like fertility, gestation, parturition, neonatal viability, lactation and post weanling growth were normal across all test groups and comparable to control.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1b
Effect level:
350 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
haematology
gross pathology
other: histopathology, organ / body weight ratios and reproductive parameters
Remarks on result:
other: No effects observed at the highest dose of 350 mg/kg bw/day (0.5% LAS in diet)

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not specified

Details on results (F2)

Mortality, body weight and organ to weight ratios: These parameters were comparable to the control group for all three treatment groups.

Hematological parameters: Red blood cell count was depressed in the F2b females from high dose test group as compared to control but was within the normal range as determined by previous experiments conducted in the same laboratory.

Gross pathological findings: No gross abnormalities were observed in any treatment group.

Histopathological findings: Pancreatic lesions consisting of acinar atrophy and tissue degeneration leading to the fibrous tissue replacement was observed in the F2b males and mild islet cell hyperplasia was also indicated. General lesions were also increased in the group receiving highest level of LAS. As similar lesions were also identified in the control males as well as control animals from the other studies, so these were not considered to be treatment related.

Reproductive parameters: General reproductive parameters like fertility, gestation, parturition, neonatal viability, lactation and post weanling growth were normal across all test groups and comparable to control.

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2b
Effect level:
350 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
haematology
gross pathology
other: histopathology, organ / body weight ratios and reproductive parameters
Remarks on result:
other: No effects observed at the highest dose of 350 mg/kg bw/day (0.5% LAS in diet)

Target system / organ toxicity (F2)

Critical effects observed:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

F3b generation:

Rats sacrificed at weaning were normal w.r.t growth and organ to body weight ratios, gross pathology and histology and did not differs from control. Although statistically significant differences were observed were in hematological values between control and test groups, these differences were small and does not indicate a pattern as these animals were young leading to more individual variation as compared to mature animal.

NOAEL for F3b generation (based on absence of effects on growth and organ to body weight ratios, gross pathology and histology parameters at the highest tested dose): 350 mg/kg bw/day

Table 1: Average body weight gain and feed utilization (initial 12 weeks) for two year feeding study in rats (Buehler et. al., 1970)

Dietary level (%)

Dose (mg/kg bw/day)

Sex

Initial weight (g)

Final weight (g)

Weight gain (g)

Feed consumption (g)

Feed efficiency

0

0

M

59.5

491.1

431.6

2117.9

0.204

F

57.2

289.4

232.2

1579.9

0.147

0.02

14

M

59.4

485.1

425.7

2095

0.203

F

57

279

222

1545.7

0.144

0.1

70

M

59.9

492.8

432.9

2163.9

0.2

F

57.2

284.5

227.3

1568.5

0.145

0.5

350

M

59.8

480

420.2

2087.9

0.201

F

57.3

275.9

218.6

1520

0.144

Table 2: Growth and organ to body ratio's for two year feeding study in rats (Buehler et. al., 1970)

Time (Months)

Dietary level (%)

Dose (mg/kg bw/day)

Sex

Body weight (g)

Organ to body weight (expressed as % of total body weight)

Liver

Kidney

8

0

0

M

575

3.45

0.59

F

349

3.28

0.64

0.5

350

M

576

3.47

0.62

F

291

3.49

0.71

15

0

0

M

623

3.08

0.6

F

375

3.05

0.67

0.5

350

M

622

2.95

0.6

F

423

2.99

0.58

24

0

0

M

645

2.91

0.77

F

460

3.16

0.64

0.5

350

M

673

2.55

0.68

F

488

3.18

0.68

Table 3: Hematologic values for two year feeding study in rats (Buehler et. al., 1970)

Time (Months)

Dietary level (%)

Dose (mg/kg bw/day)

Sex

RBC count

Hemoglobin

Hematocrit

WBC count

Differential white cells

I

II

III

IV

V

8

0

0

M

6.81

15.5

48

7800

12

86

2

0

0

F

6.26

15

45

5700

9

89

1

0

1

0.5

350

M

5.81b

15.1

45

8900

11

85

1

0

3

F

5.7

14.6

42

4950

12

86

1

0

1

15

0

0

M

9.52

17.1

51

11100

10

84

5

0

1

F

6.88

16

44

6600

12

82

4

0

2

0.5

350

M

8.43

15.9

49

7600

13

83

3

0

1

F

7.74

14.7

45

6850

16

80

2

0

2

24

0

0

M

7.14

13.6

43

10500

23

72

4

0

1

F

6.65

13.2

43

10500

21

74

5

0

1

0.5

350

M

7.24

13.9

45

12600

35

59b

5

0

1

F

6.15

13.7

44

7900

25

68

6

0

1

b: Groups significantly different from control group

Differential white cells: Neutrophil / heterophil (I), Lymphocyte (II), Monocyte (III), Basophil (IV) and Eosinophil (V)

Table 4: Summary of mating for reproduction study in rats (Buehler et. al., 1970)                                                                                                                                                                 

Generation Dietary level (%) Dose (mg / kg bw/day) 0 day number in litter 5 days 21 days
No. in litter before culling  No. in litter after culling  Total weigh, origA Total weight after cullingA Number weaned  Body weights 
Male pups Female Pups MotherB
Number WeightA Number WeightA
F1a 0 0 12.3 12.1 7.81 129.2 87.7 7.4 3.6 201.3 4.1 205.3 319.5
0.5 350 12.7 12.5 7.6 123.4 81.6 7.5 3.8 197.6 3.7 178.3 324.8
F1b 0 0 12.4 11.5 7.6 120.2 82.4 6.4 3.5 197.9 2.9 157.8 363.1
0.5 350 13.7 12.9 8 128.4 83.9 7.6 3.5 183.5 3.7 185 355.9
F2a 0 0 12.2 11.7 7.9 145.5 102.4 7.7 3.4 158.9 4.4 189.2 316.2
0.5 350 12.7 12.1 7.9 141.7 96.4 7.6 4 183.1 3.6 154.2 323.2
F2b 0 0 11.8 11.6 7.5 141.4 94.9 7.5 3.7 209.5 3.8 208.4 350
0.5 350 12.2 11.8 7.8 130.1 89.3 7.6 4.2 201.4 3.4 166.5 352.4
F3a 0 0 12.6 12.3 8 136.7 91.9 7.9 4 216.8 3.9 202.6 328.7
0.5 350 11.4 11.2 7.8 127 90.7 7.8 4 202.7 3.8 186.2 316.8
F3b 0 0 12.9 12.3 7.9 151.2 102.6 8.2 3.8 213.3 4.3 233.5 356.1
0.5 350 12.1 11.7 7.7 150.1 100.6 7.7 4.3 240.1 3.4 181.4 337.6

                                   

A: Total group weight of pups (g)

B: Weight in grams

Table 5: Summary of hematology for two year reproduction study in rats (Buehler et. al., 1970)

Generation

Dietary level (%)

Dose (mg/kg bw/day)

Sex

RBC count

Hemoglobin

Hematocrit

WBC count

Differential white cells

I

II

III

IV

V

F1b

0

0

M

7.45

16.5

50

6800

14

80

3

0

3

F

6.03

16.9

49

5800

18

77

3

0

2

0.5

350

 

M

6.23

16.4

49

6800

16

80

2

0

2

F

5.88

16

47

6900

21

74

2

0

3

F2b

0

0

M

7.78

16.1

49

7900

13

79

6

0

2

F

7.27

16.2

48

5500

14

80

5

0

1

0.5

350

M

7.15

16

-

8400

10

85

4

0

1

F

6.15b

16.2

49

6400

11

83

5

0

1

b: Groups significantly different from control group

Differential white cells: Neutrophil / heterophil (I), Lymphocyte (II), Monocyte (III), Basophil (IV) and Eosinophil (V)

Applicant's summary and conclusion

Conclusions:
Administration of linear alkylbenzene sulphonate sodium salt (Na-LAS) to male and female Charles river rats in their diet over 2 years across 3 generations at dose of 0, 14 (0.02%), 70 (0.1%) and 350 (0.5%) mg/kg bw/day resulted into NOAEL of 350 mg/kg bw/day for Po, F1, F2 and F3 generations (based on absence of marked effects on general reproduction parameters, growth, body weight, organ weight, body weight to organ weight ratio, hematology and histopathology parameters at the highest dose).

Executive summary:

The reproductive toxicity study of linear alkylbenzene sulphonate sodium salt (Na-LAS) in rats across three generations was conducted according to the accepted scientific principles.

 

Weanling rats and females after mated were housed in the individual wire bottom cages and opaque plastic boxes containing clean, dry sawdust and paper for nesting, respectively and maintained under standard laboratory conditions (temperature: 76 ± 3 °F, humidity: 50 ± 5 %, 12-hour light/12-hour dark cycle/day). The animals were allowed free access to the drinking water and food.

 

Male and female rats (Charles River strain) were used in this study. Experiment was initiated by randomizing rats into 4 groups of 50 males and 50 females each according to litter and weight and LAS was added into their diet at levels of 0 (control), 0.02, 0.1 and 0.5%. Dietary levels of 0.02, 0.1 and 0.5% corresponds to 14, 70 and 350 mg/kg bw/day, respectively. When parent animals (Po) were 107 – 112 days old and had received test diets for 84 days, groups of 20 female’s rats from each group were mated with 20 males from same group. After 17 days of initial exposure to males, females were transferred to the opaque plastic litter boxes with the clean and dry saw dust along with sufficient paper for nesting.

 

Litters were examined for deformities and number of pups were counted. On the fourth day, number of pups in each litter was limited to 8 for equalizing the stress of lactation among dams. The first litters of F1a generation were sacrificed at 21 days of age and after interval of 10 days, all females were remated with different males from the same group to obtain F1b generation. Twenty females and 20 males were selected from each group at weaning to continue their respective diets and used in further reproductive studies while and all remaining weanling rats were sacrificed and examined. These rats were housed in the individual wire bottom cages and fed the same diet. Reproduction studies on these rats were started at age of 80 - 85 days in a similar manner to obtain F2a and F2b generation and continued as such until F3b generation was weaned.

Average body weights, feed consumption and feed efficiency were recorded on weekly basis for first 8 weeks (F1b and F2b). After completion of the reproductive studies, 5 males and 5 female rats were selected from each parenteral group (F1b and F2b) for necropsy and body weight, organ to body weight ratio was recorded. Routine hematology and histology were also performed. Weanling animals from F3a generation were treated similarly. No significant effects were observed in the Po, F1b, F2b and F3b animals even at the highest dose i.e. 350 mg/kg bw/day (0.5% diet).

Based on above, administration of linear alkylbenzene sulfonate sodium salt to male and female rats by diet for three generation reproductive toxicity at dose of 0, 14 (0.02%), 70 (0.1%) and 350 (0.5%)  mg/kg bw/day resulted into NOAEL of 350 mg/kg bw/day for Po, F1, F2 and F3 generations (based on absence of marked effects on general reproduction parameters, growth, body weight, organ weight, body weight to organ weight ratio, hematology and histopathology parameters at the highest dose).

 

This reproductive toxicity study is acceptable and satisfies accepted scientific principles.