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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Limited details on test animals and environmental conditions. Not all haematological examinations were performed. Neurobehavioural and ophthalmoscopic examinations are missing due to the year when the study was conducted.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1976
Report Date:
1976

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
limited details on test animals and environmental conditions; not all haematological examinations were performed; neurobehavioural and ophthalmoscopic examinations are missing due to the year when the study was conducted.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Colworth Wistar-derived
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 43-87 g (males), 51-79 g (females)
- Housing: animals were housed in individual cages.
- Diet: semi-synthetic purified diet, ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: semi-synthetic purified diet
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): semi-synthetic purified diet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0.07 other: % nominal in diet
Dose / conc.:
0.14 other: % nominal in diet
Dose / conc.:
0.28 other: % nominal in diet
Dose / conc.:
0.56 other: % nominal in diet
Dose / conc.:
1.13 other: % nominal in diet
Dose / conc.:
2.25 other: % nominal in diet
Dose / conc.:
58 mg/kg bw/day (actual dose received)
Remarks:
Male animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
113 mg/kg bw/day (actual dose received)
Remarks:
Male animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
228 mg/kg bw/day (actual dose received)
Remarks:
Male animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
470 mg/kg bw/day (actual dose received)
Remarks:
Male animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
961 mg/kg bw/day (actual dose received)
Remarks:
Male animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
1 944 mg/kg bw/day (actual dose received)
Remarks:
Male animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
66 mg/kg bw/day (actual dose received)
Remarks:
Female animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
131 mg/kg bw/day (actual dose received)
Remarks:
Female animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
261 mg/kg bw/day (actual dose received)
Remarks:
Female animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
506 mg/kg bw/day (actual dose received)
Remarks:
Female animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
1 070 mg/kg bw/day (actual dose received)
Remarks:
Female animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
Dose / conc.:
2 218 mg/kg bw/day (actual dose received)
Remarks:
Female animals, reported daily intake of the test substance based on mean body weight and food consumption over the entire 13-week treatment period
No. of animals per sex per dose:
10 test group
20 control group
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed daily for mortality and general health conditions.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights of all animals were determined once per week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): twice weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the experimental period (13 weeks)
- Anaesthetic used for blood collection: Yes (halothane)
- How many animals: all
- Parameters checked: packed cell volume, haemoglobin level, mean corpuscular haemoglobin concentration and white cell count

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: at the end of the experimental period (13 weeks)
- How many animals: all
- Parameters checked: serum levels of sodium, potassium, calcium, magnesium, urea, creatinine, total protein, cholesterol, triglyceride, glutamic oxaloacetic transaminase, glutamic-pyruvic transaminase, lactate dehydrogenase, hydroxybutyrate dehydrogenase, cholinesterase, alkaline phosphatase and creatinine phosphokinase
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, in all animals; absolute and relative organ weights of heart, liver, spleen, kidneys, brain, adrenals, pituitary and testes were determined.
HISTOPATHOLOGY: Yes, in all animals of the control group and the two high dose groups (1.13 and 2.25% in the diet); histopathological examination was performed in heart, liver, spleen, kidneys, brain, adrenals, pituitary, testes, lung, stomach, jejunum, ileum, caecum, colon, thyroid, pancreas, urinary bladder, seminal vesicles, ovaries, uterus, skeletal muscle, thymus, salivary glands, skin, aorta, eye, mesenteric lymph node, tongue and sternum. Since liver changes were determined in the two high dose groups, histopathological examination of all other dose groups was performed.
Statistics:
All relevant parameters, except those for pathology, were statistically examined by analysis of variance for significant effects between treatment groups and controls. Statistical significance between treatment groups and controls was indicated at p < 0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
0.14 and 1.13% in the diet: each one male animal died (not treatment-related)
Mortality:
mortality observed, treatment-related
Description (incidence):
0.14 and 1.13% in the diet: each one male animal died (not treatment-related)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
2.25% in the diet: significantly lower body weight gain in males (-20%) and females (-16%) compared to controls; 1.13% in the diet: significantly lower body weight gain in males (-10%) compared to controls
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
2.25% in the diet: significantly lower food consumption in males (-9%) and females (-6%) compared to controls (non-adverse)
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
2.25% in the diet: significantly poorer food utilisation in males (-13%) and females (-10%) compared to controls; 1.13% in the diet: significantly poorer food utilisation in males (-5%) compared to controls
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
2.25% in the diet: significantly lower water consumption in females (-15%) compared to controls
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
2.25% in the diet: signif. decreased magnesium, protein and cholesterin values (m); signif. increased GOP (m), GPT (m)and AP (m/f); 1.13% in the diet: signif. incraesed AP (m) and GPT (f); 0.28 and 0.56% in the diet: signif. increased AP (m)
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
2.25%: increased in liver (m/f), brain (m/f), testes (m); decreased in spleen (m/f), kidney (m/f) and heart (m/f); 1.13%: increased in liver (m/f), brain (m), testes (m); decreased in spleen (m), kidney (f), heart (m); 0.56%: increased in liver (m/f)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
2.25% in the diet: no abdominal fat (m) and changes in colour and consistency of intestinal contents (m/f); 1.13% in the diet: changes in colour and consistency of intestinal contents (m)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
2.25% in the diet: liver (m/f), kidney (f), alimentary tract (m/f), connective tissue (m/f); 0.56-1.13% in the diet: liver (m/f); 0.28% in the diet: liver (f)
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Two male rats were killed due to ill health during this study. One of these animals, which was fed 0.14% of the test substance in the diet, was killed during Week 3 of the study due to a deformity of the posterior thorax, which is a lesion occasionally observed in Colworth Wistar rats and considered to represent injuries incurrent at birth or in the early post-partum period. The second animal, which was fed with dietary levels of 1.13%, was sacrificed during Week 6 due to rapid loss of weight and respiratory distress, which was associated with severe pulmonary oedema and emphysema and a major congenital cardiac defect, viz. patent interventricular foramen. Lesions of this type are also occasionally observed in young Colworth Wistar. Therefore, none of these lesions was considered to be related to treatment with the test substance in both animals.
No other animal died during the study period and the appearance of all 158 survivors was indicative of good health.

BODY WEIGHT AND WEIGHT GAIN (see Table 1 under “Any other information on results incl. tables”)
Males and females fed 2.25% of the test substance in diet showed significantly lower body weights and body weight gain compared to controls. This effect was also observed in males treated at dietary levels of 1.13%.

FOOD CONSUMPTION (see Table 2 under “Any other information on results incl. tables”)
The food intakes of males and females fed at a dietary dose level of 2.25% were significantly lower compared to controls, which corresponded to the decrease in body weights in both genders at this dose level.

FOOD EFFICIENCY (see Table 3 under “Any other information on results incl. tables”)
Food utilisation by male and female rats fed dietary levels of 2.25% was significantly poorer than by the control animals. This effect was also observed in males treated at dietary levels of 1.13%. The observations of poorer food utilisation corresponded well to the reported lower body weight gains in animals at dietary dose levels of 2.25 and 1.13%.

WATER CONSUMPTION
Significantly lower water consumption compared to controls was observed in females at dietary dose levels of 2.25%.

HAEMATOLOGY
No changes in haematological parameters were observed between animals of all treatment groups and controls.

CLINICAL CHEMISTRY (see Tables 4 and 5 under “Any other information on results incl. tables”)
Serum values of magnesium, total protein and cholesterol were decreased at the highest dose (2.25% in the diet) in males. In males of the same dose group, serum GOT, GPT and AP were elevated. Serum GPT was also increased in males and females receiving the second highest dose group (1.13% in the diet). Serum AP was also elevated in males receiving, 1.13, 0.56 and 0.28% of the test substance in the diet and in females receiving 1.13% and 2.25% of the test substance in the diet.

ORGAN WEIGHTS (see Tables 6 and 7 under “Any other information on results incl. tables”)
Relative liver weights (both sexes) significantly increased in the three highest dose groups (0.56%-2.25%). At the highest dietary level of feeding, the increase in relative liver weight in males and females was 19% and 42%, respectively. Furthermore, absolute weights of the liver were significantly increased in females of the two highest dose groups (1.13 and 2.25%) but not in males.
Absolute spleen weights significantly decreased in males at the two highest dose levels (1.13 and 2.25%) and in females at the highest dose. No changes in relative spleen weights were observed in any of the treated animals compared to controls.
Absolute kidney weights significantly decreased in high dose males, but relative kidney weights were normal compared to controls. Relative kidney weights significantly increased in females at the two highest dose levels (1.13 and 2.25%) while absolute kidney weights were not affected.
Relative brain weights were significantly increased in males fed the two highest dose levels (1.13 and 2.25%) and in females receiving the highest dose.
Relative weights of the testes increased at the two highest dose levels (1.13 and 2.25%).
Absolute heart weights significantly decreased in males fed the two highest dose levels (1.13 and 2.25%) and in females fed the highest dose. Relative heart weights were increased in males at the highest dose level.

GROSS PATHOLOGY
High dose males had virtually no abdominal fat and changes in colour and consistency of intestinal contents. Changes in colour and consistency of intestinal contents were also observed in one male of the 1.13% dose group. Depletion of body fat and changes in intestinal contents were less frequently noted in high dose females. All other lesions noted were not considered to be related to treatment as they were identified to represent normal background pathology in rats of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver:
Histological changes attributable to treatment with the test substance were found in liver, kidney, alimentary tract and connective tissue. The major histopathological findings were noted in liver.
At the highest dietary level (2.25%), diffuse (6/10 females, 3/10 males) and periportal (4/10 females, 7/10 males) hypertrophy, reduced cytoplasmic (glycogenic) vacuolation (10/10 females, 9/10 males) as well as reduced cytoplasmic neutral fat and hemosiderin content were prominent in the hepatic parenchyma of rats. In addition, the hemosiderin content of Kupffer cell was greatly depleted.
Diffuse hypertrophy was also identified in 5/10 females fed 1.13% of the test substance in the diet. Furthermore, periportal hypertrophy was observed in rats (5/10 females, 8/9 males) at this dose level and the next lower dose level of 0.56% (10/10 females, 5/10 males). In addition, this lesion was also observed in females (4/10 animals), but not in males, at the 0.28% dose level.
Reduced cytoplasmic (glycogenic) vacuolation, reduced cytoplasmic neutral fat, hemosiderin content and depletion of Kupffer cell hemosiderin were observed in males and/or females receiving dietary dose levels of 0.56%-2.25%.
No treatment-related histopathological changes were observed in the livers of rats (both genders) treated with dietary dose levels of 0.07 and 0.14%

Kidney:
The incidence and severity of nephrocalcinosis were significantly reduced in females (7/10 animals) treated with 2.5% in the diet. Normally, nephrocalcinosis is frequently well defined in untreated female rats of this strain and age. Further untreated animals of this age and strain also show small foci of cortical tubular athrophy, cortical interstitial fibrosis and focal lymphocytic infiltration, which are sequelae to mineral deposition in and occlusion of renal tubules. However, in females of the high those groups this three lesions were also reduced.
No changes attributable to treatment were observed in the kidney of male and female rats fed with the second highest dose level of the substance in the diet (1.13%).

Gastro intestinal tract:
Lymphatic dilation of the small intestine was more prominent at the high dose (2.5% in the diet) compared to the controls and the 1.13% dose level. The prominence was attributable to increase in the extent of dilation of individual vessels and to an increase in the number of visible lymphatic channels.At the high dose, a higher protozoan parasite colonisation in the animals compared to controls and the 1.13% dose group was observed within the mucus covering the mucosa. However, these parasites did not colonize the mucus and thus did not result in mucosal and submucosal lesions.

Connective tissue:
At the highest dose level, a reduced quantity of stromal lipid, particularly in pancreas and parotid salivary glands, was observed compared to controls and the 1.13% dietary dose group.

Other organs:
No further treatment-related histopathological changes between treated and control animals were observed in any other organ. Incidence and intensity of histopathological changes occurring in other organs were shown to be normal background pathology observed in rats of this strain and age.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
1.13 other: % in the diet
Based on:
act. ingr.
Remarks:
corresponding to 1016 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: systemic effects that could not be regarded solely as adaptive processes, i.e. increased testes weight in males
Dose descriptor:
NOAEL
Effect level:
0.56 other: % in the diet
Based on:
act. ingr.
Remarks:
corresponding to 488 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1. Average body weight gain of rats over the 13-week treatment period

Dietary level of the test substance (%)

Average body weight gain (g)

Male rats

Female rats

2.25

213*

117*

1.13

242*

135

0.56

270

131

0.28

263

133

0.14

271

140

0.07

264

141

0.0 (control)

268

140

* Statistically significant (p < 0.05) compared to controls

 

Table 2. Mean total amounts of food consumed by the rats over the 13-week treatment period

Dietary level of the test substance (%)

Mean total amount of food consumed (g)

Male rats

Female rats

2.25

1502*

1192*

1.13

1581

1250

0.56

1636

1212

0.28

1614

1220

0.14

1609

1272

0.07

1573

1270

0.0 (control)

1643

1269

* Statistically significant (p < 0.05) compared to controls

 

Table 3. Mean body weight gain (g)/mean total food consumed (g) by rats over the 13-week treatment period

Dietary level of the test substance (%)

Mean total amount of food consumed (g)

Male rats

Female rats

2.25

0.141*

0.099*

1.13

0.154*

0.108

0.56

0.165

0.108

0.28

0.163

0.109

0.14

0.169

0.110

0.07

0.168

0.111

0.0 (control)

0.163

0.111

* Statistically significant (p < 0.05) compared to controls

Table 4. Serum analysis – Changes in biochemical parameters in male rats over the 13-week treatment period (mean values)

Dietary level of the test substance (%)

Magnesium (meq/L)

Creatinine (mg/100 mL)

Total protein (g/100 mL)

Cholesterol (mg/100 mL)

GOT

(mU/mL)

GPT

(mU/mL)

AP

(mU/mL)

2.25

1.3*

0.80

6.1*

77*

73*

41*

1289*

1.13

1.4

0.80

6.4

97

60

29*

1356*

0.56

1.5

0.81

6.2

92

57

26*

1135*

0.28

1.4

0.80

6.5

113

58

20

1048*

0.14

1.5

0.76

6.4

101

63

20

838

0.07

1.7

0.78

6.6

97

63

23

941

0.0 (control)

1.6

0.80

6.5

113

61

20

868

* Statistically significant (p < 0.05) compared to controls

 

Table 5. Serum analysis – Changes in biochemical parameters in female rats over the 13-week treatment period (mean values)

Dietary level of the test substance (%)

Magnesium (meq/L)

Creatinine (mg/100 mL)

Total protein (g/100 mL)

Cholesterol (mg/100 mL)

GOT

(mU/mL)

GPT

(mU/mL)

AP

(mU/mL)

2.25

1.4

0.82

6.2

76

72

24

1350*

1.13

1.4

0.74

6.4

89

73

25*

1169*

0.56

1.4

0.82

6.3

85

61

21

957

0.28

1.5

0.82

6.4

93

72

21

1059

0.14

1.4

0.77

6.2

85

74

24

858

0.07

1.5

0.77

6.2

92

70

20

841

0.0 (control)

1.5

0.79

6.3

88

68

20

859

* Statistically significant (p < 0.05) compared to controls

Table 6. Mean absolute and relative organ weights of male rats after the 13-week treatment period

Dietary level of the test substance (%)

Final body weight (g)

Heart (g)

Liver (g)

Spleen (g)

Kidneys (g)

Brain (g)

Testes (g)

Abs.

Rel.

Abs.

Rel.

Abs.

Rel.

Abs.

Rel.

Abs.

Rel.

Abs.

Rel.

2.25

285*

0.92*

0.32*

12.5

4.4*

0.45*

0.16

2.26*

0.79

1.92

0.68*

3.10

1.10*

1.13

317*

0.95*

0.30

13.2

4.1*

0.49*

0.16

2.58

0.81

1.93

0.61*

3.21

1.02*

0.56

344

1.05

0.31

13.4

3.9*

0.55

0.16

2.69

0.79

1.95

0.57

3.20

0.93

0.28

339

1.06

0.31

12.7

3.8

0.56

0.17

2.69

0.81

1.90

0.57

3.19

0.95

0.14

348

1.05

0.30

12.9

3.7

0.57

0.16

2.71

0.79

1.98

0.57

3.19

0.92

0.07

338

1.04

0.31

12.4

3.7

0.55

0.16

2.62

0.81

1.95

0.58

3.16

0.94

0.0 (control)

342

1.03

0.30

12.6

3.7

0.55

0.16

2.72

0.79

1.92

0.56

3.19

0.94

* Statistically significant (p < 0.05) compared to controls

 

Table 7. Mean absolute and relative organ weights of female rats after the 13-week treatment period

Dietary level of the test substance (%)

Final body weight (g)

Heart (g)

Liver (g)

Spleen (g)

Kidneys (g)

Brain (g)

Abs.

Rel.

Abs.

Rel.

Abs.

Rel.

Abs.

Rel.

Abs.

Rel.

2.25

179*

0.63*

0.35

8.4*

4.7*

0.34*

0.19

1.57

0.88*

1.78

1.00*

1.13

199

0.69

0.34

8.4*

4.2*

0.38

0.19

1.66

0.84*

1.80

0.90

0.56

196

0.70

0.35

7.2

3.7*

0.40

0.20

1.57

0.80

1.79

0.91

0.28

197

0.68

0.34

6.7

3.4

0.37

0.19

1.54

0.78

1.77

0.91

0.14

204

0.70

0.35

6.9

3.4

0.39

0.19

1.62

0.80

1.78

0.88

0.07

204

0.70

0.35

6.9

3.4

0.40

0.20

1.66

0.82

1.79

0.88

0.0 (control)

204

0.71

0.35

6.7

3.3

0.40

0.20

1.60

0.79

1.80

0.89

* Statistically significant (p < 0.05) compared to controls

Applicant's summary and conclusion

Conclusions:
Since the liver as the target organ showed only adaptive responses, the NOAEL was set at 0.56% (488 mg/kg bw/day). The adaptive changes included elevated relative liver weight due to a lower body weight and reduced food consumption, hepatic periportal hypertrophy as well as increased serum alkaline phosphatase (AP) activity.
An increased serum AP activity is considered to represent a physiological adaptation resulting from changes in hepatic metabolism required for the breakdown and detoxification of the test material. Since AP is mainly localized in the hepatic parenchyma, enlargement of the hepatic parenchymal cells accompanied by an increased organ weight are an obvious consequence.
Executive summary:

C12 -15AS Na (CAS 68890 -70 -0) was tested in a 13 week feeding study on rats. Ten rats/sex/dose in the test groups and 20 rats/sex in the control group were administered dietary levels of 0.07, 0.14, 0.28, 0.56, 1.13 and 2.25% (corresponding to 58, 113, 228, 470, 961 and 1944 mg/kg bw/day in males and 66, 131, 261, 506, 1070 and 2218 mg/kg bw/day in females). The control group received the diet alone. The NOAEL was set at 488 mg/kg bw/day for males and females since only adaptive changes in liver were observed at this dose level.