Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Zoster S.A. (Raiguero, S/N, Zeneta-Murcia, Spain), lot no.1999
- Purity: > 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The sample was stored in sealed plastic bags in a cool and dry place.
- Stability under test conditions: NHDC was found to be stable in the diet under the experimental conditions applied.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
(Cpb: WU; Wistar random)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TNO Central Institute for the Breeding of Laboratory Animals, Zeist, The Netherlands.
- Weight at study initiation: males weighed around 84 g and females around 79 g.
- Housing: The rats were housed in groups of five of the same sex and treatment group in suspended stainless-steel wire-screen cages.
- Diet: cereal-based, open-formula diet (TNO-C1VO Toxicology and Nutrition Institute, the Netherlands). Food was provided ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The Institute's basal diet is analysed for both nutrients and contaminants twice a year. Typical results of analysis for calcium, phosphorus and magnesium are: Ca, 0.7-0.9%; P, about 0.6%; Mg, about 0.17%.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 40-80%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh batches of the test diets were prepared five times in the course of the study.
- Mixing appropriate amounts with (Type of food): NHDC was mixed into the Institute's cereal-based, open-formula diet, the composition (%) of which was as follows: soybean oil meal 11; fish meal 7; meat and bone scraps 4; whole ground wheat 36; whole ground maize 29.7, grass meal 3; whey powder 2; defatted bone meal 0.4; soybean oil 3; trace elements in salt 0.5; brewers' yeast 3; vitamin B preparation 0.1; vitamin ADEK mixture 0.3. Typical results of analysis for calcium, phosphorus and magnesium are: Ca, 0.7-0.9%; P, about 0.6%; Mg about 0.17%.
- Storage temperature of food: The diets were stored at about 15ºC.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Diet samples were analysed for NHDC content by HPLC, according to Fisher (1977), using an M&N 25cm x 4.6mm i.d. reverse-phase Poligosil 60-5-CI8 column. The adequacy of the mixing procedure was investigated by analysing five samples of each test diet from the first batch. The stability of NHDC in this batch of diets was examined after storage for 4 wk at room temperature. In addition, the NHDC content of the test diets was analysed monthly.
- Throughout the study, the actual levels of NHDC in the low-, intermediate- and high-dose diet were 95%, 97-99% and 94-105% of the intended levels, respectively. The quintuplicate analyses of the first batch of each of the NHDC-containing diets showed coefficients of variation of less than 5%, indicating satisfactory homogeneity.
Duration of treatment / exposure:
13 consecutive weeks.
Frequency of treatment:
Daily.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control.
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
0.2 % (w/w) in diet, ca. 150 mg/kg (males) - 166 mg/kg (females).
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
1% (w/w) in diet, ca. 757 mg/kg (males) - 848 mg/kg (females).
Dose / conc.:
4 000 mg/kg bw/day (nominal)
Remarks:
5 % (w/w) in diet, ca. 4011 mg/kg (males) - 4334 mg/kg (females).
No. of animals per sex per dose:
20 rats/sex/group.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Groups of 20 male and 20 female rats were fed diets containing 0, 0.2, 1.0 or 5.0% NHDC for 13 consecutive weeks. No further details.
- Owing to the normal decrease in food intake per unit body weight with increasing age of rats, the intake of NHDC per kg body weight became lower in the course of the study. The overall actual intakes of NHDC were roughly 150, 750 and 4000 mg/kg body weight/day for the low-, intermediate- and high-dose groups, respectively.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The rats were observed daily for external signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. Food intake was measured over 1-wk periods.

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 a drinking water study): Yes
- Time schedule for examinations: water intake was recorded in wk 7 and 12.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of the study and at wk 13.
- Dose groups that were examined: control group and high-dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: early in week 13.
- Anaesthetic used for blood collection: No
- Animals fasted: Not specified.
- How many animals: 10 rats/sex/group
- Parameters examined: haemoglobin concentration, packed cell volume, and counts of thrombocytes and red and white blood cells (including a differential white cell count).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in wk 13.
- Animals fasted: Yes
- How many animals: 10 rats/sex/group
- Parameters examined: glucose (Boehringer Glucoquant No. 245-178; Boehringer Mannheim GmbH Mannheim, FRG); alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, r-glutamyltransferase, total protein, albumin, total bilirubin, urea, cholesterol, creatinine and calcium (Cobas-Bio Centrifugal Analyzer), ornithine-carbamyl transferase (Spectro-fotometric), inorganic phosphate (Boehringer Kit No. 124-974, Boehringer Mannheim GmbH), chloride (Chloro Counter, Marius), and sodium and potassium (Electrolyte-2-Analyzer, Beckman).

URINALYSIS: Yes
- Time schedule for collection of urine: urine was collected from ten rats/sex/group during the last 16 hr of a 24-hr period of deprivation of food and water in wk 13.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: volume (calibrated tubes), density (Bellingham & Stanley refractometer), for protein, glucose, occult blood, ketones, bilirubin and urobilinogen (pooled samples; Combur test strips, Boehringer Mannheim GmbH), and microscopy of the sediment (pooled samples).

OTHER: In week 13, pH was determined in urine samples collected over 3 hr early in the morning from ten non-fasted rats/sex/group (Philips P.W. 9410 pH meter).

NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Early in wk 14, the rats were killed by exsanguination from the abdominal aorta, under light ether anaesthesia, and subjected to a thorough post-mortem examination. Any abnormalities were recorded and the adrenals, brain, caecum (with and without contents), gonads, heart, kidneys, liver, spleen, thymus and thyroid (with parathyroids) were weighed. Samples of these organs and of the aorta, axillary lymph nodes, colon, duodenum, epididymides, eyes, ileum, jejunum, lungs, mammary glands (both sexes), mesenteric lymph nodes, oesophagus, pancreas, pituitary, prostate, rectum, sciatic nerve, skeletal muscle, spinal cord, sternum, stomach (glandular and non-glandular), salivary glands (parotid, sublingual and submaxillary), trachea, urinary bladder and uterus (with cervix) were preserved in 10% phosphate buffered formaldehyde.

HISTOPATHOLOGY: Yes
- Wax-embedded 5-11 m sections of the preserved tissues from all animals in the control and high-dose group were stained with haematoxylin and eosin and examined microscopically. One female of the intermediate-dose group that died intercurrently was subjected to histopathological examination as well.
Statistics:
Data on body weights were evaluated by one-way analysis of covariance, followed by Dunnett's multiple-comparison tests. The laboratory determinations and organ weights were evaluated by one-way analysis of variance, followed by Dunnett's multiple-comparison tests, except for the differential white blood cell counts and urinary pH values which were analysed by the Mann Whitney U test. Data on food and liquid intake was evaluated by analysis of variance, followed by least significant difference tests (experimental unit: the cage). Fisher's exact probability test (two sided) was used for results of ophthalmoscopy and for pathology data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed in the low- and intermediate-dose groups. In the high-dose group, both males and females showed soft stools in wk 2 and 3.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat in intermediate dose group was found dead at day 30 but, as no additional mortality was found in any dose groups and macroscopic and microscopic examination of the animal found dead revealed renal failure not seen in any other dose groups, this death is considered a fortuitous finding.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The low and medium dose groups showed no effects. At the high-dose level, growth was depressed in males throughout the study and in females in the first 2 weeks and, accordingly, food intake slightly decreased.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The low and medium dose group showed no effects. At the high-dose level in males throughout the study and in females in the first 2 weeks food intake was slightly decreased.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
The low and medium dose group showed no effects. At the high-dose level a transient decrease in food-conversion efficiency was observed.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was not significantly affected by the administration of NHDC at any dose level.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examinations did not reveal any treatment related effects.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no significant changes in haematology among the groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical changes that might have been due to treatment, were observed in the high-dose group only: in females, plasma alkaline phosphatase activity and bilirubin concentration were increased; total plasma protein concentration was decreased in males; plasma urea concentration was relatively low in both sexes.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinary pH was decreased in both sexes of high-dose group.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- The organ-weight data showed marked enlargement of the caecum in the high-dose group in both sexes. Slight, though statistically significant increases in caecal weight occured also in males of the low- and intermediate-dose group but there was no dose-response relationship.
- The relative weights of the brain and testicles were increased in males of the high-dose group. The mean absolute weights of these organs were, however, very similar to those of the controls. This change is not ascribed to a direct effect of the test item. The other organ weights did not show treatment-related changes in either sex.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross examination at autopsy did not reveal treatment-related changes in any of the experimental groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of the organs and tissues from all rats of the control and high-dose group did not reveal any treatment-related changes.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Details on results:
- Intake of NHDC: Owing to the normal decrease in food intake per unit body weight with increasing age of rats, the intake of NHDC per kg body weight became lower in the course of the study. The overall actual intakes of NHDC were roughly 150, 750 and 4000mg/kg body weight/day for the low-, intermediate- and high-dose groups, respectively.
- In the present study feeding Wistar rats with diets containing up to 5% NHDC was associated with several changes of little, if any, toxicological significance.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
ca. 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 4 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effects were observed.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1. Body weights, food consumption and test item intakes of rats fed diets containing 0-5% NHDC for 13 wk.

Dose

(% in diet)

Body weight (g) at week

Food consumption (g/rat/wk)

Mean intake**

(mg/kg bw/d)

Males

0

1

2

3

4

8

13

1

2

3

4

8

12

0

84 ± 0.9

125 ± 1.4

166 ± 2.4

214 ± 3.7

255 ± 4.4

343 ± 7.0

392 ± 8.3

105

128

135

149

148

147

0

0.2

84 ± 0.9

125 ± 1.7

166 ± 2.8

212 ± 3.9

254 ± 4.4

341 ± 9.1

391 ± 11.0

106

128

133

148

142

146

150

1.0

84 ± 1.1

125 ± 1.8

164 ± 2.6

210 ± 3.8

249 ± 4.1

338 ± 5.6

387 ± 6.5

106

126

134

147

146

146

757

5.0

84 ± 0.9

125 ± 2.2*

146 ± 3.3*

192 ± 3.4*

232 ± 3.8*

310 ± 6.5*

355 ± 7.4

93*

111*

132

148

142

146

4011

Females

 

0

79 ± 0.9

110 ± 1.2

133 ± 1.6

156 ± 2.4

172 ± 2.4

210 ± 4.3

230 ± 5.1

92

100

103

106

111

109

0

0.2

79 ± 0.8

110 ± 1.1

135 ± 1.6

159 ± 2.3

174 ± 2.7

213 ± 3.7

234 ± 4.2

91

101

100

105

112

109

166

1.0

79 ± 1.2

109 ± 1.5

132 ± 2.0

154 ± 2.8

169 ± 2.8

206 ± 4.6

232 ± 4.5

91

102

98

106

111

111

848

5.0

79 ± 0.9

104 ± 1.6*

126 ± 2.1*

153 ± 2.7

170 ± 2.9

206 ± 4.1

227 ± 4.2

87

100

102

108

111

113

4334

**Overall means of weekly calculations from data on body weight, food intake and nominal levels of NHDC.

Body-weight values are means ± SEM for groups of 20 rats. Food consumption values are the means for four cages of five animals. Although growth and food consumption were recorded weekly, only values obtained during the first 4 weeks and subsequendly monthly are presented. The values marked with * differ significantly (body weight; Dunnett’s test, and food consumption; least significant difference test) from the control value (*P < 0.01).

Table 2. Plasma biochemical and urinary parameters of rats fed diets containing 0-5% NHDC for 13 wk.

Parameter

% NHDC in diet

0

0.2

1.0

5.0

MALES

Plasma

Alkaline phosphatase (U/L)

144.9 ± 7.0

141.0 ± 5.8

163.9 ± 7.2

150.3 ± 4.0

Total protein (g/L)

63.4 ± 0.9

61.3 ± 0.4

61.7 ± 0.5

59.6 ± 0.6**

Bilirubin (μmol/L)

0.30 ± 0.13

0.20 ± 0.08

0.02 ± 0.01

0.61 ± 0.24

Urea (mmol/L)

7.08 ± 0.29

7.68 ± 0.55

7.76 ± 0.34

6.50 ± 0.14

Urine

pH

8.0

7.5

7.8

7.2**

FEMALES

Plasma

Alkaline phosphatase (U/L)

129.1 ± 4.7

124.8 ± 8.9

142.8 ± 5.9

154.4 ± 5.1*

Total protein (g/L)

61.7 ± 0.6

61.0 ± 0.5

60.9 ± 0.8

61.6 ± 0.7

Bilirubin (μmol/L)

0.32 ± 0.13

0.25 ± 0.09

0.62 ± 0.10

1.04 ± 0.17**

Urea (mmol/L)

8.06 ± 0.75

8.52 ± 0.71

7.78 ± 0.50

6.34 ± 0.33

Urine

pH

8.0

7.9

7.9

7.3**

Values are means ± SEM for groups of ten rats; those marked with asterisks differ significantly [Dunnett’s test or Mann-Whitney U-test (for pH values)] from the corresponding control value (*P < 0.05; **P < 0.01). No other blood, plasma or urinary analysis values differed significantly from the corresponding control value.

Table 3. Relative organ weights of rats fed diets containing 0-5% NHDC for 13 wk.

Organ

Relative organ weights (g/kg bw)

0

0.2

1.0

5.0

MALES

Adrenals

0.132 ± 0.003

0.140 ± 0.004

0.145 ± 0.004

0.142 ± 0.005

Brain

5.13 ± 0.12

5.04 ± 0.14

5.12 ± 0.07

5.55 ± 0.13*

Caecum (full)

11.6 ± 0.6

14.8 ± 0.6**

14.5 ± 0.6**

20.4 ± 0.9**

Caecum (empty)

2.3 ± 0.1

2.8 ± 0.1*

2.6 ± 0.1

3.5 ± 0.2**

Heart

3.21 ± 0.06

3.32 ± 0.07

3.18 ± 0.06

3.28 ± 0.06

Kidneys

6.29 ± 0.13

6.31 ± 0.11

6.28 ± 0.12

6.33 ± 0.08

Liver

36.0 ± 0.6

36.2 ± 0.7

35.4 ± 0.5

34.1 ± 0.5

Spleen

1.60 ± 0.04

1.71 ± 0.05

1.64 ± 0.05

1.60 ± 0.04

Testes

8.55 ± 0.20

8.54 ± 0.28

8.65 ± 0.23

9.49 ± 0.23*

Thymus

0.92 ± 0.05

0.99 ± 0.05

0.85 ± 0.04

0.88 ± 0.04

Thyroid

0.063 ± 0.003

0.066 ± 0.003

0.062 ± 0.004

0.067 ± 0.003

FEMALES

Adrenals

0.286 ± 0.010

0.285 ± 0.010

0.285 ± 0.09

0.309 ± 0.009

Brain

7.90 ± 0.18

7.71 ± 0.12

7.78 ± 0.15

7.94 ± 0.13

Caecum (full)

15.5 ± 0.6

14.7 ± 0.7

16.6 ± 0.7

22.5 ± 0.8**

Caecum (empty)

3.3 ± 0.2

3.3 ± 0.2

3.5 ± 0.1

4.6 ± 0.2**

Heart

3.85 ± 0.07

3.85 ± 0.06

3.87 ± 0.09

3.90 ± 0.07

Kidneys

6.71 ± 0.09

6.58 ± 0.13

6.62 ± 0.10

6.85 ± 0.09

Liver

33.4 ± 0.3

33.3 ± 0.5

33.0 ± 0.5

33.9 ± 0.5

Spleen

1.86 ± 0.05

1.95 ± 0.05

1.84 ± 0.04

1.95 ± 0.04

Ovaries

0.363 ± 0.013

0.372 ± 0.014

0.377 ± 0.013

0.347 ± 0.010

Thymus

1.27 ± 0.05

1.31 ± 0.04

1.29 ± 0.04

1.36 ± 0.05

Thyroid

0.092 ± 0.003

0.082 ± 0.04

0.089 ± 0.004

0.094 ± 0.003

Values are means ± SEM for groups of 20 rats. Those marked with asterisks differ significantly (Dunnett’s test) from the corresponding control value (*P < 0.05; **P < 0.01).

Table 4. Major histopathological changes in rats fed NHDC for 13 wk.

Organ and lesion

Dose(%):

No. of rats affected

Males

Females

0

5.0

0

5.0

Adrenals

- Cortical vacuolation

7

4

0

0

Epididymis

- Reduced spermatogenesis

1

0

 

 

Heart

- Cartilaginous metaplasia

0

1

0

1

Kidneys

- Nephrosis

13

11

3

5

- Calcareous deposits

in the cortico-medullary layer

0

1

14

11

- Calcareous deposits in pelvis

0

0

4

0

- Urothelial hyperplasia in pelvis

0

0

3

0

- Hydronephrosis

0

1

0

1

Large intestines

- Parasites

2

1

1

3

Liver

- Aggregates of RES cells

3

4

5

5

Lung

- Perivascular lymphocytic infiltrates

1

1

9

3

Pituitary

- Cysts

1

1

1

2

- Tubular hyperplasia in pars neuralis

0

0

0

1

Spleen

- Hematopoiesis

0

1

0

1

Stomach

- Fundic glandular dilatation

0

0

3

0

Sub-lingual salivary glands

- Demi-lune cell proliferation

0

0

0

2

Testes

- Atrophy

1

0

 

 

- Tubular calcareus deposits

2

0

 

 

Urinary bladder

- Urothelial hyperplasia

0

0

0

1

Uterus

- Polyp

 

 

0

1

- Endometritis

 

 

0

2

The tissues of 20 animals were examined microscopically in all cases except that the pituitaries of only 19 control males were examined.

Applicant's summary and conclusion

Conclusions:
The test item has a sub-chronic oral NOAEL of ca. 4000 mg/kg bw/d (5% in diet) in rats, and a NOEL of ca. 750 mg/kg bw/d (1% in diet), based on clinical findings and biochemistry.
Executive summary:

A study on the sub-chronic toxicity of the test item was performed by a method similar to OECD 408, without GLP. The test item was administered to groups of 20 male and 20 female Wistar rats at dietary levels of 0 (control), 0.2, 1.0 and 5.0% for 91 days. No treatment-related ophthalmoscopical, haematological or histopathological effects were observed. In the high-dose group, a marked caecal enlargement occurred in both sexes, accompanied by soft stools in the early stages of the study, somewhat lower plasma urea concentrations and increased plasma alkaline phosphatase activity and a decreased urinary pH. This group also showed slight growth depression accompanied by transient reduction in food intake; in males the body weights remained relatively low throughout the experimental period. Furthermore, bilirubin level was increased in females and total protein level was decreased in males of the high-dose group. The above changes were considered adaptive responses or chance effects rather than manifestations of clear toxicity. Therefore the NOAEL could be set at 5% in diet (ca. 4000 mg/kg bw). The low- and intermediate-dose groups did not show any compound-related untoward effect, so it was concluded that the mid dose (1%) of about 750 mg/kg bw/d, was the NOEL.