Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
8 April 2002 to 15 July 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Reliability of 2 given since the data is based on read across, not the target substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD (SD) IGS BR (outbred albino).
- Age at arrival: Around 6 weeks.
- Weight at arrival: 180 to 190 g for the males; 113 to 161 g for the females.
- Fasting period before study: No.
- Housing: The animals were housed 2 per cage initially, in polypropylene cages (42 x 27 x 20 cm) with stainless steel grid bottoms and mesh tops. A stainless steel food hopper and a polypropylene or polycarbonate water bottle were provided for each cage. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage. Male and female cages were racked separately.
A few days prior to pairing for mating, males were transferred to individual grid-bottomed cages (59 x 38.5 x 20 cm) of similar design. Mated females were transferred to individual (42 x 27 x 20 cm) solid bottomed cages. Sterilised white wood shavings were provided as bedding and white paper tissue as nesting material where appropriate. Wooden chewsticks were provided for environmental enrichment.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): The animals had access to domestic mains water ad libitum via water bottles.
- Acclimation period: 12 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2 °C.
- Humidity (%): 50 ± 15 %.
- Air changes (per hr): Minimum of 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Automatic control of the 12 hour light cycle; light from 0700 to 1900 hours.

IN-LIFE DATES: From: 3 April 2002 To: 27 May 2002

Administration / exposure

Route of administration:
oral: feed
Vehicle:
acetone
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
An appropriate quantity of the test material was dissolved in a suitable volume of acetone. This solution was added to a suitable quantity of untreated diet and mixed for around one hour with fan assisted venting to form a dose premix. A control premix was prepared using the same proportion of acetone and untreated diet.
The diets for the intermediate and high dose groups were prepared by dilution of the dose premix with untreated diet to give the desired concentrations. The low dose diet was prepared by dilution of the high dose diet with untreated diet. The diet premixes were then placed on a Winkworth mixer for about 20 minutes.
The control diet was prepared by dilution of the control premix with untreated diet such that the diet contained the same proportion of premix as the high dose diet.
Details on mating procedure:
MATING PROCEDURE
Pairing was on a one male to one female basis, within the same treatment group. Each female was transferred to the cage of an appropriate co-group male near the end of the working day, and remained there until mating was detected.
Vaginal lavages were taken early each morning commencing on the day of pairing, until mating was detected, and the day of observation of a copulatory plug in situ and/or sperm in the lavage was designated day 0 of gestation.
Each female remained with its first designated male for a maximum of 7 consecutive nights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DIET FORMULATIONS
The analytical method was validated in a separate study at the testing facility.
Diet formulations were sampled twice during the study (weeks 1 and 4). Triplicate samples of each formulation, including control, were taken immediately after preparation. On both occasions, the analysed concentrations were found to be within ± 10 % of the nominal concentration, indicating acceptable accuracy of formulation. A low coefficient of variation (3.4 % or less) was indicative of satisfactory homogeneity.
Duration of treatment / exposure:
The males were dosed for at least 4 weeks overall, starting from 2 weeks prior to mating until termination.
The females were dosed for 2 weeks prior to mating, then through mating until termination after day 4 of lactation.
Frequency of treatment:
Continuous in the diet.
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 10 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000, 5000 and 20 000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of existing toxicological data, including a one week range finding study in the rat.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All the animals were examined for reaction to treatment on each day. The nature, onset, duration and intensity of any signs were recorded. In addition, all the animals were checked for viability early in the morning and again as late as possible on each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Male weights were recorded once during the week prior to the commencement of dosing and once weekly thereafter until termination.
Female weights were recorded once during the week prior to the commencement of dosing, and weekly thereafter until the start of the mating period, and then on day 0 of gestation (the day of detection of a positive mating sign) followed by days 7, 14 and 20 of gestation, and then days 1 and 4 of lactation (where day 0 = the day of parturition).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
For all animals, the weight of food consumed by each cage was recorded once weekly, beginning during the week prior to the commencement of dosing, until pairing for mating. All animals were fed ad libitum during mating, but following completion of mating weekly consumption was recommenced for males, until termination.
For mated females, the amount of food consumed was recorded over days 0 to 7, 7 to 14 and 14 to 20 of gestation, and days 0 to 4 of lactation.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION: Yes
- Time schedule for collection of blood: The blood samples were collected from the lateral tail vein after careful cleaning, during week 5 of dosing for males and on day 6 of lactation for females. A sample of 0.5 mL was transferred into tubes containing EDTA for haematology investigations. A further 0.45 mL was taken into tubes containing 0.05 mL 3.8 % (w/v) trisodium citrate for assessment of coagulation; the final sample volume was to be as close as possible to 0.5 mL to give a final concentration of 0.38 % (blood to citrate ratio of 9:1).
- Animals fasted: No
- How many animals: Samples were obtained from 5 males and 5 females from each dose group. For males the first 5 animals in each group were tested. For females the first 5 animals to have reared their litter to day 6 of lactation were tested.
- Parameters examined for haematology: Haemoglobin, Red Blood Cell Count, Haematocrit, White Blood Cell Count, Mean Cell Volume, Mean Cell Haemoglobin, Mean Cell Haemoglobin Concentration, Platelets, Differential White Blood Cell Count: Neutrophils; Lymphocytes; Monocytes; Eosinophils; Basophils; Large Unclassified Cells.
- Parameters examined for coagulation: Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The blood samples were collected from the lateral tail vein after careful cleaning, during week 5 of dosing for males and on day 6 of lactation for females. At least 1 mL was taken into lithium heparin tubes to be used for clinical chemistry investigations.
- Animals fasted: No
- How many animals: Samples were obtained from 5 males and 5 females from each dose group. For males the first 5 animals in each group were tested. For females the first 5 animals to have reared their litter to day 6 of lactation were tested.
- Parameters examined: Urea, Glucose, Aspartate Aminotransferase, Alanine Aminotransferase, Sodium, Potassium, Chloride, Total Protein, Albumin, A:G Ratio; Creatinine, Calcium, Phosphate, Total Bilirubin, Cholesterol, Alkaline Phosphatase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight and epididymis weight.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies and physical abnormalities.


GROSS EXAMINATION OF DEAD PUPS: Yes, for external abnormalities.
Postmortem examinations (parental animals):
GROSS PATHOLOGY
All animals were sacrificed by exposure to carbon dioxide, weighed then exsanguinated by severance of major blood vessels. All adults were subject to a detailed necropsy under the guidance of a veterinary pathologist. The necropsy consisted of an external and internal examination. All gross lesions were recorded in terms of location, size, shape, colour, consistency and number. The pups were sacrificed at the same time as their mother.

HISTOPATHOLOGY
Histological examination was conducted on control and high dose animals only, the same animals that were used for haematology, coagulation and clinical chemistry evaluations.
The following organs were removed from all animals and were weighed and preserved as appropriate: Abnormal Tissue (included local lymph nodes to masses), Adrenals, Aorta, Brain (Forebrain, mid-brain, cerebellum and pons), Ears, Epididymides, Eyes, Gastro-Intestinal Tract (Stomach; Duodenum; Jejunum; Ileum; Caecum; Colon; Rectum), Heart, Kidneys, Liver, Lung, Marrow Smear (femur), Mesenteric Lymph Node, Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Sciatic Nerve, Seminal Vesicles, Skin and Mammary Gland, Spinal Cord (Cervical, subthoracic and lumbar regions), Spleen, Sternum, Submandibular Lymph Node, Submaxillary Salivary Gland, Testes, Thymus, Thyroid with Parathyroid x 2, Trachea, Urinary Bladder, Uterus.

PROCESSING OF FIXED TISSUES
Tissues were trimmed to a maximum thickness of 3 mm for processing. Parenchymal organs were trimmed to allow the largest surface area possible for examination.
Hollow organs were trimmed and blocked to allow preparation of a cross section from mucosa to serosa.
Sections were cut at 4 to 6 µm thickness and stained with haematoxylin and eosin. An additional section from each testis was stained with Periodic Acid Schiff and haematoxylin.
Postmortem examinations (offspring):
SACRIFICE
The pups were sacrificed by injection of sodium pentobarbitone.

GROSS NECROPSY
The pups were examined for visible external abnormalities.

Statistics:
- Bodyweight and food consumption (prior to mating for females), haematology and clinical chemistry data were statistically analysed for homogeneity of variance using the ‘F-max’ test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons made via Student’s t-test using Fisher’s F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous then Kruskal-Wallis ANOVA was used.
- Organ weights were also analysed as above and by analysis of covariance (ANCOVA) using terminal kill bodyweight as covariate.
- Histology incidence data were analysed using Fisher’s Exact Probability Test.
- The following pairwise comparisons were performed against the control group: control vs low dose; control vs intermediate dose; control vs high dose.

All statistical tests were two-sided and performed at the 5 % significance level.
Reproductive indices:
The following indices of fertility were evaluated for each group.

- Fertility Index (female) = Number pregnant / Number paired

- Fertility Index (male) = Number siring a Litter / Number paired

- Gestation Index = Number bearing live pups / Number pregnant
Offspring viability indices:
The following were evaluated for each group.

- Birth Index = Total number of pups born (live and dead) / Number of implantation scars

- Live Birth Index = Number of pups live on day 0 of lactation / Total number born (live and dead)

- Viability Index = Number of pups live on day 4 of lactation / Number live on day 0

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: see below

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

BODYWEIGHTS
At 20 000 ppm there was a transient decrease in weight gain in both sexes. In males, decreased weight gain was most notable over the first week, although absolute weights were significantly lower over the first 3 weeks of treatment. In females, there was a notable decrease throughout the pre-mating phase. The resulting deficit in body weight was never regained in either sex. In pregnant females reduced weight gain was evident over days 7 to 20 of gestation, compared to the control animals.
There were no obvious effects of treatment at 5000 or 1000 ppm.

FOOD CONSUMPTION AND ACHIEVED DIETARY INTAKE
At 20 000 ppm food consumption in males was reduced for the first 2 weeks of treatment (attaining significance during week 1) and in week 4 (not recorded week 3 as paired for mating). In females, food consumption was significantly decreased during the pre-mating period. Consumption was also reduced during the first half of the gestation period, compared to the control animals.
There were no obvious effects of treatment at 5000 or 1000 ppm.
The achieved intake in the first week of treatment for males and females at 20 000 ppm was lower than the second week. For males and females at the low and intermediate dose levels, intake was higher than in the following weeks (as expected). At other times, the achieved intake was essentially proportional to the diet concentrations.

HAEMATOLOGY
At 20 000 ppm there was a non-significant decrease in white blood cells in females. Any other intergroup differences were not considered to reflect an effect of treatment.

CLINICAL CHEMISTRY
Alkaline phosphatase levels were significantly increased in females at 5000 and 20 000 ppm, and in males at 20 000 ppm. In males, there was a non-significant increase in levels at 5000 ppm and in females at 1000 ppm there was an equivocal increase, but given the small group size it was considered that the difference was too small to reflect an effect of treatment.
Total bilirubin was increased in both sexes at 20 000 ppm.
In addition, at 20 000 ppm, cholesterol levels were increased in males; albumin (and consequently total protein) were reduced in females.

ORGAN WEIGHTS
In males, at 20 000 ppm there was a decrease in bodyweights, with liver weights being essentially similar to controls. At 5000 and 1000 ppm liver weight was slightly greater than controls. Following covariance analysis, there was a dose related increase in liver weights, with the increases at 5000 and 20 000 ppm attaining statistical significance.
In females, slight non-significant increases in liver weights following covariance analysis at 5000 and 20 000 ppm were too small to attribute to treatment.
In males at 20 000 ppm, spleen weight was notably increased following variance and covariance analysis. Adrenal gland and thymus weights were slightly but significantly decreased. Following covariance analysis, adrenal gland weight was still significantly decreased, but for the thymus there was no significant difference from controls.
In females, ovary, adrenal gland and kidney weights were significantly reduced at 5000 and 20 000 ppm, with pituitary gland weight reduced at 20 000 ppm. Following covariance analysis, kidney and pituitary gland weights were essentially similar to controls, but a decrease in ovary weight at 20 000 ppm and adrenal gland weight at 5000 and 20 000 ppm was still evident, but not significant.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 20 000 ppm, decreased weight gain and food consumption and changes in liver function were seen in both sexes. At 5000 ppm increased alkaline phosphatase levels were seen in both sexes.
Dose descriptor:
NOEL
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was a marginal decrease in implant sites at 20 000 ppm with a corresponding decrease in the mean total number of pups born compared to other dose groups.
Remarks on result:
other: Generation: Reproductive Parameters (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

REPRODUCTIVE PARAMETERS
- Mating Performance and Duration of Gestation
Mating performance was not affected by treatment.
There were no obvious effects on the duration of gestation at any of the dose levels applied.

- Litter Size and Survival
At 20 000 ppm, the mean number of implant sites per pregnancy was marginally decreased and hence the mean total number of pups born was lower than that of all other dose groups. However, due to the very slight differences compared to the control group, there is some doubt as to the reproducibility of this finding.
Litter survival, as indicated by the birth index and viability index, was similar in all groups.

- Litter and Pup Weights
At 20 000 ppm mean litter weights were slightly reduced compared to the controls, reflecting the decrease in litter size.

- Abnormalities among Pups
There were no obvious external abnormalities noted in the pups at any of the dose levels applied.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1 Parental Pre-mating Mean Group Bodyweights (g)

Dose Level (ppm)

 

Males

Females

Pre-trial (days)

Treatment Period

(days)

Body-weight Gain day 0 to 28

Pre-trial (days)

Treatment Period (days)

Body-weight Gain day 0 to 14

-7

0

7

14

21

28

-7

0

7

14

 

0

Number

Mean

SD

10

249

10

10

320

17

10

374

26

10

414

34

10

447

37

10

468

45

10

149

30

10

165

8

10

194

9

10

217

13

10

237

16

10

43

9

 

1000

Number

Mean

SD

10

248

4

10

313

9

10

369

16

10

408

17

10

437

19

10

460

23

10

147

16

10

166

12

10

199

15

10

221

18

10

240

22

10

41

12

 

5000

Number

Mean

SD

10

245

6

10

316

11

10

373

16

10

415

20

10

444

26

10

471

28

10

154

20

10

167

6

10

197

8

10

219

10

10

237

10

10

40

4

 

20 000

Number

Mean

SD

10

245

8

10

313

10

10

351

13**

10

386

18*

10

413

22**

10

434

29

10

121

24*

10

156

7

10

188

9

10

200

9**

10

214

15**

10

25

12**

Significantly different from the control: *p<0.05; **p<0.01; ***p<0.001

SD = Standard Deviation

 

Table 2 Parental Bodyweights (Females) During Gestation and Lactation Mean Group Values (g)

Dose Level (ppm)

Day of Gestation

Weight Gain Days 0 to 20 of Gestation

Percentage of Control

Day of Lactation

0

7

14

20

1

4

0

245

286

329

410

165

-

298

319

1000

247

285

320

407

160

97

286

313

5000

240

275

317

388

148

90

273

300

20 000

218

256

288

345

127

77

261

281

 

Table 3 Parental Food Consumption Group Mean Values (g/animal/day)

Dose Level (ppm)

 

Males

Females

Pre-trial (day)

Treatment Period

(days)

Pre-trial (day)

Treatment Period

(days)

0

7

0

28

0

7

14

 

0

Number

Mean

SD

5

29.8

1.8

5

31.5

2.2

5

35.5

3.9

5

32.0

2.8

5

21.2

0.8

5

21.1

1.0

5

29.2

1.3

 

1000

Number

Mean

SD

5

27.9

1.1

5

30.9

2.0

5

34.1

1.9

5

31.5

1.5

5

20.5

0.7

5

21.7

1.2

5

22.6

1.1

 

5000

Number

Mean

SD

5

30.1

1.3

5

32.6

1.6

5

34.9

1.3

5

32.4

2.5

5

19.7

1.9

5

20.5

2.0

5

21.6

1.5

 

20 000

Number

Mean

SD

5

29.6

1.1

5

27.2

2.2**

5

32.1

2.4

5

29.9

1.9

5

21.0

1.7

5

17.2

1.3***

5

20.3

1.5**

Significantly different from the control: *p<0.05; **p<0.01; ***p<0.001

SD = Standard Deviation

 

Table 4 Parental Food Consumption (Females) During Gestation and Lactation Mean Group Values (g/animal/day)

Dose Level (ppm)

Day of Gestation

Day of Lactation

0 to 7

7 to 14

14 to 20

0 to 4

0

26.9

29.9

31.8

33.9

1000

26.5

30.0

32.8

31.9

5000

26.2

27.9

32.6

31.6

20 000

23.9

26.7

30.1

30.1

  

Table 5 Selected Parental Absolute Organ Weights Group Mean Values

 

 

Male

Female

Dose Level (ppm)

Dose Level (ppm)

0

1000

5000

20 000

0

1000

5000

20 000

Body Weight

(g)

Number

Mean

SD

10

470

48

10

461

24

10

469

30

10

432

28

10

329

22

10

314

17

10

306

14*

8

287

23***

Adrenal Glands (g)

Number

Mean

SD

10

0.0758

0.0106

10

0.0803

0.0097

10

0.0675

0.0154

10

0.0631

0.0070*

10

0.0944

0.0118

10

0.0849

0.0068

10

0.0784

0.0122**

8

0.0732

0.0109***

Ovaries

(g)

Number

Mean

SD

 

-

 

-

 

-

 

-

10

0.117

0.018

10

0.106

0.010

10

0.103

0.011*

8

0.093

0.014***

Kidneys

(g)

Number

Mean

SD

10

3.86

0.52

10

3.87

0.38

10

4.10

0.29

10

3.86

0.44

10

2.71

0.17

10

2.56

0.18

10

2.44

0.17**

8

2.38

0.26**

Liver

(g)

Number

Mean

SD

10

18.50

2.57

10

19.05

2.72

10

20.04

2.56

10

18.76

1.42

10

17.65

1.79

10

16.34

1.71

10

17.58

1.32

8

16.68

1.75

Pituitary Gland (g)

Number

Mean

SD

10

0.013

0.002

10

0.012

0.003

10

0.013

0.001

10

0.011

0.002

10

0.015

0.003

10

0.016

0.003

10

0.015

0.002

8

0.012

0.002**

Spleen

(g)

Number

Mean

SD

10

0.87

0.11

10

0.84

0.10

10

0.87

0.12

10

0.99

0.14*

10

0.65

0.11

10

0.64

0.08

10

0.67

0.10

8

0.66

0.08

Thymus

(g)

Number

Mean

SD

10

0.507

0.141

10

0.419

0.111

10

0.450

0.102

10

0.350

0.054**

10

0.239

0.085

10

0.240

0.076

10

0.193

0.078

8

0.192

0.072

Table 6 Group Mean Duration of Gestation and Overall Litter Performance Values

Dose Level (ppm)

Number Pregnant

Duration of Gestation (days)

No. of Females Producing a Live Litter

Gestation Index as a %

Mean No. Implant Sites Per Pregnancy (± SD)

Mean Total No. Pups Born

Mean No. of Live Pups Per Litter

(± SD)

21

22

23

Mean

Day 0 Lactation

Day 1 Lactation

Day 4 Lactation

0

10

3

7

0

21.7

10

100

13.4 ± 3.6

12.5 ± 3.5

12.4 ± 3.5

12.3 ± 3.3

12.3 ± 3.3

1000

10

4

6

0

21.6

10

100

15.4 ± 2.5

14.7 ± 2.5

14.7 ± 2.5

14.0 ± 3.9

13.1 ± 4.7

5000

10

4

5

1

21.7

10

100

14.2 ± 3.8

13.1 ± 3.4

13.1 ± 3.4

13.0 ± 3.4

13.0 ± 3.4

20 000

8

2

6

0

21.8

8

100

12.1 ± 1.5

11.8 ± 1.4

11.4 ± 1.7

11.4 ± 1.7

11.4 ± 1.7

SD = Standard Deviation

 

Table 7 Group Mean F1 Survival Indices

Dose Level (ppm)

Birth Index

Live Birth Index

Viability Index Days 1 to 4

Mean Litter Index (%)

Number Losing >2 Pups

Number of Litters

Mean Litter Index (%)

Number Losing >1 Pups

Number of Litters

Mean Litter Index (%)

Number Losing >3 Pups

Number of Litters

0

90

0

9

99

0

10

99

0

10

1000

96

0

10

100

0

10

88

1

10

5000

93

0

10

100

0

10

99

0

10

20 000

97

0

8

97

1

8

100

0

8

 

Table 8 Group Mean Litter and Pup Weight During Lactation (g) ± Standard Deviation

Dose Level (ppm)

Litter

Mean of Litter Mean Pup Weight

Day 1

Day 4

Males

Females

Day 1

Day 4

Day 1

Day 4

0

84 ± 17

126 ± 23

7.2 ± 0.9

10.8 ± 2.0

6.9 ± 1.2

10.5 ± 2.2

1000

85 ± 21

124 ± 50

6.4 ± 0.9

9.7 ± 1.9

5.9 ± 0.8

9.0 ± 2.6

5000

82 ± 16

121 ± 22

6.7 ± 1.2

10.1 ± 2.2

6.2 ± 1.0

9.4 ± 1.7

20 000

79 ± 9

117 ± 12

7.2 ± 0.8

10.7 ± 1.4

6.8 ± 0.6

10.2 ± 1.2

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, toxicity was exhibited at levels of 5000 and 20 000 ppm, but there were no clear effects of toxicity at 1000 ppm. Therefore the parental repeated dose NOEL was considered to be 1000 ppm. For reproductive parameters the NOEL was considered to be 5000 ppm.
Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422 under GLP conditions.

Four groups of 10 male and 10 female Sprague-Dawley rats received the test material via the diet at concentrations of 0, 1000, 5000 and 20 000 ppm. The males were dosed for at least 4 weeks, starting 2 weeks prior to mating. The females were dosed from 2 weeks prior to mating until at least day 6 of lactation.

The animals were monitored for clinical signs, bodyweight, food consumption, mating and litter performance. Haematology and clinical chemistry parameters were investigated; additionally, all animals were subjected to necropsy and histopathological investigations were carried out on animals in the control and 20 000 ppm dose groups. The pups were weighed and examined for gross external abnormalities.

 

At 20 000 ppm, in-life observations included decreased weight gain and food consumption in both sexes. Increased male liver weight following covariance analysis, and increases in bilirubin and alkaline phosphatase were noted in both sexes. In addition, small decreases were noted in adrenal gland weight in both sexes, and in albumin, white blood cell count and ovary weight in females; spleen weight and cholesterol were slightly increased in males.

At 5000 ppm, alkaline phosphatase levels in both sexes were increased. Female adrenal gland weight was reduced.

The only indication of reproductive toxicity was a marginal decrease in implant sites at 20 000 ppm with a corresponding decrease in the mean total number of pups born compared to all other dose groups. However, due to the differences from the control being slight, there is some doubt as to the reproducibility of this finding.

Under the conditions of this study, toxicity was exhibited at levels of 5000 and 20 000 ppm, but there were no clear effects of toxicity at 1000 ppm. Therefore the parental repeated dose NOEL was considered to be 1000 ppm. For reproductive parameters the NOEL was considered to be 5000 ppm.