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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-02-22 to 2010-09-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP study according to OECD technical guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate attached to full study report.
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain Wistar Crl:(WI)WU
- Age at study initiation: 8 weeks
- Weight at study initiation: 292 - 293g
- Housing: Makrolon, type III cages, two rats of the same sex and dose group per cage; cages and absorbing softwood bedding material were changed twice a week or more often if necessary
- Diet for the control group: (e.g. ad libitum): Ssniff V1534, Ssniff Spezialdiäten, Soest, Germany, ad libitum
- Diet for the treatment group: test item will be sent to ssniff by Fraunhofer ITEM and added to the same commercial chow in three different concentrations, ad libitum
- Water (e.g. ad libitum): Tap water from Hannover city water supplier, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12
- Air exchange rate: approx. 15 times per hour

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test item will be sent to Ssniff Spezialdiäten by Fraunhofer ITEM and added to the same commercial chow as used for the control animals (Ssniff V1534) in three different concentrations
- Food will be offered ad libitum
- Food will be changed weekly or on the dates of determination of food consumption
- For each treatment group there will be a food mixture with the corresponding concentration of the test item
- These food mixtures will be prepared and delivered once by Ssniff, Soest, Germany
- From each concentration three samples will be taken from three different sites of the first container after delivery for checking the content and homogeneity of the test item in preparation
- These samples will be investigated by repeat determination
- Based on the chemical properties of both graphite and quartz, the investigation of the stability is not regarded necessary
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Concentration and homogeneity of the test item in food will be analysed by base-digestion using 4M NaOH followed by turbidity measurement
- The content of graphite powder will be determined photometrically at a wavelength of 860nm using a calibration curve
- Determination will be according to a validated method (non-GLP) allowing an evaluation of the precision and accuracy of results
- The relative standard error for the graphite content determination is confirmed to be below +/- 20% within the operative concentrations
- Consequently, acceptance criteria for concentration and homogeneity will be set at +/- 20% of the target value
Duration of treatment / exposure:
- animals will be treated via food for two weeks before mating and during the mating period
- after successful mating, treatment of the females will be continued until day 4 post partum and subsequent sacrifice
- treatment of all males and females not successfully mated will continue until their sacrifice
Frequency of treatment:
Please see "Duration of treatent / exposure"
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1200, 3600, 12000 mg/kg food
Basis:
nominal in diet
No. of animals per sex per dose:
10 males and 10 females per dose group; in a second mating trial an additional 10 males and 10 females per dose group were used (no histopathological examination, hematology and clinical chemistry, locomotor activitiy, functional observation battery, determination of organ weight in second trial)
Control animals:
yes, plain diet
Details on study design:
DOSE SELECTION RATIONALE:
- Doses selected based on a palatability study (Fraunhofer ITEM study-no: 12N100002), were food containing 10g/kg food (estimated target dose of 1000mg/kg/day) was accepted w/o any problems.
- This dose equals the recommended limit dose given in OECD 422 and was therefore chosen as the high dose.
-Other doses were determined using a stagger of approx. three

RATIONALE FOR ANIMAL ASSIGNMENT
- Randomisation to groups based on body weight using PROVANTIS 8.2.0.8
Positive control:
No positive control inculded in this study.

Examinations

Observations and examinations performed and frequency:
INVESTIGATIONS IN F0

CLINICAL INVESTIGATIONS
- Animals will be inspected at least once daily.
- Cages in which animals exhibit unusual behavior or appearance will be tagged for inspection by the veterinarian or his designate.
- For those animals that continue to show a deterioration in health status, euthanasia and necropsy will be performed only if the animal's condition is severely influenced.
- Once per week, all animals will be inspected outside their home cages. The results of these examinations will be documented.

BODY WEIGHT
- Individual body weight of all animals will be recorded weekly to the nearest 0.1 g until sacrifice of the animals.
- After success¬ful mating, body weight of the females will be determined on days 0, 7, 14, and 20 p.c., as well as 0 and 4 post partum (p.p., day of birth = day 0 p.p.)

FOOD CONSUMPTION
- Individual food consumption of all animals will be recorded weekly to the nearest 0.1 g until sacrifice of the animals by determining the difference between initial and remaining food.
- After successful mating, food consumption of the females will be determined on days 0, 7, 14, and 20 p.c. as well as on days 0 and 4 p.p. The actual test item intake of the animals will be calculated based on food consumption.

HEMATOLOGY AND CLINICAL CHEMISTRY
- Blood sample will be taken w/o overnight fasting from the retrobulbar plexus of 5 males and 5 females per group towards the end of week two of treatment (shortly before start of the mating period)
- Blood will be collected under light isofluran ® anesthesia and collected in tubes with K2-EDTA (1.5-2mg/mL) and heparin lithium salt (>7.5 IU/mL).
- In the collected blood samples, the parameters given in table “Hematology and Clinical Chemistry” in section “Any other information on material and methods, incl. tables” will be analysed
- Clinical laboratory data will be checked using commercially available control samples.

LOCOMOTOR ACTIVITY
- Spontaneous locomotor activity over 60 minutes using the „Actimot“ computerized light-beam system (TSE, Homburg/Ts., Germany) will be determined shortly before sacrifice in males and towards the end of week two of treatment (shortly before the start of the mating period) in females.
- The data will be analyzed in 15-minutes intervals. In addition, the total values for distance, time in rest, time in movement, rearing time, and number of rearings will be determined.

FUNCTIONAL OBSERVATION BATTERY
- A functional observational battery (FOB) based on Gad (1982) and Moser et al. (1991) will be determined shortly before sacrifice in males and towards the end of week two of treatment (shortly before the start of the mating period) in females.
- In addition to the determination of forelimb grip strength (Meyer et al., 1979), the FOB will include the following endpoints: Righting reflex, body temperature, salivation, startle response, respiration, urination, mouth breathing, convulsions, pineal response, piloerection, diarrhea, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind-leg splay, tremors, extensor thrust, positive geotropism, activity and limb rotation.

MATING
- Animals will be mated beginning after two weeks of treatment for two consecutive weeks or until successful mating.
- Overnight mating will performed 1 male : 1 female of the same dose group.
- Vaginal smears will be taken the next morning.
- Mating will be considered successful if sperm and/or a vaginal plug is found.
- The day of finding sperm is considered day 0 p.c.
- Time to successful insemination (precoital time) will be determined.
- In a second mating trial an additional 10 males and 10 females per dose group were used (no histopathological examination, hematology and clinical chemistry, locomotor activitiy, functional observation battery, determination of organ weight in second trial)

POST MORTEM INVESTIGATIONS
- Males will be sacrificed after successful mating, but not before day 28 of treatment.
- Females will be sacrificed as soon as possible after day 4 p.p.
- All animals will be sacrificed by C02 overdose and subsequent exsanguination. The treatment of all animals will be continued until their sacrifice.

GROSS PATHOLOGY
- Necropsy will be performed in all F0 males and females.
- The number of corpora lutea as well as implantation sites will be determined, using ammonium sulphide staining in case of no macroscopically visible implantations
- The organs and tissues given in table “Tissues and Organs” in section “Any other information on material and methods, incl. tables” will be collected from each rat and fixed in 10% neutral buffered formalin (unless specified otherwise)

ORGAN WEIGHTS
- Weights of the following organs will be determined: liver, kidneys, adrenals, thymus, spleen, brain, heart, testes and epidydimides.

HISTOPATHOLOGY
- In 5 males and 5 females of the high dose group 4 and the control group 1, complete histopathological investigation will be performed.
- In testes, staging of the seminal epithelium will be performed.

INVESTIGATIONS IN F1

CLINICAL OBSERVATIONS
- Litters are inspected at least once daily.
- Cages in which dams or litters exhibit unusual behaviour or appearance will be tagged for inspection by the veterinarian or his designate.

SURVIVAL AND PUP WEIGHT
- Number of offspring and the occurrence of any dead pups or abnormalities will be recorded for all groups.
- Individual pup sex will be determined and individual weight of the pups will be recorded to the nearest 0.1 g on days 0 and 4 p.p.

Sacrifice and pathology:
INVESTIGATIONS IN F0

POST MORTEM INVESTIGATIONS
- Males will be sacrificed after successful mating, but not before day 28 of treatment.
- Females will be sacrificed as soon as possible after day 4 p.p.
- All animals will be sacrificed by C02 overdose and subsequent exsanguination. The treatment of all animals will be continued until their sacrifice.

GROSS PATHOLOGY
- Necropsy will be performed in all F0 males and females.
- The number of corpora lutea as well as implantation sites will be determined, using ammonium sulphide staining in case of no macroscopically visible implantations
- The organs and tissues given in table “Tissues and Organs” in section “Any other information on material and methods, incl. tables” will be collected from each rat and fixed in 10% neutral buffered formalin (unless specified otherwise)

ORGAN WEIGHTS
- Weights of the following organs will be determined: liver, kidneys, adrenals, thymus, spleen, brain, heart, testes and epidydimides.

HISTOPATHOLOGY
- In 5 males and 5 females of the high dose group 4 and the control group 1, complete histopathological investigation will be performed.
- In testes, staging of the seminal epithelium will be performed.

INVESTIGATIONS IN F1
- All pups will be humanely sacrificed as soon as possible after day 4 p.p. and carefully examined for gross abnormalities.
Other examinations:
Please see section "Observations and examinations performed and frequency" as well as section "Sacrifice and pathology".
Statistics:
- Statistical comparison of groups will be performed separately for each sex at the level of alpha=0.05.
- Body weights as well as food consumption data will be analyzed using analysis of variance.
- If the group means differ significantly with this method, the means of the treatment groups will be compared with the mean of the control group 1 using Dunnett's modification of the t-test.
- Kruskall-Wallis ANOVA and Mann-Whitney U-test will be applied in the case of non-homogeneous data.
- Qualitative data will be analyzed using the two-tailed FISHER test with Bonferroni correction or Chi-square test.
- If appropriate, other statistical tests can be applied.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
that were regarded as test item related
Mortality:
no mortality observed
Description (incidence):
that were regarded as test item related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
that were regarded as test item related
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
that were regarded as test item related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Locomotor Activity and Functional Observation Battery
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
that were regarded as test item related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL OBSERVATIONS IN F0 ANIMALS
- During gestation, one control animal showed red discharge from vagina on day 24 p.c. This animal did not give birth but showed one implantation site at necropsy.
- During Lactation, eye discharge was observed in one high dose animal on day 0 p.p.
- No other clinical signs were observed during the study, and none of the clinical observations were dose dependent so that they were not regarded as test item related.

BODY WEIGHT IN F0 ANIMALS
- No effects of test item exposure were observed on body weight or body weight gain of the animals at any time point of the study.

FOOD CONSUMPTION IN F0 ANIMALS
- No effects of test item exposure were observed on food consumption of the animals at any time point of the study.

HEMATOLOGY IN F0 ANIMALS
- In female rats in the low dose group, a marginal but statistically significant increase in the absolute and relative number of segmented neutrophiles (SEGC, SEGM) and accordingly a slight decrease in the relative lymphocyte count (LYM) were observed.
- A significant increase in the prothrombin time (PT) in females in the medium dose group is due to the high interindividual variance.
- Overall, hematological and clinical chemistry data were found in the ranges expected for the species, strain, sex and age.
- Therefore, the findings albeit significantly different from controls in individual cases, are considered to be of no toxicological relevance.

CLINICAL CHEMISTRY IN F0 ANIMALS
- Significantly decreased alanine aminotransferase (ALT) levels were observed in males in the high dose group.
- Slightly but significantly increased potassium (K) levels were found in males in the medium dose group.
- However, all values were within the normal range of this strain and age.

LOCOMOTOR ACITIVITY IN F0 ANIMALS
- No effects of test item exposure were observed, neither for any of the investigated time intervals, nor for the whole test period.

FUNCTIONAL OBSERVATIONAL BATTERY I F0 ANIMALS
- No effects of test item exposure were observed on any of the investigated endpoints.

GROSS EXAMINATION OF F0 ANIMALS
- The main findings consisted in reduced size of testes and epidydimides, which were found in all groups including the control group.
- Eight animals showed this observation, out of which 4 were able to produce a litter, while 4 were not.
- None of the macroscopic findings were considered test item induced.

ORGAN WEIGHTS IN F0 ANIMALS
- The only statistically significant differences from the control group consisted in an increase of the absolute and relative weight of the left, but not of the right, adrenal gland in the male medium dose group, an increase in relative heart weight in the male medium dose group, as well as a decrease in relative, but not absolute, liver weight in the male medium and high dose groups.
- No effects were observed in females, and none of the observed differences in males was considered an adverse effect of the test item exposure.

HISTOPATHOLOGY OF F0 ANIMALS
- Substance-related findings were not observed in the histologically examined organs of males and females of the control and high dose group.
- Several other findings in various organs of the examined experimental groups were noted which also occurred incidentally and were not unusual for this strain and age.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 813 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: NOAEL = highest dose tested, actually ingested dose recalculated from food consumption
Dose descriptor:
NOAEL
Remarks:
during premating period
Effect level:
ca. 1 067 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: NOAEL = highest dose tested, actually ingested dose recalculated from food consumption
Dose descriptor:
NOAEL
Remarks:
during gestation
Effect level:
ca. 930 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: NOAEL = highest dose tested, actually ingested dose recalculated from food consumption
Dose descriptor:
NOAEL
Remarks:
during lactation
Effect level:
ca. 1 159 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: NOAEL = highest dose tested, actually ingested dose recalculated from food consumption

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

MATING AND PREGNANCY DATA

-      Since the outcome of the first mating was insufficient, additional 10 pairs per group were added to the study for one more mating trial.

-      Consequently, each group consisted of 20 pairs for mating.

-      For the four groups, mating yielded 19, 19, 20, and 18 sperm positive females, in groups 1-4, respectively.

-      This resulted in 14, 10, 13, and 10 pregnancies; 11, 9, 13, and 10 females with liveborn offspring in F0 dams, and 11, 9, 12, and 10 females with live litters on day 4 p.p., respectively.

-      Animals no. 1102, 1107, and 2112 did not give birth, but were found pregnant at necropsy, when they showed at least one implantation site.

-      Animal no. 1101 gave birth to 3 pups, which were all stillborn.

-      Animal no. 3113 gave birth to only one live pup, which was missing on day 1 p.p.

-      The results show that mating outcome throughout all groups including controls in both mating trials was insufficient. The reasons for this phenomenon are unclear.

-      However, no effect of the test item exposure was observed on any of the observed endpoints like precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups).

LITTER SIZE, SURVIVAL AND PUP WEIGHT

-      No effect of the test item exposure could be observed on litter size and pup survival as well as pup body weight.

-      Pup clinical observations included sporadical bad condition, cold to touch and one finding of a damged tail.

-      None of these findings were considered test item related and no abnormal pups were observed.

QUALITATIVE HISTOLOGICAL EXAMINATION OF THE TESTES/EPIDIDYMIDES TAKING INTO ACCOUNT THE TUBULAR STAGES OF THE SPERMATOGENIC CYCLE

-      The randomly scanned seminiferous tubules 4/5 males of the control and 1/5 males of the high dose group showed the appropriate cell layers in their approximate normal numbers, this means that stages I-VIII contained a layer of spermatogonia, a layer of pachytene spermatocytes and several layers of round spermatids interspersed with elongated spermatids.

-      Stages IX-XIV contained a layer of spermatogonia and prepachytene spermatocytes, several layers of late pachytene or dividing (stage XIV) spermatoytesand several layers of elongated spermatids.

-      There was no evidence of degenerating spermatids or spermatocytes.

-      Very slight (multi)focal interstitial mononuclear cell infiltration was recorded in the epididymis of 3/5 males of the control and in 2/6 males of the high dose group.

-      These alterations are considered to be incidental findings.

-      Aspermia was observed in one male of the low dose and high dose group each associated with severe changes in the corresponding testis. 

-      A unilateral slight/moderate focal spermatic granuloma was detected in one male of the mid dose and high dose group each.

-      The testis of one male of the control group showed very slight degeneration/depletion of spermatocytes and spermatids associated with Sertoli-cell vacuolation and spermatid retention in one testes and a slight multifopcal tubular atrophy in the other testis.

-      In one male rat of the high dose group a very severe unilateral diffuse tubular atrophy corresponding to the gross finding reduction in size was observed.

-      One male of the low dose group was detected with very severe diffuse tubular atrophy associated with very severe germ cell degeneration in both testes and formation of multinucleated giant cells.

-      Three (3/6) males of the high dose group were seen with very slight focal/multifocal degeneration of germ cells and 2/6 males with very slight unilateral focal/multifocal vacuolation of Sertoli cells.

-      The lesions found in 1male of the control, in one male of the low dose group and in 5/6 males of the high dose group are considered to be incidental.

-      The severe changes of one male of the low and high dose group each is likely to be spontaneous as single case as well as the very minimal lesions found in the 4 males of the high dose group. 

 

ANALYSIS OF TEST ITEM CONCENTRATION IN FOOD

-      The results of the analyses of the test item concentration in food of group 2-4 are summarized in Table “Food Analysis”.

-      The mean substance intake calculations are summarized in Table “Substance Intake”.

-      The analyses showed that preparations contained 108, 103, and 96 % of the target concentration of the three dose groups, respectively.

-      Based on mean body weights and food consumption data, this resulted in the following actual substance intake: 91, 261, and 813 mg/kg body weight/day for males; 120, 343, and 1067 mg/kg body weight/day for females in the premating period; 106, 309, and 930 mg/kg body weight/day for females during gestation; and 111, 370, and 1159 mg/kg body weight/day for females during lactation, respectively.

TABLE: SUMMARY

Observations

Values

Dosage (mg/kg food)

0

1200

3600

12000

 

 

 

 

 

Pairs started (N)

20

20

20

20

Females showing evidence of copulation (N)

19

19

20

18

Females achieving pregnancy (N)

14

10

13

10

Conceiving days 1-5 (N)

19

18

19

18

Conceiving days 6-14 (N)

0

1

1

0

Pregnancy = 21 days (N)

0

0

0

0

Pregnancy=22 days (N)

4

3

2

3

Pregnancy => 23 days (N)

8

6

11

7

Dams with live young born (N)

11

9

13

10

Dams with live young at day 4 p.p. (N)

11

9

12

10

Corpora lutea/dam (mean)

13.2

12.0

13.2

13.5

Implants/dam (mean)

11.9

9.8

12.5

13.1

Live pups/dam at birth (mean)

10.9

9.8

10.7

11.2

Live pups/dam at day 4 (mean)

10.8

8.8

10.4

11.1

Sex ratio (m/f) at birth (mean)

61

39

62

61

Sex ratio (m/f) at day 4 (mean)

60

34

61

61

Litter weight at birth (mean)

67.9

60.6

66.8

70.2

Litter weight at day 4(mean)

115.8

92.4

115.7

109.1

Pup weight at birth (mean)

5.9

6.4

6.3

6.4

Pup weight at day 4(mean)

10.1

10.7

10.7

10.1

ABNORMAL PUPS

Dams with 0

11

9

13

10

Dams with 1

0

0

0

0

Dams with 2

0

0

0

0

Dams with 3

0

0

0

0

LOSS OF OFFSPRING

Pre-implantation (corpora lutea minus implantations)

Females with 0

8

3

5

5

Females with 1

2

3

4

4

Females with 2

1

2

3

0

Females with 3>

3

2

1

1

Pre-natal (implantations minus live births)

Females with 0

2

3

4

2

Females with 1

6

5

3

1

Females with 2

0

1

1

3

Females with 3>

3

1

5

4

Post-natal (live births minus alive at post natal day 4)

Females with 0

10

6

9

9

Females with 1

1

1

4

1

Females with 2

0

1

0

0

Females with 3>

0

1

0

0

TABLE: FOOD ANALYSIS

 

Low Dose

Medium Dose

High Dose

Target concentration in food, mg/kg food

1200

3600

12000

Chemically determined concentration, mg/kg food (% of target value)

1300 (108%)

3700 (103%)

11500(96%)

TABLE: SUBSTANCE INTAKE

Week no.

Group 2 (Low dose)

Group 3 (mid dose)

Group 4 (high dose)

 

Mean BW [g]

Mean FC [g/animal/d]

SI [mg/kg BW/day]

Mean BW [g]

Mean FC [g/animal/d]

SI [mg/kg BW/day]

Mean BW [g]

Mean FC [g/animal/d]

SI [mg/kg BW/day]

0

293

 

 

292

 

 

292

 

 

1

325

26

104

323

26

298

323

26

926

2

351

26

96

347

26

277

347

26

862

3

365

22

78

361

22

225

360

22

703

4

384

25

85

378

25

245

378

25

761

 

 

 

 

 

 

 

 

 

 

Mean

 

25

91

 

25

261

 

25

813

SD

 

2

12

 

2

32

 

2

100

N

 

4

4

 

4

4

 

4

4

 

 

 

 

 

 

 

 

 

 

Analytical Concentration
[mg/kg food]

1300

 

 

3700

 

 

11500

 

 

 

 

 

 

 

 

 

 

% target dose

91

 

 

87

 

 

81

Applicant's summary and conclusion

Conclusions:
Based on the results described above, the NOAEL for this study was determined as the high (limit) dose level of 11.500 mg/kg food. This corresponds to the following actual substance intake: 813 mg/kg body weight/day for males; 1067 mg/kg body weight/day for females in the premating period, 930 mg/kg body weight/day for females during gestation and 1159 mg/kg body weight/day for females during lactation, respectively.
Executive summary:

SUMMARY AND CONCLUSION

-      The aim of this study was to evaluate possible adverse effects of Expanded Graphite Powder after repeated exposure especially on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and parturition after exposure via food.

-      The study was conducted following the OECD Guideline 422 and in compliance with the Principles of Good Laboratory Practice as well as with the German Animal Protection Law.

-      The dose levels were based on a palatability study (Fraunhofer ITEM study no. 12N10002), in which the animals accepted food containing 10 g/kg food (estimated target dose of 1000mg/kg/day) without any problems.

-      The dose of 1000 mg/kg/day is also recommended as limit dose by OECD Guideline 422.

-      Consequently, this dose was chosen as the high dose in the present study, and the actual concentration in food was determined based on actual body weights and food consumption data, which resulted in a value of 12 g/kg food.

-      The other doses were determined using a stagger of approx. 3. The analyses showed that preparations contained 1300, 3700, and 11.500 mg/kg food (108, 103, and 96 % of the target concentration) of the three dose groups, respectively.

-      Based on mean body weights and food consumption data, this resulted in the following actual substance intake: 91, 261, and 813 mg/kg body weight/day for males; 120, 343, and 1067mg/kg body weight/day for females in the premating period; 106, 309, and 930 mg/kg body weight/day for females during gestation; and 111, 370, and 1159 mg/kg body weight/day for females during lactation, respectively.

-      Wistar rats (Crl:WU) were used in this study.

-      The study commenced with 40 male and 40 female rats which were randomly assigned to the control or one of the test item exposed groups.

-      Since the outcome of the first pairing was insufficient, a second subset of 40 female and 40 male animals was randomly assigned to the four groups for one more mating trial.

-      In the control group, a closed formula in pellet (ssniff Spezialdiäten) form was used as the diet for this study.

-      For the treatment groups, the test item was sent to ssniff by Fraunhofer ITEM and added to the same commercial chow in three different concentrations.

-      The animals were treated via food for 2 weeks before mating and during the mating period.

-      After successful mating (day of finding sperm in vaginal smears = day 0 post conceptionem [p.c.]), treatment of the females was continued until day 4 post partum (p.p., day of birth = day 0 p.p.) and subsequent sacrifice.

-      Treatment of all males and females not successfully mated was continued until their sacrifice.

-      After successful mating, body weight and food consumption of the females was determined on days 0, 7, 14, and 20 p.c., as well as 0 and 4 p.p. 

-      A blood sample was taken without overnight fasting from the retrobulbar plexus of 5 males and 5 females per group towards the end of week two of treatment (shortly before the start of the mating period).

-      In the collected blood samples hematology and clinical chemistry was investigated.

-      Spontaneous locomotor activity over 60 minutes was determined shortly before sacrifice in males and towards the end of week two of treatment (shortly before the start of the mating period) in females.

-      A functional observational battery (FOB) was applied shortly before sacrifice in males and towards the end of week two of treatment (shortly before the start of the mating period) in females.

-      Animals were mated beginning after two weeks of treatment for two consecutive weeks or until successful mating.

-      Overnight mating was performed 1 male: 1 female of the same dose group.

-      Vaginal smears were taken the next morning.

-      Mating was considered successful if sperm and/or a vaginal plug was found.

-      The day of finding sperm was considered day 0 p.c.

-      Time to successful insemination (precoital time) was determined.

-      Litters were inspected at least once daily. Number of offspring and the occurrence of any dead pups or abnormalities was recorded for all groups. Individual pup sex was determined and individual weight of the pups was recorded on days 0 and 4 p.p. All pups were humanely sacrificed as soon as possible after day 4 p.p. and carefully examined for gross abnormalities.

-      Males were sacrificed after successful mating, but not before day 28 of treatment.

-      Females were sacrificed as soon as possible after day 4 p.p. All animals were sacrificed by C02 overdose and subsequent exsanguination. The treatment of all animals was continued until their sacrifice.

-      Necropsy was performed in all F0 males and females.

-      The number of corpora lutea as well as implantation sites was determined, using ammonium sulphide staining in case of no macroscopically visible implantations.

-      Organ weight was determined for specified organs.

-      In 5 males and 5 females of the high dose group 4 and the control group 1, complete histopathological investigation according to OECD Guideline 422 was performed.

-      In testes, staging of the seminal epithelium was performed.

RESULTS

-      Please see “Summary Table”

-       None of the sporadically observed clinical findings regarded as test item related.

-      No effects of test item exposure were observed on body weight, body weight gain or food consumption of the animals at any time point of the study.

-      Overall, haematological and clinical chemistry data were found in the ranges expected for the species, strain, sex and age. Therefore, sporadical findings, albeit significantly different from controls in individual cases, are considered to be of no toxicological relevance.

-      No effects of test item exposure were observed on locomotor activity, neither for any of the investigated time intervals, nor for the whole test period.

-      No effects of test item exposure were observed on any of the investigated endpoints of the Functional Observational Battery.

-      Since the outcome of the first mating was insufficient, additional 10 pairs per group were added to the study for one more mating trial. Consequently, each group consisted of 20 pairs for mating. For the four groups, mating yielded 19, 19, 20, and 18 sperm positive females, in groups 1-4, respectively. This resulted in 14, 10, 13, and 10 pregnancies; 11, 9, 13, and 10 females with liveborn offspring in F0 dams, and 11, 9, 12, and 10 females with live litters on day 4 p.p., respectively. The results show that mating outcome throughout all groups was insufficient. The reasons for this phenomenon are unclear.

-      However, no effect of the test item exposure was observed on any of the observed endpoints like precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups).

-      No effect of the test item exposure could be observed on litter size and pup survival as well as pup body weight.

-      None of the sporadically observed clinical findings in pups were considered test item related and no abnormal pups were observed. The main necropsy findings consisted in reduced size of testes and epidydimides, which were found in all groups including the control group. Sporadically observed statistically significant differences in organ weights were not considered an adverse effect of the test item exposure.

-      Substance-related findings were not observed in the histologically examined organs of males and females of the control and high dose group.

CONCLUSION

Based on the results described above, the NOAEL for this study (parental, reproductive and developmental) was determined as the high (limit) dose level of 11.500 mg/kg food. This corresponds to the following actual substance intake: 813 mg/kg body weight/day for males; 1067 mg/kg body weight/day for females in the premating period, 930 mg/kg body weight/day for females during gestation and 1159 mg/kg body weight/day for females during lactation, respectively.