Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
24 September 2012 - 08 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 421)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
addition of haematology and blood biochemistry analysis
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material: Ethoxylated bisphenol A diacrylate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: on the first day of treatment, the males were approximately 10 weeks old and the females were approximately 9 weeks old
- Mean body weight at study initiation: on the first day of treatment, the males had a mean body weight of 373 g (range: 327 g to 401 g) and the females had a mean body weight of 214 g (range: 176 g to 248 g)
- Fasting period before study: no
- Housing: the females were individually housed, except during pairing and lactation, in polycarbonate cages. The males were individually housed except during pairing, in wire-mesh cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 7 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 02 October 2012 to 02 December 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
The test item was administered as a suspension in the vehicle. It was mixed with the required quantity of vehicle. No correction factor was applied.
The frequency of dose formulation preparation was on a daily basis. The dose formulations were delivered to the study room at room temperature and protected from light.

VEHICLE
- Justification for use and choice of vehicle: homogenous suspension with test item and corn oil which is commonly used in this type of study
- Concentration in vehicle: 10, 50 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred or 14 days has elapsed in the latter case, then pairing with a second male until mating occurred within 7 days
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: homogenous
Stability: not assessed, dose formulation prepared daily
Duration of treatment / exposure:
In the males:
− 2 weeks before mating,
− during the mating period,
− until sacrifice (at least 5 weeks in total),

In the females:
− 2 weeks before mating,
− during the mating period,
− during pregnancy,
− during lactation until day 5 post-partum inclusive,
− until sacrifice for females which had not delivered.
Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 250 and 1000 mg/kg/day
Basis:
actual ingested
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 by the Sponsor, following the results of a previous 4 week toxicity study performed in the same species and strain. In this study, three groups of five males and five females received the test item by gavage at 100, 300 or 1000 mg/kg/day as a suspension in corn oil. Another group of five males and five females received the vehicle only. There were no premature deaths and the only test item-related clinical sign was ptyalism at 1000 mg/kg/day. There were no toxicologically significant effects on mean body weight and food consumption. Some slight effects were seen at hematology and biochemistry investigations, such as lower mean alkaline phosphatase activity in females treated at 1000 mg/kg/day, dose related increase in cholesterol level from 300 mg/kg/day or minimally higher mean red blood cell parameters in males treated at 1000 mg/kg/day. At necropsy, there was a higher mean liver weight particularly at 1000 mg/kg/day in males and females.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required).

Examinations

Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT:
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating, on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: Males: on the first day of treatment, then once a week until pairing. Females: on the first day of treatment, then once a week until pairing, on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

HAEMATOLOGY, CLINICAL CHEMISTRY:
- Time schedule: at the end of the treatment period.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups),
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups),
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups).
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs.

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals after the end of the mating period after at least 5 weeks of treatment
- Female animals: all surviving animals = day 6 post-partum or, for female which had not delivered yet, day 26 post-coitum.

GROSS NECROPSY
Macroscopic post-mortem examination of principal thoracic and abdominal organs.

HISTOPATHOLOGY
- epididymides, liver, ovaries, testes, thyroid with parathyroids from all animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period,
- liver from low- and intermediate-dose groups (groups 2 and 3) sacrificed at the end of the treatment period,
- all macroscopic lesions of all groups.

ORGAN WEIGHTS: epididymides, liver, ovaries, testes, thyroid with parathyroids.
Postmortem examinations (offspring):
SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Statistics:
Body weight, food consumption and reproductive data : Data are compared by one-way analysis of variances and Dunnett test (mean values being
considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
Citox software (version D.6) was used to perform the statistical analysis of hematology and blood biochemistry.
PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantation sites
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
but no mortality
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
(only for food consumption)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
(only for food consumption)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY:
There were no unscheduled deaths.

CLINICAL SIGNS (see Table 1 below):
Ptyalism, round back and piloerection were considered to be test item-related.

Ptyalism was observed with a dose-relationship in incidence and duration in males at all dose levels. At 1000 mg/kg/day, all females were affected throughout the whole study.

Round back and piloerection were noted in males at 1000 mg/kg/day and in females at 250 and 1000 mg/kg/day. Round back was noted from day 12 or 13 of the pre-mating period and lasted 3 to 5 days at 1000 mg/kg/day. Animals treated at 1000 mg/kg/day had piloerection from day 17 (males) or 12-13 (females) of treatment and for 3-4 days (males) or up to 6 days (females). These clinical signs were also observed in females treated with 1000 mg/kg/day at the beginning of gestation only. At 250 mg/kg/day in females, piloerection and round back were noted during the premating period mainly on one day only and were thus considered not to be adverse.

To conclude, clinical signs (round back and piloerection) were considered to be adverse at 1000 mg/kg/day in male and female rats, because they were observed in the majority of the animals and lasted several days.

Four males showed loud breathing during the study, one male treated at 50 mg/kg/day and three males treated at 1000 mg/kg/day. There was no clear relationship between test item treatment and this finding: no dose-relationship as not observed at 250 mg/kg/day, and no clear pattern in appearance and duration (one out of the three animals affected at 1000 mg/kg/day experienced loud breathing for 6 days at the beginning of the treatment period, another one at the beginning of the treatment period but for 1 day only, the third male about 2 weeks later for 1 day only).

Incidental clinical signs included reflux at dosing in isolated females.

BODY WEIGHT:
Males given 1000 mg/kg/day of test item had a statistically significantly lower mean body weight gain in the first week of treatment compared with controls but this was transient, had no toxicologically relevant effects on mean body weight and was therefore considered of no toxicological relevance.

There were no toxicologically relevant effects during pre-mating and lactation period in females. The same trend (at 1000 mg/kg/day) as in males in the first week of the pre-mating period was observed in females but the difference was lower than in males, with no statistical significance. During gestation, females had a lower mean body weight gain over the whole gestation period when compared with controls. As the difference was slight and did not impact significantly the mean body weight, this was considered to be of non toxicological importance.

FOOD CONSUMPTION (see Table 2 below):
Food consumption recorded in week 2 for four control males and one male of the 1000 mg/kg/day group was higher (between two to three times) than what is commonly observed for this strain of rats at about the same age (about 28 g/male/day) despite the absence of observation of food spillage. This led to high mean food consumption in the control group.
However, when excluding these abnormal data from mean calculations, mean food consumption at 1000 mg/kg/day remained strongly lower than in controls in week 2. However, this was considered to be non adverse as it had no relevant impact on mean body weight and on animal survival.

The slight higher mean food consumption noted at 250 and 1000 mg/kg/day in females during the second week of pre-mating was considered to be of doubtful toxicological importance as no statistical level, slight differences and in an opposite way compared to the effects seen in males.

REPRODUCTIVE PERFORMANCE:
There were no test item-related effects on pairing, mating and fertility data.
All pairs mated in a maximum of 4 days except one control and one 1000 mg/kg/day pairs. In both cases, the female mated (with a second male which had already mated) after a total of 20 (control) or 16 (1000 mg/kg/day) days after the beginning of the treatment period, hence the slightly high mean pre-coital time in controls and at 1000 mg/kg/day. This was considered to be incidental as in isolated pairs per group and also in controls.
All females were pregnant except one in the control group which was sacrificed on day 26 p.c. for absence of delivery.
There were no relevant effects on duration of gestation and on number of corpora lutea and implantation sites. The gestation lasted 21 or 22 days, except for one female treated with 50 mg/kg/day (23 days).
There were no relevant effects on delivery data. The slightly higher mean number of pups delivered in controls when compared to the other groups was particularly due to one litter with 21 pups (mean of 14.4 pups delivered when excluding this litter). Three litters showed a lower number of live pups, one at 50 mg/kg/day (7 pups) and two at 1000 mg/kg/day (9 and 8 pups). It was due in two of these females to low numbers of corpora lutea (9 or 10). These findings were considered to be of no toxicological relevance as not dose-related, not severe and without significant impact on mean data.
Pre- and post-implantation losses in test item-treated groups were comparable with the individual values of the control group.

HEMATOLOGY (see Table 3 below):
In both sexes and when compared with controls, there was a slight but statistically significant lower mean white blood cell count at 250 and 1000 mg/kg/day in males and at 1000 mg/kg/day in females, mainly due lower mean lymphocyte numbers. As the differences were slight they were considered to be non adverse and of minor toxicological importance.
In females, there were statistically significant lower mean red blood cell parameters (count, hemoglobin and/or hematocrit/PCV) from 250 mg/kg/day when compared with controls. In view of the low amplitude of differences from control values and the absence of similar findings in males, these findings were considered to be of limited toxicological importance.
The lower mean platelet levels observed in males in test item-related groups were considered to be of limited toxicological relevance as there was no dose-relationship, no similar findings in females, and the differences from the control mean were slight.

BLOOD BIOCHEMISTRY (see Table 4 below):
There was a higher mean cholesterol level from 250 mg/kg/day when compared with controls. This effect was considered to be adverse at 1000 mg/kg/day in view of the amplitude of differences from control and historical control data.
In females, there were statistically significant lower mean concentrations of inorganic phosphorus from 50 mg/kg/day and higher mean urea blood concentration at 1000 mg/kg/day. In view of the low severity of the changes and the fact that data were in the range of the historical control data, these findings were considered not to be adverse and of minor toxicological importance.
Higher mean ALAT activity was noted at 1000 mg/kg/day in females when compared with controls, while at the dose of 50 mg/kg/day a lower mean ALAT activity was observed in females and no statistical changes were observed in males in any group. Despite the fact that about half of the individual data were outside the range of the historical control data, the differences from controls and/or historical control data were slight and they were not associated with correlating adverse findings.
Higher mean calcium blood level was noted at 1000 mg/kg/day in males. All the individual data were outside the range of the historical control data, but the differences from these controls were slight.
These variations (ALAT activity and calcium blood level) were thus considered to be of limited toxicological importance and non-adverse.
The statistical significances observed for mean sodium (250 mg/kg/d in males and females), glucose (50 mg/kg/day in males), chloride (250 mg/kg/d in males) and calcium (50 and 250 mg/kg/day in females) levels were considered to be fortuitous as there was no dose-relationship, the amplitudes of variations from controls were low and/or not associated with histopathological correlates.

ORGAN WEIGHTS (see Table 5 below):
The mean terminal body weight was slightly lower in males and females given 1000 mg/kg/day (-8% and -4%, respectively).
The mean absolute and relative liver weights were statistically higher in both sexes at 1000 mg/kg/day (p<0.05 for the absolute weight, p<0.01 for the relative weight in both sexes). Similar trend was seen in males at 250 mg/kg/day (p<0.05 for the relative weight).
The mean absolute and relative weights of the thyroids were higher in females given 1000 mg/kg/day, reaching significant value for the relative weight (p<0.05). This was mainly due to individual values. As these variations were of opposite trend in males and in the absence of histopathological correlates this difference was considered to be of no toxicological relevance.
The mean absolute weight of epididymides in males was statistically lower at 1000 mg/kg/day (p<0.05). In the absence of a similar trend for the relative weight and of test item-related changes at histopathological examination, this difference was considered to be without toxicological significance, probably secondary to the slightly lower terminal body weight.
There were no test item-related changes in the mean ovaries weights.

GROSS PATHOLOGY:
There were no test item-related changes at necropsy. Red discoloration correlating with sinusal red blood cells seen in the mandibular lymph nodes of two low dose males (50 mg/kg/day) was considered to be part of the normal background commonly seen in rats and therefore incidental.
No other macroscopic findings were observed in this study.

HISTOPATHOLOGY (see Table 6 below):
Test item-related changes were confined to the liver in both sexes at 250 and 1000 mg/kg/day.
Minimal to slight centrilobular hypertrophy was observed in males given 250 mg/kg/day and in both sexes at 1000 mg/kg/day, but particularly in males. At this dose-level, this was associated with minimal or slight vacuolation of the Kupffer cells which had a foamy appearance. These changes correlated with the higher liver weights noted at necropsy and in the absence of any degenerative changes they were considered to be non-adverse.
There were no histopathological correlates with the higher mean absolute and relative thyroid weights in females or with the lower absolute epididymides weight in males.
Careful histopathological examination of testes and epididymides in males and of ovaries in females did not show any test-item related changes.

Pathology conclusion
Daily oral administration of the test item in rats at the dose-levels of 250 or 1000 mg/kg/day from before mating, through mating in males and, for females, through gestation until day 5 post partum induced centrilobular hypertrophy in males at 250 mg/kg/day and in both sexes at 1000 mg/kg/day. At this dose-level, this was associated in both sexes with vacuolation of Kupffer cells in the liver. These liver changes correlated with the higher liver weight noted at necropsy and in the absence of associated degenerative changes they were considered to be non-adverse.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Parental toxicity.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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)

MORTALITY/VIABILITY:
There were no test item-related deaths in pups.

CLINICAL SIGNS:
There were no test item-related clinical signs in pups.
The most relevant clinical signs were noted in the only 3 live pups of one female (50 mg/kg/day) which were cold to the touch throughout the lactation period, and in one pup (1000 mg/kg/day) which showed several clinical signs and did not take bodyweight throughout lactation (all the others pups of the same litter didn’t show clinical signs).
This was considered to be incidental as not dose-related and in isolated litters and/or pups.
Other incidental clinical signs of pups included hematoma (hindlimb, back, head), necrosed tail, cutaneous lesions or scabs (abdomen, thorax or forelimb). The nature and incidence of these signs can be commonly observed in pups of this age or was observed with low or similar incidence as controls, and were considered not toxicologically relevant.

BODY WEIGHT:
There were no effects on mean pup body weights and mean pup body weight gains.

GROSS PATHOLOGY:
There were no test item-related macroscopic findings at pup necropsy.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Toxic effects on progeny

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

 Table 1 - Clinical signs:

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Pre-mating (females) or whole study (males)

 

 

 

 

 

. Round back

 

 

1

7

 

 

7

9

. Piloerection

 

 

 

10

 

 

2

4

. Loud breathing

 

1

 

3

 

 

 

 

. Ptyalism

1

5

10

10

 

1

 

9

Number of animals affected

1/10

6/10

10/10

10/10

0/10

1/10

7/10

10/10

Gestation

 

 

 

 

 

 

 

 

. Round back

 

 

 

 

 

 

 

1

. Piloerection

 

 

 

 

 

 

 

4

. Ptyalism

 

 

 

 

 

 

2

10

Number of animals affected

/

/

/

/

0/10

0/10

2/10

10/10

Lactation

 

 

 

 

 

 

 

 

. Ptyalism

 

 

 

 

 

 

1

10

Number of animals affected

/

/

/

/

0/9

0/10

1/10

10/10

/: not applicable.

Table 2 - Food consumption: 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Pre-mating

 

 

 

 

 

 

 

 

Days 1 - 8

26

26

27

24

17

16

16

16

Days 8 -15

44

28

22*

17**

17

13

21

23

when excluding
the abnormal data
(no statistics performed)

25

28

22

10

/

/

/

/

Gestation

 

 

 

 

 

 

 

 

Days 0 - 7p.c.

/

/

/

/

21

21

21

21

Days 7 - 14p.c.

/

/

/

/

23

24

23

25

Days 14 - 20p.c.

/

/

/

/

25

26

25

25

Lactation

 

 

 

 

 

 

 

 

Days 1- 5p.p.

/

/

/

/

37

37

36

35

/: not applicable; statistically significant:*: p<0.05; **: p<0.01.

Table 3 - Hematology:  

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

White blood cell count (G/L)

10.89

8.16

7.76*

7.79*

13.73

11.31

12.01

10.13*

Lymphocytes +
Large unstained cells (G/L)

9.34

6.96

6.28*

6.61

9.18

7.13

7.63

6.02**

Monocytes (G/L)

0.26

0.15*

0.17*

0.17*

0.56

0.44

0.41*

0.25**

Eosinophils (G/L)

0.12

0.09

0.13

0.10

0.13

0.11

0.10

0.09*

Basophils (G/L)

0.05

0.02

0.03

0.03

0.07

0.03*

0.03**

0.02**

Red blood cell count (T/L)

9.16

8.86

9.12

8.62

7.63

7.19

7.21

6.94**

Hemoglobin (g/dL)

15.8

15.2

15.9

15.3

14.2

13.6

13.4**

13.0**

Packed cell volume (PCV) (L/L)

0.49

0.47

0.49

0.47

0.44

0.42

0.41*

0.40**

Platelets (G/L)

835

656*

675*

690

1358

1302

1307

1265

Statistically significant from controls: *: p<0.05, **: p<0.01.

Table 4 - Blood biochemistry:

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Cholesterol (mmol/L)

1.4

1.3

2.0**

3.8**

1.6

1.7

2.1

3.6**

Inorganic phosphorus (mmol/L)

2.14

2.14

2.10

2.13

2.84

2.53*

2.49*

2.30**

Urea (mmol/L)

4.4

4.3

4.5

4.8

6.5

6.4

7.2

8.1*

Alanine aminotransferase (ALAT) (IU/L)

41

45

42

45

86

65**

78

117**

Calcium (mmol/L)

2.74

2.70

2.77

2.93**

2.95

2.83*

2.80**

2.95

Glucose (mmol/L)

7.72

8.79*

7.89

7.69

6.35

6.47

6.54

6.02

Sodium (mmol/L)

149.0

149.6

150.9**

149.8

142.8

144.6

145.0*

143.7

Chloride (mmol/L)

103.9

104.8

105.7*

105.3

98.9

100.3

100.1

99.5

Statistically significant from controls: *: p<0.05; **: p<0.01.

Table 5 - Organ weights:  

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

50

250

1000

50

250

1000

Number of animals

10/10

10/10

10/10

10/10

10/10

10/10

Body weight

0

-2

-8

+3

+1

-4

- Liver

 

 

 

 

 

 

  . absolute

+6

+11

+19*

+3

+5

+16*

  . relative

+6

+14**

+29**

+1

+4

+22**

- Thyroids

 

 

 

 

 

 

  . absolute

-6

+2

-14

+5

+24

+31

  . relative

-6

+4

-6

+2

+22

+37*

- Epididymides

 

 

 

 

 

 

  . absolute

-3

-3

-10*

 

 

 

  . relative

-2

-1

-2

 

 

 

Statistically significant from controls: *: p<0.05, **: p<0.01

The significance concerned the organ weights values and not the percentages.

Table 6 - Microscopic examination: 

Sex

Males

Females

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Number of animals

10

10

10

10

10

10

10

10

Hypertrophy; hepatocytes

Minimal

-

-

6

6

-

-

-

1

Slight

-

-

-

4

-

-

-

2

Total

0

0

6

10

0

0

0

3

Vacuolation, Kuffer cells

Minimal

-

-

-

8

-

-

-

3

Slight

-

-

-

2

-

-

-

-

Total

0

0

0

10

0

0

0

3

-: not observed.

Applicant's summary and conclusion

Conclusions:
The test item was administered daily by oral gavage to male and female Sprague Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 5 p.p., at dose-levels of 50, 250 or 1000 mg/kg/day.
At 1000 mg/kg/day, round back and piloerection noted in both sexes in weeks 2/3, as well as the higher mean cholesterol level at the end of the treatment period were considered to be adverse.
There were no adverse findings at 50 and 250 mg/kg/day.

Therefore, based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 250 mg/kg/day,
- the NOAEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day,
- the NOAEL for toxic effects on progeny was considered to be 1000 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum (p.p.), based on the OECD guideline No. 421, 27 July 1995.

This study provides information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by oral (gavage) from before pairing, through pairing and, for the females, through gestation until day 5 p.p. The test item was administered as a suspension in the vehicle,corn oil, at dose-levels of 50, 250 or 1000 mg/kg/day.

Another group of ten males and ten females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage‑volume of 5 mL/kg/day was used.

 

The concentration of the dose formulation was checked in study weeks 1, 3 and 6.

 

The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Body weight and food consumption were recorded at leastonce a week. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p. The litter sizes and numbers of pups per sex were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities, and weighed on days 1 and 5 p.p.

Hematology and blood biochemistry were carried outfor all males and all females sacrificed on day 6 p.p.

The males were sacrificed after 5 weeks of treatment and the dams on day 6 p.p. Final body weights and selected organs weights (epididymides, liver, ovaries, testes, thyroid with parathyroids) were recorded and a macroscopic post‑mortem examinationof the principal thoracic and abdominal organswas performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from the control and high-dose groups (epididymides, liver, testes, thyroid with parathyroids), on the liver from low- and intermediate-dose groups and on all macroscopic lesions.

The pups were sacrificed on day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

 

Results

 

The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 6 were within the acceptance criteria (± 15% of the nominal concentrations) and no test item was found in control samples.


There were no unscheduled deaths. Ptyalism was observed in males at all dose-levels (half of the animals at 50 mg/kg/day, all animals at 250 and 1000 mg/kg/day) and in all females at 1000 mg/kg/day. Round back was noted from the end of week 2 in females (7/10) at 250 mg/kg/day and in both sexes at 1000 mg/kg/day (7/10 males, 9/10 females). Piloerection was also observed at 250 mg/kg/day in females (2/10) and at 1000 mg/kg/day in all males and 4/10 females. At 1000 mg/kg/day, round back and piloerection lasted 3 to 6 days and were considered to be adverse. During gestation, one female still had round back on the first day of gestation, and four females piloerection at the beginning of gestation only.

There were no toxicologically significant effects on mean body weight gain and mean body weight. When compared with controls, mean food consumption at 1000 mg/kg/day was lower in males; this change was considered not to be adverse because of no relevant impact on mean body weight and on animal survival.

 

At hematology, there was a slight lower mean white blood cell count than in controls at 250 and 1000 mg/kg/day in males and at 1000 mg/kg/day in females, mainly related to lower mean numbers of lymphocytes. This was considered to be non adverse and of minor toxicological importance.

At blood biochemistry and when compared with controls, there was a dose-related higher mean cholesterol level from 250 mg/kg/day. This effect was considered to be adverse at 1000 mg/kg/day in view of the amplitude of differences from control and historical control data. In females, there was also a dose-related lower mean concentration of inorganic phosphorus from 50 mg/kg/day and a slightly higher mean urea blood concentration at 1000 mg/kg/day. These changes in mean inorganic phosphorus and urea levels were considered not to be adverse and of minor toxicological importance.

 

There were no test item-related macroscopic changes or effects on mean thyroid/parathyroid, epididymides, testis and ovary weights at necropsy. The microscopic examination and mean organ weights showed test item-related changes in liver only. Administration of the test item in rats at 1000 mg/kg/dayinduced mild centrilobular hypertrophy and vacuolation of Kupffer cells in the liver which correlated with the higher mean liver weight at necropsy. Centrilobular hypertrophy alone was also observed in males at 250 mg/kg/day. In the absence of any associated degenerative liver changes, these observations were considered not to be adverse.

 

There were no test item-related effects on pairing, mating and fertility data. There were no toxicologically relevant effects on delivery data.

 

In pups, there were no test item-related deaths or clinical signs andno test item-related effects on mean pup body weights, mean pup body weight gains,on the percentage of male pups at birth and no test item-related findings noted at necropsy.

 

Conclusion

The test item was administered daily by oral gavage to male and female Sprague‑Dawley rats, for 2 weeks before pairing, during pairing, gestation and until day 5p.p., at dose-levels of 50, 250 or 1000 mg/kg/day.

 

At 1000 mg/kg/day,round back and piloerection noted in both sexes in weeks 2/3, as well as the higher mean cholesterol level at the end of the treatment period were considered to be adverse.

There were no adverse findings at 50 and 250 mg/kg/day.

 

Therefore, based on the experimental conditions of this study:

.         the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 250 mg/kg/day,

.         the NOAEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day,

.         the NOAEL for toxic effects on progeny was considered to be 1000 mg/kg/day.