Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test guideline (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 November 2008 - 22 October 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to recent OECD guideline but histopathological examination was not performed in the low and intermediate doses therefore no NOAEL could be identified for systemic toxicity for parents.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010
Reference Type:
other: Statement of purity for Amber core
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
yes
Remarks:
histopathological examination was not performed in the low and intermediate doses therefore no NOAEL could be identified for systemic toxicity for parents.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Amber core (P#620)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Weight at study initiation: (P) Males: 147-213 g; Females: 182-248 g
- Fasting period before study: none
- Housing: in groups of four by sex in solid floor polypropylene cages
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 November 2008 To: 24 March 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % Carboxy methylcellulose/Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 4, 20 and 100 mg/mL for low, intermadiate and high dose, respectively
- Amount of vehicle (if gavage): 5 mL/kg/day
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 21 days maximum
- Proof of pregnancy: sperm within the vaginal smear and/or vaginal plug in situ
- After successful mating each pregnant female was caged individually during the period of gestation and lactation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of Amber Core (P#620) in the test material formulations was determined by gas chromatography (GC) using an external standard technique.
The homogeneity of formulations at 2 and 200 mg/mL was confirmed and stability of these formulations was demonstrated for up to 15 days storage at approximately 4 °C in the dark.
Dosing formulations (4, 20 and 100 mg/mL) analysed during the study were between 88 - 105 % of nominal concentration.
Duration of treatment / exposure:
Males were treated for at least 10 weeks prior to pairing with treatment continuing until termination (at least 18 weeks of treatment), females were treated for at least 2 weeks prior to pairing and then throughout mating, gestation and lactation until weaning (Day 21 of lactation).
Frequency of treatment:
Daily (except during parturition for females)
Details on study schedule:
None
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
20 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on a 28-day toxicity study in the rat. In this study no effects on bodyweight performance or food consumption were observed at dosages of up to 1000 mg/kg bw/day. However, histopathological findings relating to the liver and kidney were also apparent at 1000 mg/kg bw/day and it was considered that these histopathological changes precluded the use of 1000 mg/kg bw/day in the one-generation study, in view of the longer dosing period employed.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week; immediately before and after dosing and one hour after dosing at weekends and public holidays.
- Cage side observations: overt signs of toxicity, ill-health or behavioural change

BODY WEIGHT:
- Time schedule for examinations: for males, Day 1 (prior to treatment) and then weekly for males until termination; for females, Day 1 of treatment and at weekly intervals during the pre-mating phase, then daily throughout the mating period. Mated females were weighed on Days 0, 7, 14 and 21 post coitum and on Days 1, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION:
- for males: weekly
- for females: weekly before mating, Days 0 to 7, 7 to 14 and 14 to 21 post coitum and Days 1 to 4, 4 to 7, 7 to 14 and 14 to 21 of lactation.

WATER CONSUMPTION:
- Time schedule for examinations: daily during the pre-pairing phase for both sexes, daily for females during gestation
Oestrous cyclicity (parental animals):
During mating period, a vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded daily.
Sperm parameters (parental animals):
None
Litter observations:
After parturition, the number of live and dead offspring was recorded as well as the number of offspring born, the daily number of offspring alive, the sex of individual offspring (on Days 1, 4, 7, 14, 21 postpartum), the clinical condition of offspring from birth to weaning, the individual offspring and total litter weights on Days 1, 4, 7, 14 and 21 post partum and necropsy findings.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after the majority of females had reared their young to weaning
- Maternal animals: All surviving animals, at Day 21 or 22 post partum

GROSS NECROPSY
The corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: epididymides, prostate, kidneys, seminal vesicles (with coagulating gland and fluids), liver, testes, ovaries, uterus (including oviducts and cervix) and pituitary gland.
The following tissues were prepared for microscopic examination: coagulating gland, pituitary gland, epididymides, prostate, kidneys, seminal vesicles, liver, testes, ovaries and uterus (with oviducts and cervix).
Postmortem examinations (offspring):
Offspring were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
For females and offspring, food consumption, water consumption, organ weight (absolute and relative to terminal bodyweight), bodyweight gain, litter weights and offspring bodyweights, data were assessed for dose response relationships by linear regression analysis, followed by Levene's test for homogeneity of variance. Where variances were shown to be homogenous, one way analysis of variance (ANOVA) followed by pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test. Implantation loss and offspring sex ratio were analysed using the stated non-parametric methodology.
Histopathology data were analysed to determine significant differences between control and treatment groups for the individual sexes using Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater, or Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Reproductive indices:
Pre-coital interval: time elapsing between initial pairing and the observation of positive evidence of mating
Fertility indices: Mating Index (%) = Number of animals mated / Number of animals paired x 100 ; Pregnancy Index (%) = Number of pregnant females / Number of animals mated x 100
Gestation Length: number of days of gestation including the day for observation of mating and the start of parturition
Parturition Index (%) = Number of females delivering live offspring / Number of pregnant females x 100
Pre-implantation loss = (Number of Corpora Lutea - Number of implantation sites) / Number of corpora lutea x 100
Post-implantation loss = (Number of implantation sites - Number of offspring) / Number of implantation sites x 100
Offspring viability indices:
Live Birth Index (%) = Number of offspring alive on Day 1 / Number of offspring born x 100
Viability Index 1 (%) = Number of offspring alive on Day 4 / Number of offspring alive on Day 1 x 100
Viability Index 2 (%) = Number of offspring alive on Day 7 / Number of offspring alive on Day 4 x 100
Viability Index 3 (%) = Number of offspring alive on Day 14 / Number of offspring alive on Day 7 x 100
Viability Index 4 (%) = Number of offspring alive on Day 21 / Number of offspring alive on Day 14 x 100
Viability Index 5 (%) = Number of offspring alive on Day 21 / Number of offspring alive on Day 1 x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 500 and 100 mg/kg bw/day, dosing was associated with a transient increase in post-dosing salivation. No unscheduled mortality occured during the study.

BODY WEIGHT (PARENTAL ANIMALS)
At 500 mg/kg bw/day, bodyweight gains of males were generally slightly lower than control, with differences often attaining statistical significance, particularly during the early treatment period (i.e. to Week 8). However, despite this lower bodyweight gain, absolute bodyweight after 18 weeks of treatment was still 92% of that of the control. (See table 1)

WATER CONSUMPTION (PARENTAL ANIMALS)
At 500 mg/kg bw/day in males, there was a slight increase in water consumption from the second week of treatment, compared to control, although differences only attained statistical significance during Weeks 3 and 4. At 500 mg/kg bw/day in females, increased water consumption was observed throughout the pre-pairing and gestation phases of the study, with differences from control attaining statistical significance. The increase in water intake was considered consistent with the dosing of an unpalatable, or slightly irritant, test material. (See table 2)

ORGAN WEIGHTS (PARENTAL ANIMALS)
At 500 mg/kg bw/day, differences in organ weights considered to be of toxicological significance were restricted to increases in male and female liver weights and male kidney weights; with differences from control attaining statistical significance for both absolute and bodyweight relative values.
At 100 mg/kg bw/day, a similar but smaller increase in male absolute and bodyweight relative liver weights were considered to be of toxicological significance; differences from control attaining statistical significance. There was however no corresponding statistically significant increase in liver weight for females at this dosage.
Absolute and bodyweight weight relative pituitary weights were lower for treated males than control; although differences attained statistical significance there was no dosage-relationship, values, particularly when adjusted for bodyweight, were all within one standard deviation and there was no similar increase amongst treated females. In the absence of any histopathological findings for this organ, the differences in organ weight were considered to be incidental and unrelated to treatment. Additionally, at 500 mg/kg bw/day, absolute and bodyweight relative prostate weight was lower than control, with differences attaining statistical significance. Absolute values were within one standard deviation and in the absence of any histopathological findings for this organ or corresponding effects on fertility, this finding was considered not to be of toxicological significance. (See table 3)

HISTOPATHOLOGY (PARENTAL ANIMALS)
Centrilobular hepatocyte enlargement was seen in relation to treatment for animals of either sex treated with 500 mg/kg bw/day. A greater incidence and severity of globular accumulations of eosinophilic material were observed in the tubular epithelium of males treated with 500 mg/kg bw/day. An associated greater incidence and severity of groups of basophilic tubules was also seen among male rats at this treatment level. Treatment-related tubular necrosis was observed for ten males treated with 500 mg/kg bw/day and affected isolated tubules in the outer medulla immediately adjacent to the renal cortex. (See tables 4 and 5)

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No relevant effect from Amber core. It is assumed that the changes observed in the liver and the kidney resulted from a strong cellular adaptation phenomena and a species specificity, respectively.

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)

No effect of toxicological relevance was observed in offspring.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects related to treatment were observed even at the highest dose tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean bodyweight gains in males (g)

Week numbers relative to start date

From:

To:

1

2

2

3

3

4

4

5

5

6

6

7

7

8

8

9

9

10

10

11

11

12

12

13

13

14

14

15

15

16

16

17

17

18

18

19

1

19

Control (n=24)

Mean

38.3

31.6

24.9

22.9

18.3

19.7

13.3

12.2

9.9

7.6

3.5

7.7

6.4

9.6

7.6

9.8

8.3

1.7

253.3

S.D.

4.8

4.9

4.8

3.9

4.0

4.0

3.3

3.0

4.4

4.4

5.7

5.4

3.5

4.6

5.1

4.8

3.7

4.1

29.9

500 mg/kg bw/day (n=24)

Mean

36.7

27.7*

21.3**

20.5

14.7**

15.0**

8.5**

8.8**

7.1

7.0

7.3*

5.0

2.3**

11.5

8.2

6.6*

9.5

0.2

217.7

S.D.

4.6

7.3

4.5

5.9

4.2

4.6

5.0

5.1

6.7

4.8

4.7

5.2

4.8

4.3

4.6

4.8

5.0

7.3

28.2

 

*            Significantly different from control group P≤0.05

**         Significantly different from control group P<0.01

Table 2: Mean daily water consumptions in females

Day numbers relative to start date

 

Pre-Mating

Gestation

From:

To:

1

8

8

15

0

7

7

14

14

21

Control

Mean

22.7

22.1

30.3

33.1

40.0

S.D.

 

 

4.9

5.1

6.0

N

6

6

23

23

23

500 mg/kg bw/day

Mean

28.0***

26.6***

39.2***

39.8***

49.6**

S.D.

 

 

8.3

7.8

11.5

N

6

6

23

23

23

S.D. not calculated for gang-housed animals

**        Significantly different from control group P<0.01

***       Significantly different from control group P<0.001

Table 3: Mean organ weights with corresponding relative (% of bodyweight) organ weights

 

 

0

Control

20

mg/kg bw/day

100

mg/kg bw/day

500

mg/kg bw/day

 

 

MALES (n=24)

Terminal Bodyweight

Mean

442.5

434.0

448.2

409.9

S.D.

33.2

36.8

50.6

33.3

Kidneys

Mean

2.32348

2.25048

2.35785

2.40308**

S.D.

0.24981

0.20535

0.22925

0.20768

Liver

Mean

12.9961

12.7320

13.8428**

15.5342**

S.D.

1.26480

1.26329

1.56312

1.53478

Pituitary Gland

Mean

0.00642

0.00484*

0.00550*

0.00444**

S.D.

0.00263

0.00237

0.00253

0.00227

Prostate

Mean

0.61125

0.55620

0.56081

0.49537**

S.D.

0.08015

0.10649

0.10723

0.13310

 

 

FEMALES

 

 

n = 24

n = 23

n = 22

n = 22

Terminal Bodyweight

Mean

286.2

290.7

288.8

289.0

S.D.

16.6

15.5

15.0

16.9

Liver

Mean

13.22465

13.48687

13.89770

17.41297***

S.D.

1.23862

1.01370

1.38246

1.90273

**        Significantly different from control group P<0.01

***       Significantly different from control group P<0.001

Table 4: Summary incidence of histopathological findings in males

Histopathological Finding

 

Dose Level (mg/kg bw/day)

 

0

500

Total

Kidneys

Groups of basophilic tubules

Number of lesions

8

17

26

Number of animals

24

24

50

P

 

+

 

Globular accumulations of

eosinophilic material

Number of lesions

8

21

31

Number of animals

24

24

50

P

 

+++

***

Tubular necrosis

Number of lesions

0

10

11

Number of animals

24

24

50

P

 

+++

**

 

Liver

Centrilobular hepatocyte

Number of lesions

2

12

14

Number of animals

24

24

50

P

 

++

**

**        Significantly different from control group P<0.01

***       Significantly different from control group P<0.001

Table 5: Summary incidence of histopathological findings in females

 

Histopathological finding

 

Dose Level (mg/kg/day)

 

0

500

Total

Liver

Centrilobular hepatocyte enlargement

Number of lesions

1

18

19

Number of animals

24

22

46

P

 

+++

***

 

Uterus/Cervix

Dilatation horn 1

Number of lesions

7

1

8

Number of animals

24

22

46

P

 

 

Dilatation horn 2

Number of lesions

6

1

7

Number of animals

24

22

46

P

 

(-)

(*)

Peripheral foam cells/ haemorrhage/ pigment

Number of lesions

21

22

43

Number of animals

24

22

46

P

 

(+)

(*)

*            Significantly different from control group P≤0.05

***       Significantly different from control group P<0.001

Applicant's summary and conclusion

Conclusions:
It is assumed that the highest dose of 500 mg/kg bw/d is a NOAEL for systemic toxicity for adults within this study. A clear NOEL for reproductive toxicity was established at 500 mg/kg bw/day.
Executive summary:

The effects of Amber core (P#620) on reproductive function and pre-natal and post­natal developments were assessed according to OECD guideline 415. The test material was administered by gavage to three groups of 24 male and 24 female Wistar rats each, at dose levels of 20, 100 and 500 mg/kg bw/day. A control group of 24 males and 24 females was dosed with vehicle alone (0.5% sodium carboxymethyl cellulose/0.5% Tween 80).

Clinical signs, bodyweight, dietary intake and water consumption were monitored. After ten weeks of treatment for males and two weeks of treatment for females, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size. Litter weights and individual offspring weights were also recorded on specific days post partum. All surviving females and offspring were terminated on Day 21 post partum with the males being terminated after the majority of the females and litters had been killed. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

There was no unscheduled death, no relevant clinical sign, no effect on bodyweight and food consumption. Increased water intake was observed for both sexes at 500 mg/kg bw/day and males at 100 mg/kg bw/day (likely to reflect unpalatable, or slightly irritant, test material formulation). Mating performance, fertility and gestation length were considered to have been unaffected by treatment. No treatment-related macroscopic abnormalities were detected for adults or offspring.

Histopathological examinations of adult tissues revealed centrilobular hepatocyte enlargement for both sexes at 500 mg/kg bw/day and a greater incidence and severity of globular accumulations of eosinophilic material in the tubular epithelium of male rats at 500 mg/kg bw/day, with an associated greater incidence and severity of groups of basophilic tubules also being present. Additionally tubular necrosis and affected isolated tubules in the outer medulla immediately adjacent to the renal cortex was observed for 10 males at this dosage level.

It is assumed that the centrilobular hepatocyte enlargement observed, occurs as an induction of the microsomal drug metabolizing enzyme systems caused by the treatment of several compounds and is considered to be cellular adaptation phenomena. In the case of Amber core, the adaptative response is important as it was already demonstrated in the 28 -day repeated oral dose toxicity study described in this dossier (see § 7.5.1).

Moreover, the changes of both the renal tubular epithelium and basophilic tubules are lesions known to be spontaneous in male rats only and are not observed in other species. Therefore, this effect is specific to male rat, and is not relevant for the risk assessment in human.

There was no obvious adverse effect of treatment of the corpora lutea and implantation counts, litter size at birth and subsequent survival and bodyweight of the offspring to weaning or on sex ratio.

Therefore, it can be assumed that no test substance related effect was observed in the highest dose group of adult animals considering that the changes observed in the liver and in the kidney resulted from cellular adaptation phenomena and species specificity, respectively.

Hence, histopathological examinations of liver and kidneys at 20 or 100 mg/kg bw/day were not performed even if increased absolute and relative liver weights in males were recorded assuming that in the absence of any other effect, the liver weight changes was likely related to cellular adaptation phenomena. Therefore, it is assumed that the highest dose (500 mg/kg bw/d) is a NOAEL for systemic effect in the adult animals.

Moreover, there were no effects observed for reproductive parameters and a clear NOEL for reproductive toxicity including the survival, growth and development of the offspring was established at 500 mg/kg bw/day.