Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines, which do not affect the quality of the relevant results.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

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

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Acacia mearnsi, ext., reaction products with ammonium chloride and formaldehyde
EC Number:
285-077-0
EC Name:
Acacia mearnsi, ext., reaction products with ammonium chloride and formaldehyde
Cas Number:
85029-52-3
Molecular formula:
Not necessary (UVCB substance)
IUPAC Name:
Acacia mearnsi, ext., reaction products with ammonium chloride and formaldehyde
Test material form:
other: liquid
Details on test material:
Physical State at room temperature: liquid form
Colour: dark brown
Density: liquid= 1.1-1-2 g/cm3
Molecula weight of base form: UVCB
pH: Liquid= 1.7-2.3
Boiling point: 98oC
Active Components (%): 100%
Purity: 100%
Storage condition: room temperature, protection from light
Other specification: UVCB= Substance of unknown or variable composition, complex reaction products or biological materials.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 3°C
- Relative humidity: 55 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0702)
- Free access to tap water, (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 010812)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Number and Sex of the Animals
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Experimental Groups and Doses
A dose range finding study was performed to choose the dose (BSL Study number 122037) for the main study. Based on dose range finding study in which no overt toxicological changes were observed at 1000 mg/ kg bw/day and in consultation with the sponsor the following doses (Table 1) were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.

The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same dose volume.

Administration of Doses
The test item and vehicle were administered at a single dose to the animals by oral gavage using a gavaging cannula. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured on week- based.
Details on mating procedure:
Mating was performed in a ratio of 1:1 (male to female). The vaginal smears of the females were checked every morning after the start of the mating period to confirm the copulation. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.
Copulation index, fertility index and delivery index was calculated for each group. The calculations were made using the formula as below,
Copulation Index (%) = (No. of rats copulated / No.of pairs) X 100
Fertility Index (%) = (No. of females pregant / No.of females copulated) X 100
Delivery Index (%) = (No. of dams with live newborns / No.of pregnant dams) X 100
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparation of the Test Item and Control Formulations
The test item was weighed into a tared plastic vial on a suitable precision balance and the sterile water was added to give the appropriate final concentration of the test item. The formulation was placed on Vortex machine for short period to ensure proper homogenistation of the formulation.
The vehicle has been selected as suggested by the sponsor and on the basis of the test item’s characteristics.
The test item formulation was prepared freshly on each administration day before the administration procedure.

Dose Formulation Analysis
Each dosing concentration were analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and the low dose preparation in study week 1 and 5.
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high and low dose preparations.
All formulation samples were preserved at -20 oC until the analysis. The samples were analyzed at Spectral Service AG, Emil-Hoffmann-Str. 33, 50996 Köln, Germany. The contents of the reaction products of acacia mearnsi with ammonium chloride and formaldehyde in the solutions will be determined by 1H-NMR using caliberation curve of test item and internal standards. This phase of the study was performed as standalone GLP study and the procedure followed for the sample analysis was described in detail in the study plan issued by Spectral Service.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days (5 males from each group received dose applications for 28 days and the rest 5 animals from each group received dose applications for 29 days) was completed.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same dose volume.
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 / 100 / 300 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:
According to the results of the dose range finding study and in consultation with the sponsor three selected doses were tested for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
Positive control:
No positive control

Examinations

Parental animals: Observations and examinations:
Clinical Observations
General clinical observations were made once a day in all animals. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 selected males and on day 3 of the lactation period in 5 selected females (only lactating females were evaluated) from each of control and treated groups outside the home cage using a functional observational battery of tests :
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).
Oestrous cyclicity (parental animals):
The vaginal smears of the females were checked every morning after the start of the mating period to confirm the copulation. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
Sperm parameters (parental animals):
Reproductive organs (including epididymis) and macroscopic changes were evaluated in all study animals.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Litter observations:
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours off parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
Postmortem examinations (parental animals):
Gross Pathology:
All male animals were sacrificed after the completion of the mating period (minimum dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 (maximum dosing period: 54 days) using an anaesthesia (ketamine/xylazin, 3:1, medistar Arzeneimittel,and Serumwerk) was used. Pups were killed on PND 4 by using high dose of sodium pentobarbitol.

Females showing no evidence of copulation up to 14 days of the mating period and non pregnant females were sacrificed on day 26 after the last day of the mating period or the from the confirmed day mating, respectively.
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for eyes, testes and epididymides which were fixed in modified Davidson’s Solution for 24 hours before transferring to 10% neutral buffered formalin.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.


Reproductive organs (ovary, uterus, cervix, vagina, testis, epididymis, prostate gland, seminal vesicle and coagulating gland) and macroscopic changes were evaluated in all study animals.
Postmortem examinations (offspring):
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Statistics:
Evaluation of Results and Statistical Analysis. The findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings. Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights are also presented as figures. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and are presented as percentage.A statistical assessment of the results of the body weight. food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).
Reproductive indices:
Copulation index, fertility index and delivery index was calculated for each group. The calculations were made using the formula as below,
Copulation Index (%) = (No. of rats copulated / No.of pairs) X 100
Fertility Index (%) = (No. of females pregant / No.of females copulated) X 100
Offspring viability indices:
Delivery index was calculated as follow:

Delivery Index (%) = (No. of dams with live newborns / No.of pregnant dams) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
see "Details on results"
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
see "Details on results"
Reproductive performance:
no effects observed
Description (incidence and severity):
see "Details on results"

Details on results (P0)

Clinical observation:

There were clinical signs namely nasal discharge (Males C: 3/10, MD: 2/10; HD: 1/10; females MD: 2/10), alopecia (Males LD: 1/10; Females HD: 1/10), chromodacryorrhea (Males C: 1/10), injury (Males HD: 2/10; Females C: 1/10), moving the bedding (Males MD: 1/10), abnormal breathing (Males HD:1/10; Females LD: 1/10, MD: 1/10, HD: 2/10), salivation (Females MD: 1/10, HD: 3/10), piloerection (Females MD: 2/10, HD: 2/10), wasp waist (Females MD: 1/10), reduced spontaneous activity (Females MD: 1/10), trembling before application (females HD: 1/10) recorded in male or female animals of treated or control groups. These clinical signs were noted in few isolated animals occasionally and transiently. These findings were not considered to be of toxicological relevance.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

Functional Observations
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development
In both males and females, no treatment related changes were noted for body weight and body weight gain during the study period. Statistically there were no significant differences noted between the treated and corresponding controls. However, there was substantial decrease (lower by 138.29 % over control group) in body weight gain noted in females between the lactation days 0-4 in HD group. In the absence of statistical significance this finding was not considered to be of toxicological relevance.

Precoital Interval and Duration of Gestation
No treatment related changes were noted for the precoital interval and duration of gestation in treated groups when compared to control. All pregnancies resulted in normal births, except two animals in LD group (animals 52 and 59). At necropsy there was a fully developed single dead fetus noticed in uterus and in animals 59 implantation sites were observed. The reason for these two findings could not be determined based on the results of this study, but was considered unrelated to the test item as a dose relationship was lacking.
Successful mating resulted in 100% pregnancy rates in C, LD and HD groups, 90% pregnancy rate in MD group. In the absence of dose response pattern the decrease in pregnancy rate of MD group was not considered to be related to treatment.

Pre- and Post-Natal Data
No treatment related changes were noted for number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and percentage of pre and post implantation loss in treated groups when compared to control. The statistical evaluation of data revealed no significant differences between the values of treated and control groups.
However, there was substantial increase in pre implantation loss in LD and MD groups and post implanation loss in LD group when compared to corresponding control. There was no dose response pattern noted for these findings. Hence, in the absence of dose response patter and/or absence of statistical significance the changes were not considered to be related to treatment.

Reproductive Indices
There were no treatment related changes noted for copulation index (%), fertility index (%) and delivery index (%) in treated groups when compared to corresponding control group.
Among the females, one rat of MD group was found not to be pregnant. This was not dose-related and therefore considered to be unrelated to the treatment. One female animal was recorded with vaginal atresia, which went unnoticed during the detailed examination of animals before the initiation of study. This finding was incidental in origin.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
see "Details on results"
Mortality / viability:
no mortality observed
Description (incidence and severity):
see "Details on results"
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see "Details on results"
Sexual maturation:
no effects observed
Description (incidence and severity):
see "Details on results"
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see "Details on results"
Gross pathological findings:
no effects observed
Description (incidence and severity):
see "Details on results"
Histopathological findings:
no effects observed
Description (incidence and severity):
see "Details on results"

Details on results (F1)

Pup Survival Data
No treatment related changes were noted for survival of the pups from PND 0 to PND 4 in any treated group when compared to controls. However, there was 1 missing pup in LD group and 2 found dead pups in MD group recorded between PND 0-4. The missing pup is assumed to be cannibalized by dam. These findings not being in a dose related manner, this was considered to be incidental. No treatment related changes were considered for viability index (%).

Pup External Findings
No treatment related gross external findings were observed in the treated groups. However, there were few isolated findings namely dark head, dark snout and small size noted in few isolated pups of treated or control groups, which were considered to be incidental.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean Precoital Interval and Duration of Gestation

 

Group

Precoital Interval (Days)

Duration of Gestation (Days)

C

Mean

1.78

22.11

SD

0.83

0.60

N

9

9

LD

Mean

3.30

21.88

SD

3.56

0.83

N

10

8

MD

Mean

2.00

22.00

SD

0.87

0.50

N

9

9

HD

Mean

2.50

21.80

SD

0.97

0.42

N

10

10

 

Table 2: Pre- and Post-Natal Data

Group

No. of Corpora Lutea (CL)

No. of Implantation Sites (IS)

No. of Live Pups on PND 0

No. of Live Pups on PND 4

Pre Implantation Loss (%)

Post Implantation Loss (%)

C

Mean

11.44

11.00

10.22

10.22

3.82

7.34

 

SD

1.13

1.12

2.11

2.11

4.57

16.37

N

9

9

9

9

9

9

LD

Mean

10.20

9.10

8.20

8.10

14.49

24.56

 

SD

2.70

3.84

4.42

4.36

26.17

40.21

N

10

10

10

10

10

10

MD

Mean

11.44

10.22

10.00

9.78

10.68

4.70

 

SD

1.88

1.92

1.87

1.99

8.53

5.34

N

9

9

9

9

9

9

HD

Mean

11.90

11.30

11.00

11.00

4.70

2.32

 

SD

1.79

1.49

1.15

1.15

5.34

3.74

N

10

10

10

10

10

10

 

Table 3: Mean Reproductive Indices

Group

Viability Index (%)

C

Mean

100.00

SD

0.00

N

9

LD

Mean

97.73

SD

4.21

N

8

MD

Mean

97.78

SD

6.67

N

9

HD

Mean

100.00

SD

0.00

N

10

Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100

 

Table 4: Mean Pup Survival Data (days 3 and 4)

Group

Post Natal Day

Total Mortality (PND 1-4) %

3

4

No. of alive Males

No. of alive Females

Total

No. of Mortality

% Mortality

No. of alive Males

No. of alive Females

Total

No. of Mortality

% Mortality

C

Mean

5.56

4.67

10.22

0.00

0.00

5.56

4.67

10.22

0.00

0.00

0.00

SD

0.88

1.66

2.11

0.00

0.00

0.88

1.66

2.11

0.00

0.00

0.00

N

9

9

9

9

9

9

9

9

9

9

9

LD

Mean

6.13

4.00

10.13

0.00

0.00

6.13

4.00

10.13

0.00

0.00

1.14

SD

1.55

1.31

0.99

0.00

0.00

1.55

1.31

0.99

0.00

0.00

3.21

N

8

8

8

8

8

8

8

8

8

8

8

MD

Mean

4.67

5.11

9.78

0.00

0.00

4.67

5.11

9.78

0.00

0.00

2.22

SD

1.00

1.83

1.99

0.00

0.00

1.00

1.83

1.99

0.00

0.00

6.67

N

9

9

9

9

9

9

9

9

9

9

9

HD

Mean

5.00

6.00

11.00

0.00

0.00

5.00

6.00

11.00

0.00

0.00

0.00

SD

1.41

1.25

1.15

0.00

0.00

1.41

1.25

1.15

0.00

0.00

0.00

N

10

10

10

10

10

10

10

10

10

10

10

 

 

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of the Acacia mearnsi, ext., reaction products with ammonium chloride and formaldehyde to the male (minimum 28 days; 5 males/ group treated for 28 days and rest 5 males/ group treated for 29 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight/ day revealed neither mortalities nor findings of toxicological relevance in male and female animals. There were also no toxicologically relevant findings noted for reproductive and developmental parameters.

Based on the data generated from this “Combined Repeated Dose Oral Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test with Acacia mearnsi, ext., reaction products with ammonium chloride and formaldehyde, the no observed adverse effect level (NOAEL) for systemic and reproductive and developmental toxicity is considered to be 1000 mg/kg body weight/ day.
Executive summary:

No mortality occurred in the control or treated groups during the treatment period of this study.

In both males and females, no treatment related changes were noted for body weight, body weight gain and food consumption during the study period. No treatment-related changes were noted for number of still births, number of runts, total number of pups born on PND 0 and number of male and female pups, sex ratio, live pups on PND 0 and PND 4. No treatment related changes were noted for the mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 in treated groups when compared to corresponding control. No treatment related changes were noted for the precoital interval and duration of gestation in treated groups when compared to control.

No treatment related changes were noted for number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and percentage of pre and post implantation loss in treated groups when compared to control.

There were no treatment related changes noted for copulation index (%), fertility index (%) and delivery index (%) in treated groups when compared to corresponding control group.

No treatment related changes were noted for survival of the pups from PND 0 to PND 4 in any treated group when compared to controls. No treatment related gross external findings were observed in the treated groups.

There were no changes considered to be of toxicological relevance noted for organ weight in both males and females when compared to corresponding control.