Registration Dossier

Toxicological information

Toxicity to reproduction: other studies

Currently viewing:

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 August 2011 to 04 February 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP study; haematology, clinical chemistry and ophthalmological examination not performed
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

GLP compliance:
no
Remarks:
The study was conducted in a GLP laboratory.
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: 01/2011
Purity: 93.6% (sum of the three main constituents)
Name of test material (as cited in study report): TERPINEOL MULTICONSTITUENT
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 23 June 2013

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore, India.
- Age at study initiation: 12 weeks
- Weight at study initiation: G1: 371.07 ± 12.60 g; G2: 369.06 ± 10.11 g
- Housing: One rat per cage was housed in sterilized suspended polysulfone cages (size: approximately L 425 mm x W 266 mm x H 175 mm) with stainless steel top
- Diet: Ssniff rats/mice pellet food and powder food – maintenance meal - manufactured by Ssniff spezialdiäten GmbH, Ferdinand-Gabriel-Weg 16, D-59494 SÖest, Germany was provided ad libitum
- Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25 °C
- Humidity: 60-67 %
- Air changes: 12-14 air changes/hour
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
DIET PREPARATION
- The required quantities of test item (G2- 12.0 g) was weighed, dissolved in 30 mL corn oil and mixed with approximately 300 g of Ssniff powder food for 2 minutes in stainless steel drum using a stainless steel spoon to prepare the premix. The premix was added in portions to the remaining bulk food (approximately 700 g) and mixed in a stainless steel drum using spoon for approximately 5 to 8 minutes.
- For the negative control group (G1), 1000 g of Ssniff powder food was mixed with 30 mL corn oil and mixed manually in a stainless steel drum using a spoon for 7-10 minutes.
A similar procedure was followed for the subsequent mixing sessions. The leftover formulated diet was sent for incineration.
- Rate of preparation of diet (frequency): Test item fortified food was prepared daily.

Stability and homogeneity of the test item
The Sponsor confirmed 2 days ambient storage stability and 8 days frozen at 100 and 20000 ppm, when the test material was first dissolved in corn oil to improve stability (Thacker C., 2010).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Test item fortified diet was fed daily ad libitum throughout the treatment period (13 weeks)
Duration of test:
13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
12000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 males/dose
Control animals:
yes, plain diet
Details on study design:
DOSE SELECTION RATIONALE: A dose level of 12000 ppm for terpineol multiconstituent No. 2 was selected by the Sponsor. In addition to test dose, a concurrent negative control group (0 ppm) was included.

RATIONALE FOR ANIMAL ASSIGNMENT: Rats were randomly distributed to different groups by body weight stratification method using ProvantisTM software. Grouping was done one day prior to initiation of treatment.

CAGE SIDE OBSERVATIONS:
Time schedule: General clinical signs were observed once daily. All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon.

DETAILED CLINICAL OBSERVATIONS:
Time schedule: Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter during treatment period.

BODY WEIGHT:
Time schedule for examinations: Individual body weights were recorded before the start of treatment and at weekly intervals thereafter except for Week 13 wherein the animals were weighed on Day 6 of that week. Fasting body weights were recorded on Day 91.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption was measured daily during the treatment period. The average food consumption (g/rat/day) was calculated.
- Mean food intake (g/kg bw/day) and test item intake (mg/kg bw/day) was calculated from food intake and body weight data.
Food Intake (g/kg bw/day):
Net food intake (g/kg bw/day) = (Mean food intake / Mean Body weight) x 1000
Test Item Intake (mg/kg bw/day) = (Dose (ppm) / 1000) x Net food intake

GROSS PATHOLOGY: Rats sacrificed by design (terminal necropsy) were euthanized by exsanguination under isoflurane anaesthesia. All rats in the study were subjected to necropsy after overnight fasting (water allowed).

HISTOPATHOLOGY:
Tissue Collection: On completion of the gross pathology examination, the tissues listed in the table 7.5.1/1 were collected and weighed from all the animals. The organ weight ratios as percentage of body weight were also determined. All the collected tissues were preserved in 10 % neutral buffered formalin.
Histopathological examination of testes and epididymides were performed for all the animals. The tissues were processed for routine paraffin embedding and 5 µm sections stained with Mayer’s Haematoxylin and Eosin stain.

SPERM EVALUATION
Sperm evaluation was carried out at termination for all males. At necropsy, right testes and corresponding epididymis were collected and frozen for sperm count and the right vas deferens was collected for evaluation of sperm motility and sperm morphology. Sperm motility was evaluated for all the groups using Hamilton- Thorne TOX-IVOS sperm analyzer.
Statistics:
The Data was captured using ProvantisTM for the parameters namely body weights and organ weights, and were analysed using built-in statistical tests.
The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package version 12.0. All quantitative variables like body weight, sperm parameters, organ weights and organ weight ratios data were tested for homogeneity of variances (Levene’s test) within the group before performing One-way analysis of variance (ANOVA).
For the non-optimal data (non-normal or heteroschedastic), ANOVA was done after suitable transformation of the data.
Comparison of means between the treatment group and the control group was done using Dunnett’s test, where ‘F’ test found significant.
All analyses and comparisons were evaluated at the 5% (P≤0.05) level.
Statistically significant differences (P≤0.05), indicated by the aforementioned tests were designated by the superscripts throughout the report as stated below:
+/-: Significantly higher/lower than the control group

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Remarks:
male fertility
Effect level:
>= 12 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no testicular and epididymal toxicity was evidenced in animals receiving Terpineol Multi-constituent No. 2 at 12000 ppm for 90 days

Observed effects

CLINICAL SIGNS AND MORTALITY
No treatment related mortality or signs of toxicity were noted.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight on Day 8 was lower when compared to the initial body weight (3.6 % of mean body weight loss) in the treated group. After Day 15 the mean body weights were increased when compared to the initial body weight but remained significantly lower when compared to control group (5-6 % lower than controls). The net weight gain was markedly reduced during the first week of treatment and during the Days 57-64.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food intake was markedly reduced during the first week of treatment in animal receiving 12000 ppm of test item (up to 80-90 % decrease during the first 3 days when compare to control group) indicating the non-palatability of the test item, and then remained significantly lower during most of treatment period (13 % decrease in mean food intake when compare to controls).
Thus, the decreased body weights at 12000 ppm dose group were associated with the decrease in the food intake throughout the treatment period.

ORGAN WEIGHTS
- There were no test item-related changes in the terminal fasting body weights. The lower terminal fasting body weight (6 %) noted in the test item treated group was considered incidental.
- Increased liver weights (absolute- 13 % and relative-20 %) noted in the treatment group was considered to be test item-related. Slight increase in relative weights (paired and unpaired) of testes and epididymides observed in the treatment group were considered to be incidental, as it was not associated with any microscopic change.
- All other weight changes noted in treated males which attained statistical significance were of low magnitude and/or were comparable to historical data, hence considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
- There were no test item-related histological changes observed in testis and epididymis.

OTHER FINDINGS
A slight significant increase in the percentage of abnormal (4.8%) sperms was noted in treatment group as compared to the control group. However, the change was considered incidental as it was well within range of normal biological variation noted among male rats [the range of the in-house historical control data for mean percentage of abnormal sperms: 0.1- 7.4%] . The sperm motility remained unaffected by dietary administration of test item. There were no test item-related changes observed in cauda epididymal weight/sperm count and testicular weight/spermatid count.

Any other information on results incl. tables

Table 7.8.3/2: Calculated mean food and test item intake

Group No.

Dose (ppm)

G1

0

G2

12000

Food Intake

g/animal/day

23.99

20.97

g/animal/91 days

2183.34

1908.63

Net Food Intake

g/kg bw/day

55.94

51.89

g/kg bw/91 days

5090.69

4721.76

Test Item Intake

mg/kg bw/day

0.00

622.65

mg/kg bw/91 days

0.00

56661.15

Table 7.5.1/3: Summary of Terminal Body Weights and Organ Weights (Day 92)

Group No.

Dose (ppm)

Terminal

Fasting BW

(g)

 

Heart

(g)

Liver

(g)

Prostate

(g)

Seminal vesicles

& coagulating

glands

(g)

Spleen

(g)

Adrenals

(g)

Epididymides

(g)

Kidneys

(g)

Testes

(g)

G1

0

449.12 ± 22.56

1.415 ± 0.108

12.850 ± 0.999

1.399 ± 0.170

1.9188 ± 0.1937

0.845 ± 0.095

0.053 ± 0.007

1.437 ± 0.072

2.666 ± 0.161

3.977 ± 0.225

G2

12000

422.26 ± 14.55 (-)

1.311 ± 0.082 (-)

14.468 ± 0.894 (+)

1.246 ± 0.163

1.7579 ± 0.2106

0.858 ± 0.091

0.057 ± 0.009

1.464 ± 0.089

2.676 ± 0.110

4.072 ± 0.186

%Diff

6

7

13

$

$

$

$

$

$

$

N = 10

1 Way ANOV (Automatic)

$: Not calculated as difference is not statistically significant.

+/–: Significantly higher/lower than the control group at P0.05

Table 7.5.1/4: Summary of Terminal Body Weights andOrgan to Body Weight Ratios (Day 92)

Group No.

Dose (ppm)

Terminal

Fasting BW

(g)

 

Heart

(%)

Liver

(%)

Prostate

(%)

Seminal vesicles

& coagulating

glands

(%)

Spleen

(%)

Adrenals

(%)

Epididymides

(%)

Kidneys

(%)

Testes

(%)

G1

0

449.12 ± 22.56

0.3152 ± 0.0203

2.8592 ± 0.1207

0.3123 ± 0.0415

0.4281 ± 0.0481

0.1884 ± 0.0225

0.012 ± 0.002

0.321 ± 0.022

0.5944 ± 0.024

0.887 ± 0.052

G2

12000

422.26 ± 14.55 (-)

0.3103 ± 0.0136 

3.4271 ± 0.1922 (+)

0.2951 ± 0.0382

0.4165 ± 0.0491

0.2033 ± 0.0216

0.013 ± 0.002

0.347 ± 0.027 (+)

0.634 ± 0.017 (+)

0.965 ± 0.049 (+)

%Diff

6

$

20

$

$

$

$

8

7

9

N = 10

1 Way ANOV (Automatic)

$: Not calculated as difference is not statistically significant.

+/–: Significantly higher/lower than the control group at P0.05

Table 7.5.1/5: Summary of Terminal Body Weight and Paired Organ Weights (Day 92)

Group No.

Dose (ppm)

Terminal

Fasting BW

(g)

 

Left Adrenal

(g)

Right

Adrenal

(g)

Left

Epididymis

(g)

Right

Epididymis

(g)

Left Kidney

(g)

Right Kidney

(g)

Left Testis

(g)

Right Testis

(g)

G1

0

449.12 ± 22.56

0.027 ± 0.004

0.027 ± 0.004

0.725 ± 0.049

0.712 ± 0.034

1.327 ± 0.099

1.339 ± 0.075

1.974 ± 0.115

2.004 ± 0.115

G2

12000

422.26 ± 14.55 (-)

0.028 ± 0.005

0.029 ± 0.004

0.743 ± 0.044

0.721 ± 0.052

1.324 ± 0.072

1.352 ± 0.065

2.042 ± 0.081

2.029 ± 0.109

%Diff

6

$

$

$

$

$

$

$

$

N = 10

1 Way ANOV (Automatic)

$: Not calculated as difference is not statistically significant.

+/–: Significantly higher/lower than the control group at P0.05

Table 7.5.1/6: Summary of Terminal Body Weight andPaired Organ to Body Weight Ratios (Day 92)

Group No.

Dose (ppm)

Terminal

Fasting BW

(g)

 

Left Adrenal

(%)

Right

Adrenal

(%)

Left

Epididymis

(%)

Right

Epididymis

(%)

Left Kidney

(%)

Right Kidney

(%)

Left Testis

(%)

Right Testis

(%)

G1

0

449.12 ± 22.56

0.006 ± 0.001

0.006 ± 0.001

0.162 ± 0.013

0.159 ± 0.011

0.295 ± 0.018

0.298 ± 0.008

0.440 ± 0.028

0.447 ± 0.024

G2

12000

422.26 ± 14.55 (-)

0.007 ± 0.001

0.007 ± 0.001(+)

0.176 ± 0.013 (+)

0.171 ± 0.015

0.313 ± 0.012 (+)

0.320 ± 0.014 (+)

0.484 ± 0.020 (+)

0.481 ± 0.029 (+)

%Diff

6

$

14

9

$

6

7

10

8

N = 10

1 Way ANOV (Automatic)

$: Not calculated as difference is not statistically significant.

+/–: Significantly higher/lower than the control group at P0.05

Table 7.5.1/7:Summary of Vas Deferns Sperm Motility, Cauda Epididymidal Count and Detergent and Homogenization Resistant Testicular Spermatid Counts

 

Group No.

Dose (ppm)

Motility

Cauda Epididymal Sperm Counts

Detergent and Homogenization Resistant Testicular Spermatid Counts

Percentage of

progressive

motile sperms

Percentage

of motile

sperms

Cauda

epididymis

weight (g)

No. of sperms

per cauda

epididymis

(x 106)

No. of sperms

per gram of

cauda

epididymis

(x 106)

Parenchyma

weight (g)

No. of

spermatids

per testis

(x 106)

No. of

spermatids per

gram of

parenchyma (x 106)

G1

0

88.3 ± 4.00

66.3 ± 5.98

0.265 ± 0.022

224.73 ± 22.70

850.74 ± 69.17

1.792 ± 0.107

233.65 ± 19.72

130.58 ± 11.24

G2

12000

87.8 ± 4.16

65.3 ± 5.81

0.280 ± 0.010

230.20 ± 25.31

822.50 ± 79.23

1.815 ± 0.102

224.63 ± 20.28

123.89 ± 10.89

%Diff

$

$

$

$

$

$

$

$

N = 10

$: Not calculated as difference is not statistically significant.

Applicant's summary and conclusion

Conclusions:
No testicular and epididymal toxicity was evidenced in animals receiving Terpineol multiconstituent No. 2 at 12000 ppm for 90 days to Sprague-Dawley rats.
Executive summary:

In a repeated dose oral dietary toxicity study terpineol multiconstituent No. 2 was administered to Sprague-Dawley rats for 90 days.

The test item was dissolved in corn oil, mixed in Ssniff powder feed at the dose level of 12000 ppm and fed to male Sprague-Dawley rats (10/dose) daily ad libitum for 13 weeks. Rats in the control group were fed basal diet only without any test item admixtures. All rats were observed for clinical signs, mortality, and changes in the body weights and food intake. Sperm evaluations were conducted at termination for all the males from each group. Sperm motility, count and morphology were evaluated for all the groups. All rats were subjected to detailed necropsy at termination and organs were weighed. Histopathological examination of the testes and the epididymides were carried out.

 

No treatment related mortality or signs of toxicity were noted. The body weights were significantly reduced in rats receiving test item at 12000 ppm. This decrease was associated with a decrease in the food intake throughout the treatment period. The food consumption was significantly reduced in males receiving test item at 12000 ppm dose during the treatment period. The calculated mean daily test item consumption was 0 and 622.65 mg/kg bw/day corresponding to 0 and 12000 ppm, respectively.

 

A slight significant increase in the percentage of abnormal (4.8%) sperms was noted at 12000 ppm as compared to the control group. However, the change was considered incidental as it was well within the range of normal biological variation noted among male rats [the range of the in-house historical control data for mean percentage of abnormal sperms: 0.1- 7.4%]. The sperm motility remained unaffected by dietary administration of test item. There were no test item-related changes observed in cauda epididymal weight/sperm count and testicular weight/spermatid count.

 

There were no test item-related changes in the terminal fasting body weights. Increased liver weights (absolute-13 % and relative-20 %) were noted in the treatment group. Increased relative weights (paired and unpaired) of testes and epididymides were observed in the treatment group. There were no test item-related histological changes observed in the testis and the epididymis.

 

No testicular and epididymal toxicity was evidenced in Sprague-Dawley rats receiving terpineol multi-constituent No. 2 at 12000 ppm for 90 days.