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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 08 November 2013 to 14 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 414 without deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
other: audited draft report
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: 148465
Purity: 82.9% (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: 09 September 2013

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Number of animals ordered: 88 females; spare animals were removed from the study room after treatment commenced.
- Age of the animals at the start of the study (day 0 of gestation): at least 70 days old.
- Weight range of the animals at the start of the study (day 0 of gestation): 235 to 286 g.

HOUSING
- Cages: cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
- Number of animals per cage during acclimatisation up to four animals; during pairing one (stud) male and one female; during gestation: one female.
- Diet: ad libitum. SDS VRF1 certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Ad libitum.
- Duration of acclimatisation: five days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23ºC.
- Humidity: 40-70%.
- Air changes: approximately 15 to 20 per hour.
- Air supply: filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (artificial lighting): 12 h dark / 12 h light
- rodent facility: full barrier - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
METHOD OF PREPARATION
Vehicle was gradually added to the required amount of test substance and mixed to produce a pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
The use of plastic or rubber equipment/storage containers was avoided.

VEHICLE
- Concentration in vehicle: determined for each formulation.
- Amount of vehicle (if gavage): 5 mL/kg body weight.
- Justification for use and choice of vehicle (if other than water): substance only slightly soluble in water.
- Formulation frequency: weekly and prepared in advance of the first day of use. The homogeneity and stability of formulations in the concentration range 1 to 200 mg/mL were confirmed for 15 days following refrigerated storage (2-8˚C) and for 2 days following ambient storage (nominally 21˚C) as part of another study, Huntingdon Life Sciences (OAD0004).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration in test formulations:
- Verification: on Day 1 of dosing and the last day of dosing, freshly prepared test formulations were sampled (4 × 1 mL accurately weighed, 2 × 3 mL accurately weighed for controls) and submitted for analysis. Duplicate samples were analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency. These were discarded following acceptable results.
- Results: the mean concentrations of Terpineol multiconstituent in test formulations analysed for the study were within 4% of nominal concentrations, confirming accurate formulation.

Analytical procedure:
The analytical method involved extraction and dilution in acetone followed by gas chromatography analysis with flame ionisation detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 5 μg/mL to 25 μg/mL.
Details on mating procedure:
- Male/female ratio per cage: 1:1 with identified stock males.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- Day 0 of gestation: When positive evidence of mating was detected.

- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data

- Allocation: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Day 6 to 19 after mating, inclusive.
Frequency of treatment:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Duration of test:
21 days (Days 0-20 post coitum)
No. of animals per sex per dose:
20 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 60, 200 and 600 mg/kg bw/day were selected in agreement with the Sponsor.
- Volume dose: 5 mL/kg body weight.
- Rationale for animal assignment, method: To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.
- Identification of animals: Each animal was assigned a number and identified uniquely within the study by a tail tattoo.
- Identification of cages: Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Examinations

Maternal examinations:
CAGE SIDE & CLINICAL OBSERVATIONS: Yes
Time schedule:
- Mortality or signs of morbidity: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.
- Clinical signs: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each adult was recorded on Days 0, 3 and then daily from Days 6 to 20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

POST-MORTEM EXAMINATIONS: Yes
- Time schedule: animals were killed on Day 20 after mating

OTHER:
Preservation of tissues: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (including cervix and ovaries), for pregnant females surviving to term
- Number of corpora lutea: Yes, for all females
- Number of implantations: Yes, for all females
- Number of early resorptions: Yes, for all females
- Number of late resorptions: Yes, for all females
- Other: fetuses (live and dead), for all females
- For apparently empty uterine horns: The number of uterine implantation sites were checked after staining with ammonium sulphide
Fetal examinations:
EXAMINATION OF FETUSES:
- Number of all live fetuses: Yes
- Number of dead fetuses (fetuses at term without spontaneous movements and breathing): Yes
- Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
- Body weight of fetuses: Body weight of each live fetus was recorded.
- Sex of fetuses: Sex of each fetus was determined.
- External examinations: Yes; each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.
- Soft tissue examinations: Yes, 50% of the live fetuses in each litter were sexed internally and eviscerated.
- Skeletal examinations: Yes [all the remaining litters].
- Head examinations: Yes, cleft lip, protruding tongue, craniorachischisis, open eye lid(s), short snout,
- Skin examination: shinny, subcutaneous oedema
Statistics:
- The computer systems that were used on this study to acquire and quantify data include: Liberate, in-house system used for reporting in-life, necropsy, fetal pathology and statistics.
- Pre/post implantation loss and sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect
- Each treated group was compared to control using a Wald chi-square test.
- For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test
- The mean values: group mean values and SD were calculated using individual litter mean values.
Indices:
- Pre-implantation loss (%): [(Number of corpora lutea - Number of implantations) / Number of corpora lutea] X 100
- Post-implantation loss (%): [(Number of implantations - Number of live fetuses) / Number of implantations] X 100
- Sex ratios of the foetuses were calculated as the percentage of males per litter.
- All group values and SD (as appropriate) were calculated from the individual litter values.
- For litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed.
Historical control data:
The results of 8 groups corresponding to historical control data are presented in Annex 3 of the report.
We can find the number of fetuses/litters examined, the number of incompletely ossified/unossified skeletals (5th/6th sternebrae, other sternebrae, total number of affected fetuses/litters, mean % fetuses per litter with affected sternebrae).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Signs in relation to dose administration were limited to a low incidence of salivation or chin rubbing among females receiving 600 mg/kg/day.
Mortality:
no mortality observed
Description (incidence):
No mortality and no relevant clinical signs or signs of reaction to treatment were noted in treated females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
-Following the commencement of treatment on Day 6 of gestation, females in the 600 mg/kg/day group showed statistically significantly low mean body weight gain compared to Controls between Day 6 and 8 of gestation (3g versus 6g among Controls). A similar statistically significant decrease in mean body weight gain was evident among these females between Day 12 and Day 15 of gestation (17g versus 21g among Control), and mean weight gain from Day 15 to Day 20 of gestation was 0.93X Control although statistical significance was not attained for this difference. As a consequence, overall mean weight gain from Day 6 to Day 20 of gestation for females receiving 600 mg/kg/day was statistically significantly lower than Control (0.92X Control). There was no effect of treatment with Terpineol on the weight gain of females receiving 60 or 200 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Following the commencement of treatment on Day 6 of gestation, mean food intake among females in the 600 mg/kg/day group was slightly lower than Control throughout the treatment period, with statistical significance attained for the differences during Days 6-17 of gestation. Mean food intake for females in the 60 or 200 mg/kg/day groups was unaffected by treatment with Terpineol.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At scheduled termination on Day 20 of gestation, the mean gravid uterine weight of females in the 600 mg/kg/day group was slightly lower than Control (0.94X) and when overall mean body weight gain was adjusted for the contribution of the gravid uterus, net body weight gain during gestation was also lower than Control (0.85X); these differences from Control did not, however, attain statistical significance. Mean gravid uterine weight and net mean body weight gain were similar to Control for females receiving 60 or 200 mg/kg/day and no effect of treatment was inferred.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- There were no treatment-related macroscopic abnormalities detected at scheduled termination at any dose level investigated.
- Two females in the 200 mg/kg/day group (No’s. 47 and 49) were noted to have a misshapen liver with a raised area protruding through the diaphragm, resulting in a diaphragmatic hernia; these abnormalities were deemed to be congenital and unrelated to treatment with Terpineol.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
- With the exception of one female in the 200 mg/kg/day group (No. 52) which had a total litter resorption, all females were found to be pregnant with live young at scheduled termination on Day 20 of gestation.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg/day marginally low mean male and female fetal weight was apparent, with statistical significance attained for mean female fetal weight when compared to Control (0.95X), resulting in marginally but statistically significantly low overall mean fetal weight (0.96X Control). In addition, mean placental weight in this dose group was slightly low (0.91X Control) with differences attaining statistical significance. Mean placental, litter and fetal weights at 60 or 200 mg/kg/day were unaffected by maternal treatment with Terpineol.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment with Terpineol.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 600 mg/kg/day there was a slightly higher incidence of incompletely ossified or unossified 5th and/or 6th sternebrae compared to concurrent control and the Historical Control Data range. In the absence of any related abnormalities this finding was considered to reflect a minor delay in development, indicated by the marginally decreased mean fetal body weight in this group, rather than a direct effect of treatment on fetal development.
Visceral malformations:
no effects observed
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 7.8.2/2: Clinical signs – group distribution of observations 

Days 0 - 20

Number of animals affected

Group / sex

1F

2F

3F

4F

Category

Observations

Number in group

20

20

20

20

Coat

Hair loss, forelimbs

 

1

2

4

6

Table 7.8.2/3: Signs associated with dosing - group distribution of observations

Sign

Day number

Group/sex and number of animals showing sign

1F

2F

3F

4F

Behaviour

 

 

Chin Rubbing

17

0

0

0

3

 

 

18

0

0

0

7

 

 

19

0

0

0

6

Salivation

7

0

0

0

3

 

 

8

0

0

0

7

 

 

11

0

0

0

1

 

 

12

0

0

0

2

 

 

13

0

0

0

2

 

 

14

0

0

0

2

 

 

15

0

0

0

1

Table 7.8.2/4: Food consumption - group mean values (g/animal/day) during gestation

 

 

 

Group/Sex

 

Day 0-2

Day3-5

Day 6-9

Day 10-13

Day 14-17

Day 18-19

Statistical test

 

Av

Av

Wi

Wi

Wi

Wi

1F

Mean

25

26

23

25

27

26

 

SD

2.0

2.4

2.5

3.1

2.4

2.5

 

N

20

20

20

20

20

20

2F

Mean

25

26

24

25

28

27

 

 

SD

1.9

2.0

1.7

2.1

2.3

3.2

 

 

N

20

20

20

20

20

20

 

3F

Mean    

25

26

22

25

28

28

 

 

SD

2.3

2.3

1.5

2.3

2.3

3.2

 

 

N

19

19

19

19

19

19

 

4F

Mean

24

25

20**

23**

25*

25

 

 

SD

2.1

2.1

1.8

2.0

2.1

2.4

 

 

N

20

20

20

20

20

20

 

Table 7.8.2/5: Organ weights - group mean unadjusted and adjusted values (g)

Group/Sex

 

Terminal Body Weight

Liver weight

 

Unadjusted Means

Statistical test:

 

Wi

 

 

1F

Mean

420

18.17

 

 

SD

20

1.47

 

 

N

20

20

 

2F

Mean

425

18.48

 

 

SD

18

1.45

 

 

N

20

20

 

3F

Mean    

420

18.37

 

 

SD

23

1.44

 

 

N

19

19

 

4F

Mean

404*

19.14

 

 

SD

18

1.56

 

 

N

20

20

 

Adjusted Means

Statistical test:

 

 

Wi

 

1F

Mean

 

18.02

 

2F

Mean

 

18.06

 

3F

Mean

 

18.22

 

4F

Mean

 

19.84**

 

Table 7.8.2/6: Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g)

 

 

Group/Sex

 

Body weight on Day 6

Terminal Body weight on Day 20

Body weight Change Days 6-20

Gravid Uterine Weight

Adjusted Body weight Day 20

Adjusted Body weight Change Days 6-20

Statistical test

 

Av

Wi

Wi

Wi

Wi

Wi

1F

Mean

292

420

128

95

325

33

 

SD

11.1

20.4

13.6

10.4

16.9

8.8

 

N

20

20

20

20

20

20

2F

Mean

294

425

131

95

330

37

 

 

SD

10.1

18.1

13.2

11.6

12.8

8.3

 

 

N

20

20

20

20

20

20

 

3F

Mean    

291

420

129

94

326

35

 

 

SD

12.9

11.9

14.5

14.6

19.4

10.3

 

 

N

19

19

19

19

19

19

 

4F

Mean

286

404*

118*

89

315

28

 

 

SD

11.7

18.1

11.6

11.9

14.6

8.6

 

 

N

20

20

20

20

20

20

 

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) of Terpineol multiconstituent was considered to be 600 mg/kg bw/day for maternal toxicity and 600 mg/kg bw/day for developmental toxicity in Sprague-Dawley rats.
Executive summary:

In a GLP-compliant prenatal developmental toxicity study performed according to OECD guideline 414, Terpineol multiconstituent diluted in corn oil was administered by gavage to groups of mated female Sprague-Dawley rats (20 mated females/dose) at the dose levels of 0, 60, 200, 600 mg /kg bw/ day from Days 6 to 19 after mating. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Detailed observations were recorded daily at the following times in relation to dose administration. A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health. The weight of each adult was recorded on Days 0, 3 and and then daily from Days 6 to 20 after mating. The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

On Day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterine weight, number of implantations, live and dead fetuses, early and late resorptions and corpora lutea were recorded. Gross evaluation of the placenta was also performed.

Fetuses were sexed, weighed and examined for external, soft tissue and skeletal malformations.

With the exception of one female in the 200 mg/kg bw/day group (No. 52) which had a total litter resorption, all females were found to be pregnant with live young at scheduled termination on Day 20 of gestation. No mortality was observed.

At scheduled termination on Day 20 of gestation, the adjusted mean liver weight of females receiving 600 mg/kg bw/day was significantly higher than Control (1.10X Control). There were no treatment-related macroscopic abnormalities detected. No relevant clinical signs or signs of reaction to treatment were noted in treated females. Females receiving 60 or 200 mg/kg bw/day showed no treatment-related changes in clinical condition, body weight performance, food intake, liver weight or macropathology.

There was no effect of maternal treatment with Terpineol multiconstituent at any dose level investigated on litter data. Sex ratio, as assessed by the percentage of males per litter, was generally comparable in all groups and in line with expectations. Embryo-fetal growth was slightly reduced by maternal treatment at 600 mg/kg bw/day.

It was considered that there was no adverse effect of maternal treatment on embryo-fetal development; the incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment with Terpineol multiconstituent.

In the 600 mg/kg bw/day group there was a slightly higher incidence of incompletely ossified or unossified 5th and/or 6th sternebrae compared to concurrent control and the Historical Control Data range.

It was considered that this minor finding did not constitute an adverse effect on development.

On the basis of the results obtained in this study, the dosage of 600 mg/kg bw/day was considered to be the NOAEL (No Observed Adverse Effect Level) for maternal toxicity. The NOAEL for developmental toxicity was considered to be 600 mg/kg bw/day.