Exo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data from peer reviewed journal

Data source

Reference

Reference Type:

publication

Title:

Developmental toxicity of d- camphor in New Zealand white (new) rabbits.

Author:

U.S. National Toxicology Program (NTP)

Year:

1992

Bibliographic source:

U.S. National Toxicology Program (NTP), 1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

Reproduction and developmental toxicity evaluation of d-camphor administered by gavage to new zealand white (new) rabbits on gestational days 6 through 19

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(+)-bornan-2-one

EC Number:

207-355-2

EC Name:

(+)-bornan-2-one

Cas Number:

464-49-3

Molecular formula:

C10H16O

IUPAC Name:

1,7,7-Trimethylbicyclo[2.2.1]heptan-2-one

Details on test material:

- Name of test material (as cited in study report):d-camphor
- Molecular formula : C10H16O
- Molecular weight : 152.235g/mol
- Substance type:Organic
- Physical state: Solid

Specific details on test material used for the study:

- Name of test material (as cited in study report):d-camphor
- Molecular formula : C10H16O
- Molecular weight : 152.235g/mol
- Substance type:Organic
- Physical state: Solid

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

female

Details on test animals or test system and environmental conditions:

Details on test animals and env. conditions
TEST ANIMALS
- Source: Hazelton Research Products, I n c . , Denver. PA
- Age at study initiation: (P) approximately 6 mos
- Weight at study initiation: Females: 2490-4440g
- Fasting period before study: No data available
- Housing: Animals were individually hosed in stainless steel cage with mesh flooring (Hoeltge. In c .Cincinnat,, OH).
- Use of restrainers for preventing ingestion (if dermal):no
- Diet (e.g. ad libitum): Purina certified Rabbit chow@ (#5322) pellets , ad libitum
- Water (e.g. ad libitum): deionized/filtered water ad libitum
- Acclimation period:14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):18.33-18.66°C
- Humidity (%):58%-64%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): lights were on from 07.00h to 19.00h.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Details on exposure
PREPARATION OF DOSING SOLUTIONS:
Test materials dissolved in corn oil. The corn oil used i n the dose formulations contained less than 2 mill equivalents of peroxide/kg corn oil.
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material dissolved in corn oil and stable throughout the study.
- Concentration in vehicle: 0, 50,200,400mg/kg bw/day
- Amount of vehicle (if gavage): 2ml/kg bw
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

- M/F ratio per cage:A artificially inseminated female animals were used
- Length of cohabitation: No data available
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:The day of insemination was designated as
Gestational day (gd) 0

- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)]No data available
- After successful mating each pregnant female was caged (how): No data available
- Any other deviations from standard protocol:No data available

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 days ( gestational days 6-19)

Frequency of treatment:

daily

Details on study schedule:

No data available

Doses / concentrations

Remarks:

0, 50, 200, 400 mg/kg bw/day

No. of animals per sex per dose:

Total:104
0 mg/kg bw/day:26
50mg/kg bw/day:26
200mg/kg bw/day:26
400 mg/kg bw/day:26

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The designated dose level were based upon Preliminary dose rang – finding study.
- Rationale for animal assignment (if not random):
- Other:

Positive control:

No data available

Examinations

Parental animals: Observations and examinations:

Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS:

Time schedule: Animals were observed daily before (gd 0-5). During (gd 6-19). and after (gd 20-30) dosing
For clinical signs of toxicity .

BODY WEIGHT: Yes
Time schedule for examinations: animals were weighed on the mornings of gd 0, 6 through 19,25 and 30
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes Food was weighed on gd 0,3,6,9,12,15,18,19,22,25,28 and 30.
Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Time schedule for examinations:

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

No data available

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded. No data available

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies were observed
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:No data available

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:No data available

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]No data available
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]The animals were killed with an injection of sodium pentobarbital (Barber Veterinary Supply. Fayetteville NC) in the marginal ear vein on gd 30


GROSS NECROPSY: yes
At gross necropsy Liver and intact uterus was weighed and corpora lutea were counted. Uterine contents were examined to determine the number of implantation sites, resorptions, dead foetuses and live foetuses. Uteri which had no visible implantation sites was stained with ammonium sulphide (10%) to detect very early resorptions

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE:live foetuses were dissected from the uterus and also anesthetized with sodium pentobarbital.

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY: yes
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

General Linear Models (GLM) procedures were applied for the analyses of variance (ANOVA) of maternal and fetal parameters. Prior to GLM analysis ,an arcsine-square root transformation was performed on a litter- derived percentage data (Snedecor and Cochran. 1967) and Bartletts test for homogeneity of variance was performed on all data to be analysed by ANOVA (Winer. 196'2). GLM analysis determined the significance of dose-response relationships and the significance of dose effects. Replicate effects and dose x replicate interactions. When ANOVA revealed a significant (p<0.05) dose effect. Williams' and Dunnett's Multiple Comparison Tests (Williams, 1971: 1972: Dunnett. 1955: 1964) compared CAM-exposed to control groups. One-tailed tests were used for all pairwise comparisons except maternal body and organ weights. Maternal food consumption. fetal body weight and percent males per litter. Non-significant (p>0.05) dose x replicate effects on selected fetal parametric measures were considered justification for pooling data across replicates for nonparametric analysis on related measures. When a significant (p<0.05#) dose x replicate' interaction occurred, the data for that endpoint and f0.r any related nominal scale data were analysed separately for dose effects within each replicate in the study. As well as for all replicates combined. Nominal scale measures were analysed by a x2 test for independence and by a test 'f0.r linear trend on proportions. When x2 test showed significant group differences. a one tailed
Fisher's exact probability test was used for pairwise comparisons of CAM and control groups.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There were no dose related maternal deaths during the study, but two does were removed from the 400 mg/kg/day dose group because of dosing error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal body weight before, during and after treatment period in the treated animals was also comparable to control weights at all gestational ages.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption in all dose -treated groups was similar to control throughout the study

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

reproductive performance

Remarks on result:

other: No maternal toxicity was observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

Live litter size were unaffected .

Body weight and weight changes:

no effects observed

Description (incidence and severity):

average fetal body weight were unaffected

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No external ,skeletal and visceral malformations were observed .

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

No incidence of anatomical variation or defects were observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

viability

body weight and weight gain

gross pathology

Remarks on result:

other: No developmental toxicity was observed at given dose level

Target system / organ toxicity (F1)

Critical effects observed:

no

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Maternal toxicity in New Zealand white rabbits exposed to d- Camphor on gestational day 6 through 19

	d- Camphor (mg/kg/day)
	0	50	200	400
Subjects
Total treated	26	26	26	26
No . removed or dead^e	0	0	0	2
No. (%) pregnant at sacrifice	23(88)	20(77)	21 (81)	20(83)
Maternal body weight gain (g)^a.b
Treatment period (gd 6 to 19)	165±38$	144± 22	157±32	67 ± 33
Gestation period (gd 0 to 30)	529± 36	577 ± 43	533 ± 44	506 ± 46
Corrected gestation wt. gain^c	81± 54	67± 59	41 ± 58	11 ± 55
Gravid uterine weight^a	474 ± 33	509± 33	492 ± 24	495 ± 30
Maternal liver weight^a.b
Absolute (g) relative (% body weight)	108± 4.7 2.7 ± 0.1	114 ± 6.2 2.9 ±0.1	114 ± 4.2 2.8 ± 0.1	111 ± 5.9 2.8 ±0.1
Relative food consumption^a.b(g/kg /day)
Gd 0 to 6	50.4 ± 2.3	53.9 ± 2.4	56.3 ±2.2	54.2 ± 1.6
Gd 6 to 9	37.1 ± 3.2	37.3 ± 3.2	37.2± 2.9	31.1 ±2.8
Gd 9 to 12	48.2 ±2.5	48.1 ± 2.0	48.6± 2.1	43.7 ±2.0
Gd 12 to 15	34.9 ±2.7	35.0 ±3.0	34.8 ±3.1	30.8 ± 2.7
Gd 15 to 19	33.4 ±3.1	30.4 ± 3.0	34.2 ±2.8	28.3 ±3.3
Gd 19 to 25	44.1 ±1.9	42.7 ±1.2	43.3 ±2.7	43.2 ±2.1
Gd 25 to 30	35.8 ±2.2	36.5 ± 1.7	35.0 ±2.0	35.9 ±2.2

$Test for Liner Trend .p<0.05

A includes all dams pregnant at sacrifice. Mean ± SEM.

B Maternal gestational and corrected weight changes and relative organ weight were calculated using body weight at the time of sacrifice an gd 30

C Weight gain during gestation minus gravid uterine weight.

D body weight was recorded in morning of each designated gestational day. Relative food intake was calculated using these weights.

E Two does were removed in the 400mg/kg bw dose group because of gavage error.

Results of the Uterine Examination of New Zealand White Rabbits

Exposed to d-Camphor on Gestational Days 6 Through 19

	D – camphor
	0	50	200	400
All litters (no.)^a	23	20	21	20
No corpora lutea per doe ^c	9.2±0.6	9.4±0.6	10.1±0.7	9.2 ±0.5
No. implantation sites per litter^c	6.9 ±0.6	7.4 ±0.7	7.1 ±0.5	7.1±0.6
% resporption per litter ^c	4.7±1.8	5.9 ±2.9	5.1± 1.8	5.6±2.1
% litters with one or more resorption	26	25	33	30
%late fetal death per litter^c	0.9 ±0.6	0.8±0.6	0.0±0.0	2.3±1.6
% litters with one or more late fetal death	9	10	0	10
% nonlive implants per litter^c d	5.6±1.8	6.7 ±3.1	5.1±1.8	7.8±2.4
% litters with one or more nonlive implants^d	35	25	33	40
% litters with 100% nonlive implants^d	0	0	0	0
% adversely affected implants per litter^c e	8.1±2.3	8.4± 3.3	5.1±1.8	8.3±2.4
% litters with one or more adversely affected implants^e	43	30	33	45
No . live litters^b	23	20	21	20
No. live foetuses per litter^c	6.5±0.6	6.7 ±0.5	6.7±0.5	6.5±0.6
Av. Fetal body wt. (g) per litter^c
Male foetuses	51.9±1.6	54.2±1.7	51.8± 1.6	53.3± 1.5
Female foetuses	49.9 ±1.4	53.7±1.4	52.5±2.0	53.3±1.4
% male foetuses per litter^c	49 ±6	51±5	44± 5	55±6
Malformations^b
% foetuses malformed per litter^c
Both sexes	2.5±1.8	1.7 ±1.7	0.0±0.0	0.5±0.5
Male foetuses	1.2 ±1.2	2.6±2.6	0.0 ±0.0	0.0 ±0.0
Female foetuses	4.8 ±4.8	0.0±0.0	0.0±0.0	0.8 ±0.8
Variation^b
% fetuse with variations per litter ^c	61.5±6.9	54.0±8.6	54.3±8.2	60.4 ±6.8
% litter with one or more variations per litter	91	80	86	95

a Includes all does pregnant at sacrifice ; litter size = no. implantation site per doe.

b Includes only does with live foetuses: litter size = no. live fetuses per doe.

c Mean±SEM.

d Non live = resorptions and late fetal deaths.

e Adversely affected = non live and malformed.

Morphological Abnormalities Observed i n New Zealand White Rabbit Fetuses Following Maternal

Exposure to d-Camph or on Gestational Days6through 19: Listing by Defect

	D – camphor
	0	50	200	400
Total No. of Fetuses Examined^b	149	134	140	129
Total No. of Litters Examined^d	23	20	21	20
Any malformation
No of fetuese with any malformations^c	2	1	0	1
% fetuses with any malformations	1.3%	0.7%	0.0%	0.8%
No of litters with any malformations^e	2	1	0	1
% litters with any malformations	8.7%	5.0%	0.0%	5.0%
External malformation
No of fetuese with external malformations^c	0	0	0	0
% fetuses with external malformations	0.0%	0.0%	0.0%	0.0%
No of litters with external malformations^e		0.0%	0.0%	0.0%
% litters with external malformations	0.0%	0.0%	0.0%	0.0%
Skeletal malformation
No of fetuese with skeletal malformations^c	1	0.0%	0.0%	0.0%
% fetuses with skeletal malformations	0.7%	0.0%	0.0%	0.0%
No of litters with skeletal malformations e	1	0.0%	0.0%	0.0%
% litters with skeletal malformations	4.3%	0.0%	0.0%	0.0%
Fused sternebrae : III and IV	1
Visceral malformation
No of fetuese with visceral malformations^c	1	1	0 1
% fetuses with visceral malformations	0.7%	0.7%	0.0%	0.8%
No of litters with visceral malformations e	1	1	0	1
% litters with visceral malformations	4.3%	5.0%	0.0%	5.0%
Left subclavian Arising from the apparent ductus arteriosis	1
reversed Aorta	1
Gall bladder: small and round		1		1
Any variation
No of fetuese with any variations^c	95	75	77	79
% fetuses with any variations	63.8%	56.0%	55.0%	61.2%
No of litters with any variations	21	16	18	19
% litters with any variations	91.3%	80.0%	85.7%	95.0%
External variations
No of fetuese with external variations^c	0	0	3	0
% fetuses with external variations	0.0%	0.0%	2.1%	0.0%
No of litters with external variations	0.0%	0.0%	1	0.0%
% litters with external variations	0.0%	0.0%	4.8%	0.0%
Hematoma : most or All of body			3
Skeletal variations
No of fetuese with Skeletal variations^c	92	67	71	71
% fetuses with Skeletal variations	61.7%	50.0%	50.7%	55.0%
No of litters with Skeletal variations	20	13	16	16
% litters with Skeletal variations	87.0%	65.0%	76.2%	80.0%
Rib on lumbar I : Bilateral full	71	52	55	52
Left full	9	2	8	8
Right full	4	7	1	2
Bilateral rudimentary	3	5	4	6
Left rudimentary	1	1	2	3
Right rudimentary	8		3	2
Visceral variations
No of fetuese with Visceral variations^c	4	8	7	10
% fetuses with Visceral variations	2.7%	6.0%	5.0%	7.8%
No of litters with Visceral variations	3	5	6	6
% litters with Visceral variations	13.0%	25.0%	28.6%	30.0%
Agenesis of the innominate Artery abnormal number of papillary muscles :2 in right ventricle			1
4 in right ventricle	5		1	6
5 in right ventricle	1
6 in right ventricle			1
Bifurcated papillary muscle : 1 in left ventricle			1
1 in left ventricle
2 in left ventricle	1	4	2	1
Trifurcated papillary muscles : 1 in right ventricle			1
Right kidney slightly displaced and ureter shorter than normal	1
gall bladder: Half normal size	2			1
Half normal size with a constriction at the fundus			1
Twice normal size				1

A incidence of individual defects is expressed a s the number of individual fetuses exhibiting defect. Thus a single fetus may be represented more than once in listing defects. Approximately 50% of the fetuses were decapitated prior to skeletal staining: heads were fixed in Bouin's solution and examined using a free-hand section method.

B Only live fetuses were examined.

C Fetuses with one or more malformations/variations.

D Includes only litters with live fetuses.

E Litters with one or more malformed/variant fetuses

Applicant's summary and conclusion

Conclusions:

No Observed Adverse Effect Level (NOAEL) for maternal and foetal toxicity was considered to be 400 mg/kg/day. When female new Zealand white rabbits were treated with d-camphor (464-49-3) orally.

Executive summary:

The reproductive and developmental toxicity study ofd-camphor(464-49-3) was performed in new Zealand white rabbits.104 rabbits were divided as 26/ dose group. The test material dissolved in corn oil and administered in dose volume 2ml /kg in concentration0, 50, 200, 400 mg/kg bw/day by oral gavage routeon gestational day 6 through 19.The designated dose level were based upon

Preliminary dose rang – finding study. A artificially inseminated female animals were used and the day of insemination was designated as Gestational day (gd) 0. Animals were observed daily before (gd 0-5),during (gd 6-19) and after (gd 20-30) dosing for clinical signs of toxicity.Animalswere weighed on the mornings of gd 0, 6 through 19,25and 30.Food was weighed on gd 0,3,6,9,12,15,18,19,22,25,28 and 30. At necropsy,The animals were killed with an injection of sodium pentobarbital(Barber Veterinary Supply. Fayetteville NC)in the marginal ear vein on gd 30. Liver and intact uterus was weighed and corpora lutea were counted. Uterine contents were examined to determine the number of implantation sites, resorptions, dead foetuses and live foetuses. Uteri which had no visible implantation sites was stained with ammonium sulphide (10%) to detect very early resorptions.live foetuses were dissected from the uterus and also anesthetized with sodium pentobarbital.The foetuses were weighed, sexed and subjected to external, soft tissue or skeletal examinations.Half of the foetuses were decapitated prior to dissection, the heads were fixed in bouins solution and then examined by free hand sectioning technique . All fetal carcasses (50% without heads) were stained with alcian blue/ alizarin red S and examined for skeletal malformations

 

There were no dose related maternal deaths during the study, but two animals were removed from the 400 mg/kg/day dose group because of dosing error. Food consumption in all dose -treated groups was similar to control throughout the study. Maternal body weight before, during and after treatment period in the treated animals was also comparable to control weights at all gestational ages. Maternal weight gain during the dosing period tended to decrease with increasing dose. Maternal weight gain relative to control animals was reduced 13%, 5% and59%in the 50, 200 and 400 mg/kg/day dose groups respectively. Gravid uterine and absolute and relative maternal liver weights were similar to vehicle control values.

Results of the uterine examination revealed that test material was not developmental toxic effects even at the highest dose. No effect was seen for the percent resorption per litter. The percent late fetal deaths per litter , the percent non live implants per litter or the percent adversely affected implants per litter. The proportion of litters with one or more resorption , late fetal deaths, nonlive implants or adversely affected implants were not affected by dose exposure. Live litter size and average fetal body weight were also unaffected .No external , skeletal and visceral malformations were observed . No incidence of anatomical variation or defects were observed. HenceNo Observed Adverse Effect Level (NOAEL) for maternal and foetal toxicity was considered to be 400 mg/kg/day.When femalenew Zealand white rabbits were treated withd-camphor(464-49-3)orally.