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Toxicological information

Toxicity to reproduction

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 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 animals)

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
Description (incidence and severity):
Gravid uterine and absolute and relative maternal liver weights were similar to vehicle control values.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
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 deade

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. gainc

81± 54

67± 59

41 ± 58

11 ± 55

Gravid uterine weighta

474 ± 33

509± 33

492 ± 24

495 ± 30

Maternal liver weighta.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 consumptiona.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 litterc

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 litterc d

5.6±1.8

6.7 ±3.1

5.1±1.8

7.8±2.4

% litters with one or more nonlive implantsd

35

25

33

40

% litters with 100% nonlive implantsd

0

0

0

0

% adversely affected implants per litterc e

8.1±2.3

8.4± 3.3

5.1±1.8

8.3±2.4

% litters with one or more adversely affected implantse

43

30

33

45

No . live littersb

23

20

21

20

No. live foetuses per litterc

6.5±0.6

6.7 ±0.5

6.7±0.5

6.5±0.6

Av. Fetal body wt. (g) per litterc

 

 

 

 

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 litterc

49 ±6

51±5

44± 5

55±6

Malformationsb

 

 

 

 

% foetuses malformed per litterc

 

 

 

 

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

Variationb

 

 

 

 

% 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 Examinedb

149

134

140

129

Total No. of Litters Examinedd

23

20

21

20

Any malformation

 

No of fetuese with any malformationsc

2

1

0

1

% fetuses with any malformations

1.3%

0.7%

0.0%

0.8%

No of litters with any malformationse

2

1

0

1

% litters with any malformations

8.7%

5.0%

0.0%

5.0%

External malformation

 

No of fetuese with external malformationsc

0

0

0

0

% fetuses with external malformations

0.0%

0.0%

0.0%

0.0%

No of litters with external malformationse

 

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 malformationsc

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 malformationsc

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 variationsc

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 variationsc

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 variationsc

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 variationsc

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.