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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study is conducted according to OECD TG 414, without deviations that influence the quality of the results, in compliance with GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997
Reference Type:
publication
Title:
Developmental Toxicity Studies of Four Fragrances in Rats.
Author:
Christian, M.S., Parker, R.M., Hoberman, A.M., Diener, R.M.|and Api, A.M.
Year:
1999
Bibliographic source:
Toxicology Letters, 111: 169-174.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Information regarding the activity, identity, strength, composition and stability of the test article is on file with RIFM.
- Name of test material (as cited in study report): HHCB (>95% purity, in corn oil)
- Physical state: a clear viscous liquid
- Analytical purity: >95%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
female
TEST ANIMALS
- Source: Charles River Laboratories, Inc, Portage, Michigan
- Age at study initiation: approximately 66 days at arrival
- Weight at study initiation: 208 to 256 gr
- Fasting period before study: no
- Housing: individually, except during the cohabitation period. During cohabitation each pair of male and female rats was housed in the male rats's cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 26 (+/- 2 %)
- Humidity (%): 40% to 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Storage temperature of food: room temperature: no data


VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 5ml/kg bw
- Lot/batch no. (if required): 827AG
- Purity: not tested
Analytical verification of doses or concentrations:
no
Details on mating procedure:
no data
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 5 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
days 7 -17 of gestation
Frequency of treatment:
Daily
Duration of test:
approximately 4 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150 and 500 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were selected on the basis of a dosage-range study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
for viability: twice during acclimation and on Day of Gestation (DG) 0.
clinical observations: after dosage (DGs 7 through 17)

BODY WEIGHT: Yes
- Time schedule for examinations:
twice during acclimation
On DG 0
Daily during dosage and postdosage periods

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No (oral gavage)
- 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: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution
Continuous data (e.g., maternal body weights, body weight changes, feed consumption values and litter averages for percent male fetuses, percent
resorbed conceptuses, fetal body weights, fetal anomaly data and fetal ossification site data) were analyzed using Bartlett's Test of Homogeneity of
Variances and the Analysis'of Variance, when appropriate [i.e., Bartlett's Test was not significant (p>0.05)]. If the Analysis of Variance was significant (p≤0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (p≤0.05)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the ind vidual groups. If there were greater than 75% ties, Fisher's Exact Test was used to analyze the data.
Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described for the Kruskal-Wallis Test
Historical control data:
Historical control data were included in the report. These include reproductive indices, maternal necropsy observations, fetal external alterations, fetal soft tissue alterations, fetal skeletal alterations, fetal ossification sites. Period: June 1994 - June 1996

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The 500 mg/kg bw/day dosage group had four to nine (p -<0.01) rats with excess salivation (9 animals), urine-stained abdominal fur (7 animals), red or brown substance on the forepaws (4 animals) and alopecia (6 animals). Dams on 500 and 150 mg/kg bw/day showed statistically significant dosage-dependent reductions in maternal body weight gains for the entire dosage period (days 7 to 18 gestation), to 78% and 91% of body weight gain in controls, respectively. These reductions in weight gain reflected significant weight loss (-10.9 g compared to +10.5 g in the control group) on days 7 to 10 at 500 mg/kg bw per day, while on days 10 to 12 the weight gain was increased (+17.9 g compared to +11.8 g in the control group)At 150 mg/kg bw/day, a significantly reduced weight gain (+5.5 g) was also found on days 7 to 10. For the remainder of the study, body weight gains at 150 and 500 mg/kg bw per day were comparable to the control group values . Body weights were significantly reduced compared to controls on GD20 by 2.5, 3,3 and 4.6% for 50, 150 and 500 mg/kg bw/day, respectively.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Foetuses in the 500 mg/kg bw/day dosage group showed significantly reduced body weights, 3.24 g compared to 3.48 g in the control group (7% reduction). Significant increases in foetal incidences of skeleton (vertebral/rib) malformations were found (2.0 compared to 0 in the control group). In addition, significant increases in foetal incidences of incomplete ossification and/or no ossification of sternal centra and a significantly decreased number of ossification sites in the metatarsals were seen at 500 mg/kg bw/day (5.4 compared to 0.5 in the control group).For litters, these incidences were also increased, 14.3 compared to 0 in the control group for the skeleton malformations, and 23.8 compared to 4.0 in the control group for the ossification problems. No other Caesarean-sectioning and litter parameters were affected by administration of HHCB to the dams at doses as high as 500 mg/kg bw/day. The litter averages for corpora lutea, implantations, litter sizes, live/dead foetuses, early and late resorptions, percent resorbed conceptuses and percent live male/female foetuses were comparable among the four dosage groups and did not differ significantly. No dam had a litter consisting of only resorbed conceptuses and there were no dead foetuses.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

The effects were decreased mean bodyweights in mid and high-dose dams and axial skeleton (vertebral/rib malformations in high dose offspring.  

Table 1 Maternal body weights
DOSAGE GROUP I II III IV
DOSAGE (MG/KG/DAY)a 0 (vehicle) 50 150 500
RATS TESTED N 25 25 25 25
PREGNANT N 25 24 24 21
MATERNAL BODY WEIGHT (G)
DAY 0 MEAN±S.D. 241,5 ± 8,4 232,9 ± 9,60 ** 232,2 ± 9,5 ** 233,6 ± 9,1 **
DAY 7 MEAN±S.D. 275,2 ± 9,5 266,2 ± 10,40 269,2 ± 12,90 270,2 ± 14,1
DAY 8 MEAN±S.D. 276,3 ± 10,9 263,8 ± 10,9 ** 266 ± 13,3 ** 259,8 ± 14,1 **
DAY 9 MEAN±S.D. 280,1 ± 10,2 269,2 ± 11,20 ** 269,2 ± 12,4 ** 254,7 ± 17,4 **
DAY 10 MEAN±S.D. 285,7 ± 10,1 275,1 ± 12,10 214,8 ± 13,20 259,3 ± 18,5
DAY 11 MEAN±S.D. 291,6 ± 10,5 281,8 ± 12,90 * 282,1 ± 14,30 * 269,4 ± 18,3 **
DAY 12 MEAN±S.D. 297,8 ± 11,3 286,9 ± 12,90 * 286,5 ± 15,40 ** 277,2 ± 16,1 **
DAY 13 MEAN±S.D. 301,3 ± 10,7 291,6 ± 12,30 * 290,8 ± 14,90 ** 202,7 ± 14,2 **
DAY 14 MEAN±S.D. 304,2 ± 11,4 300,8 ± 14,90 295,4 ± 15,20 * 288,9 ± 14,7 **
DAY 15 MEAN±S.D. 313,7 ± 12,5 307,3 ± 12,70 303,8 ± 17,10 * 295,7 ± 14,8 **
DAY 16 MEAN±S.D. 326,6 ± 14,6 316,5 ± 15,40 * 313,6 ± 18,70 ** 306,8 ± 16 **
DAY 17 MEAN±S.D. 340,5 ± 16,8 330 ± 16,30 * 326,5 ± 19,80 ** 321,3 ± 16 **
DAY 18 MEAN±S.D. 355 ± 18,9 347,7 ± 16,70 341,6 ± 21,30 * 333,2 ± 17,5 **
DAY 19 MEAN±S.D. 372,8 ± 21,4 364 ± 17,40 357,4 ± 23,70 * 349,5 ± 17,3 **
DAY 20 MEAN±S.D. 391,9 ± 21,6 362,2 ± 19,20 ** 379,1 ± 27,20 * 373,7 ± 21,2 **
DAY - DAY OF GESTATION
a. Dosage occurred on days 7 through 17 of gestation
* Significantly different from the vehicle control group value (p ≤ 0,05)
**  Significantly different from the vehicle control group value (p ≤ 0,01)

The 500 mg/kg bw/day dosage group had four to nine (p -< 0.01) rats with excess salivation (9 animals), urine-stained abdominal fur (7 animals), red or brown substance on the forepaws (4 animals) and alopecia (6 animals). Dams on 500 and 150 mg/kg bw/day showed statistically significant dosage-dependent reductions in maternal body weight gains for the entire dosage period (days 7 to 18 gestation), to 78% and 91% of body weight gain in controls, respectively. These reductions in weight gain reflected significant weight loss (-10.9 g compared to +10.5 g in the control group) on days 7 to 10 at 500 mg/kg bw per day, while on days 10 to 12 the weight gain was increased (+17.9 g compared to +11.8 g in the control group)At 150 mg/kg bw/day, a significantly reduced weight gain (+5.5 g) was also found on days 7 to 10. For the remainder of the study, body weight gains at 150 and 500 mg/kg bw per day were comparable to the control group values . Body weights were significantly reduced compared to controls on GD20 by 2.5, 3,3 and 4.6% for 50, 150 and 500 mg/kg bw/day, respectively.

Foetuses in the 500 mg/kg bw/day dosage group showed significantly reduced body weights, 3.24 g compared to 3.48 g in the control group (7% reduction). Significant increases in foetal incidences of skeleton (vertebral/rib) malformations were found (2.0 compared to 0 in the control group). In addition, significant increases in foetal incidences of incomplete ossification and/or no ossification of sternal centra and a significantly decreased number of ossification sites in the metatarsals were seen at 500 mg/kg bw/day (5.4 compared to 0.5 in the control group). For litters, these incidences were also increased, 14.3 compared to 0 in the control group for the skeleton malformations, and 23.8 compared to 4.0 in the control group for the ossification problems. No other Caesarean-sectioning and litter parameters were affected by administration of HHCB to the dams at doses as high as 500 mg/kg bw/day. The litter averages for corpora lutea, implantations, litter sizes, live/dead foetuses, early and late resorptions, percent resorbed conceptuses and percent live male/female foetuses were comparable among the four dosage groups and did not differ significantly. No dam had a litter consisting of only resorbed conceptuses and there were no dead foetuses.

Applicant's summary and conclusion

Conclusions:
The test material was not more toxic to the conceptus than to the dam. Therefore, unclassified according to Annex VI Directive 67/548/EEC, Section 4.2.3.3 "comments regarding the categorisation of substances toxic to reproduction"
Executive summary:

Based on a range-finding study (supporting study, Christian et al., 1997, 1999), HHCB (purity not reported in report, confirmed to be >95% pure undiluted material, IFF personal communication with RIFM) in corn oil was administered by gavage to groups of 25 female Sprague-Dawley rats on days 7 through 17 of presumed gestation at dosages of 0, 50, 150 and 500 mg/kg bw/day in a GLP compliant study. The dams were observed for signs of toxicity and body weights and feed intake were recorded. On day 20 of gestation, the dams were sacrificed and gross necropsy was performed. The number of corpora lutea in the ovaries was recorded and the uteri were examined for pregnancy, number and distribution of implantations, live and dead foetuses and early and late resorptions and the placenta were examined. All foetuses were weighed and examined for sex and gross external abnormalities. One half of the foetuses in each litter were examined for soft tissue alterations. The remaining foetuses were examined for skeletal alterations.

The 500 mg/kg bw/day dosage group had four to nine (p ≤ 0.01) rats with excess salivation (9 animals), urine-stained abdominal fur (7 animals), red or brown substance on the forepaws (4 animals) and alopecia (6 animals). Dams on 500 and 150 mg/kg bw/day showed statistically significant dosage-dependent reductions in maternal body weight gains for the entire dosage period (days 7 to 18 gestation), to 78% and 91% of body weight gain in controls, respectively. These reductions in weight gain reflected significant weight loss (-10.9 g compared to +10.5 g in the control group) on days 7 to 10 at 500 mg/kg bw per day, while on days 10 to 12 the weight gain was increased (+17.9 g compared to +11.8 g in the control group). At 150 mg/kg bw/day, a significantly reduced weight gain (+5.5 g) was also found on days 7 to 10. For the remainder of the study, body weight gains at 150 and 500 mg/kg bw per day were comparable to the control group values. Body weights were significantly reduced compared to controls on GD20 by 2.5, 3,3 and 4.6% for 50, 150 and 500 mg/kg bw/day, respectively.

Foetuses in the 500 mg/kg bw/day dosage group showed significantly reduced body weights, 3.24 g compared to 3.48 g in the control group (7% reduction). Significant increases in foetal incidences of skeleton (vertebral/rib) malformations were found (2.0 compared to 0 in the control group). In addition, significant increases in foetal incidences of incomplete ossification and/or no ossification of sternal centra and a significantly decreased number of ossification sites in the metatarsals were seen at 500 mg/kg bw/day (5.4 compared to 0.5 in the control group). For litters, these incidences were also increased, 14.3 compared to 0 in the control group for the skeleton malformations, and 23.8 compared to 4.0 in the control group for the ossification problems. No other Caesarean-sectioning and litter parameters were affected by administration of HHCB to the dams at doses as high as 500 mg/kg bw/day. The litter averages for corpora lutea, implantations, litter sizes, live/dead foetuses, early and late resorptions, percent resorbed conceptuses and percent live male/female foetuses were comparable among the four dosage groups and did not differ significantly. No dam had a litter consisting of only resorbed conceptuses and there were no dead foetuses.

Based on a reduction in maternal body weight gains for the dosing period (days 7 to 18 of gestation), the maternal no-observable-adverse effects level (NOAEL) for HHCB was concluded to be 50 mg/kg bw/day. Based on a reduction in foetal body weight, increased incidences of foetal-skeletal (vertebral/rib) malformations, and decreased ossification of sternal centra and metatarsals seen at 500 mg/kg bw/day, the developmental NOAEL was 150 mg/kg bw/day. Because adverse effects on development occurred only at dosages that produced toxic effects in the dams, HHCB is likely not selectively toxic to development. This study was conducted in accordance with GLP and evaluated ICH Harmonized Tripartite Guideline stages C and D (Christian et al., 1997; 1999).