Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
developmental neurotoxicity
Remarks:
based on test guideline (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1995 - January 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is performed similar to OECD TG 426 and in compliance with GLP. The highest dose used does not induces toxicity and less neuropathological examinations have been performed when compared to the guideline.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
Deviations:
yes
Remarks:
less neuropathological examinations performed. highest dose no adverse effects.
Qualifier:
according to
Guideline:
other: International Conference on Harmonisation (ICH) Guideline on Detection of Toxicity to Reproduction for Medicinal Products
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD BR VAF/Plus strain
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK ltd, Manston Road, Margate, Kent
- Age at study initiation: (P) 8 - 10 weeks
- Weight at study initiation: (P) Females: 181 - 264g
- Fasting period before study: no data
- Housing: prior to parturation, F0 animals were housed four to a cage in suspended galvanised metal cages (Biotech) equipped with solid sides and wire grid front, back, floor and top. Each cage measured 26 cm wide, 55 cm deep and 26 cm hight.
On day 20 of pregnancy, F0 animals were rehoused in individual plastic breeding cages (North kent Plastics, RB3-R type) for birth and rearing of young. suitable nesting material was provided
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: From:day of arrival To: day 22 post partum
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Oral Gavage to pregnant and lactating dams, oral via milk to F1

PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil (no justification given)
- Concentration in vehicle: 0,4; 1,2; 4,0 mg/ml
- Amount of vehicle (if gavage): 0,5 ml/100gr bw
- Purity:not reported
Details on mating procedure:
No data. Animals were time mated with males from the same strain
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At specified intervals during the study, representative samples (approximately 20 ml) of freshly prepared test formulations were submitted by personnel for analysis. The test formulations were thoroughly mixed by vigorous shaking and duplicate sub-samples (1 ml) were analysed in accordance with the analytical procedure.
The mean concentrations of test formulations analysed during the toxicity study were within 6 % of the nominal concentration confirming the accuracy of preparation. The results also confirm that test formulations were true solutions and stable during ambient temperature storage for 24 hours, a period representing the maximum time from preparation to completion of dosing.
Duration of treatment / exposure:
Exposure period: From the 3rd week (day 14) of pregnancy through the weaning of the F1 offspring (day 21 post partum).
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until 9 weeks after selected from the F1 litters
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals (F1) in the study: 12 weeks
Remarks:
Doses / Concentrations:
2,6, and 20 mg/kg/day.
Basis:
actual ingested
by females (F0)
No. of animals per sex per dose:
28 females per dose group
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: no data
Parental animals: Observations and examinations:
Observations included surface righting reflex, pupil reflex, and, after maturity, accelarating rotarod test, passive avoidance test and assessment of reproductive capacity.
CAGE SIDE OBSERVATIONS: Yes



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations:
All animals were weighed on arrival (Day 1; batch A, Day 2; batch B) and on Days 2 (batch A), 7, 12, 14, 16, 18 and 20 post coitum, following completion of parturition onDay 0/1 and on Days 1, 7, 14 and 21 postpartum.


OTHER: food consumption (no feeding study)
Food consumption was recorded for each cage of animals from weighday to weighday between allocation to groups (on Day 12) and Day 20 post coitum. Following rehousing and birth of litters, food consumption was recorded on an individual basis from weighday to weighday commencing on Day 1 postpartum. Food consumption was not continued after Day 20 post coitum for animals which failed to give birth.
Individual consumption is presented for all periods as recorded. Group mean values are presented only for those periods for which data relevant to characterising any response to treatment have been generated. Group mean values are not presented for females between Day 14 and 20 postpartum since the intake is a combination of both the adult female and her litter.
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in F1 male parental generations: testis weight, epididymis weight,
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 offspring: number and sex of pups, weight, physical or behavioural abnormalities, stillbirths, live births


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

After parturition, the young were counted, sexed, weighed and examined for external abnormalities. On day 4 post partum the pups were weighed and all litters containing more than 8 pups were culled to 8 retaining, where possible 4 males and 4 females. During the pre-weaning period, all pups were examined to determine the age of reaching certain developmental stages by examining surface righting reflex, startle reflex, air righting reflex and pupil reflex. This F1 generation was also evaluated for behavioural effects by examining changes in motor coordination and balance, activity and avoidance. When the F1 generation reached approximately 84 days of age (having been continuously observed for signs of adverse health) they were mated one male to one female avoiding brother-sister pairings. The females were examined before and after mating to determine time of pregnancy, marked anomalies of the oestrous cycle, median pre-coital time, whether pregnancy had occurred and terminated and duration of pregnancy.
The offspring (F2 generation) were examined for abnormalities at parturition and periodically until day 21 post partum at which time the study was terminated
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving F1 animals after the last possible date of birth of F2 pups
- Maternal animals: All surviving animals after day 22 post partum (F0) and on or shortly after day 21 post partum (F1)


GROSS NECROPSY
Animals were examined externally and internally for abnormalities. Abnormal tissues were preserved at the discretion of the post mortem pathologist. Sex of the pups was confirmed by gonadal inspection. The uterus of each female which gave birth was visually inspected for implantation sites
and the number of sites was recorded. Uteri of apparently non-pregnant females were examined for evidence of implantation using a modified Salewski technique (Salewski, 1964). Testes (with epididymides) from males which failed to induce pregnancy were weighed and preserved but not examined further.
Postmortem examinations (offspring):
no data
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [22/21] days of age.
- These animals were subjected to postmortem examinations (macroscopic) as follows: Abnormal tissues were preserved at the discretion of the post mortem pathologist. Sex of the pups was confirmed by gonadal inspection.


Statistics:
Significance tests, employing analysis of variance followed by an intergroup comparison with the control, were performed on the following parameters : Food consumption, bodyweight change, litter data, sex ratios, pre-weaning development, post weaning behavioral tests, sexual maturation.

Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance , followed by Williams' test) or non-
parametric tests (Kruskal-Wallis followed by Shirley's test) were used to analyze the data, as appropriate. For litter data and pre-weaning development, the basic sampling unit was the litter and, due to the preponderance of non-normal distributions nonparametric analyses were routinely used.

All significant (p=<0.05) intergroup differences from the control are reported.

Where 75 % or more of the values for a given variable were the same, a Fishers' exact test was used, when considered necessary.

For the passive avoidance test, Day 1 times were analyzed using the Kruskal-Wallis test. Intergroup differences were made using Shirley's test. For Day 2, the number of animals showing maximum avoidance behavior were analyzed using Fisher's exact test.
Reproductive indices:
In assessing litter parameters, implantation loss was calculated from the formula:
[ (No. of implantation sites - total young born)/(No. of implantations)]* 100

Pup loss on completion of parturition was calculated as a percentage from the formula:
[(Total no. of young at birth - no. of live young)/(Total no. of young at birth)] *100

Pup loss at Day 4 prior to the cull was calculated as a percentage from the formula:
[(Total no. of young at birth - no. of live young at Day 4 pre-cull)/(Total no. of young at birth)] * 100

Sex ratios at birth will be calculated from the formula:
(Total no. of males)/(Total no. of young) * 100


at Day 4 pre-cull, where appropriate, the formula will be:
(No. of males at Day 4)/(Total no. of young at Day 4) * 100

At weaning the formula will be:.
(No. of males at weaning)/(Total no. of young at weaning) * 100



Offspring viability indices:
For litters derived from Fl adult animals, cumulative pup loss from birth was calculated from the formula:

[(Total no. of young at birth - no. of live young at Day x)/ (Total no. of young at birth)] * 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Occasional and isolated instances of salivation were noted after dosing amongst some treated animals but generally not among controls.

There were no other clinical signs noted that were considered to be related to treatment.

There were no mortalities.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

Following the start of treatment on Day 14 of pregnancy, there were no treatment-related effects on bodyweight gain prior to parturition or on the pattern of bodyweight change during lactation.

There were no treatment-related effects on food intake following the start of treatment or through to lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
no effects


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

The mean duration of pregnancy was not affected by treatment. There were no indications of any intergroup differences in the length of parturition.

ORGAN WEIGHTS (PARENTAL ANIMALS)
not examined

GROSS PATHOLOGY (PARENTAL ANIMALS)

There were no treatment-related changes in the incidence of minor abnormalities noted at post mortem examination of the females following weaning of their offspring.

Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Sex:
female
Basis for effect level:
other: Treatment of the pregnant rat during the peri and post natal period at dosages of HHCB up to 20 mg/kg/day was without adverse toxic effect.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
not specified
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
no effects

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs noted amongst the F1 adults that were considered to be related to treatment of the FO parent female.

There were no mortalities


BODY WEIGHT (OFFSPRING)

Mean weekly bodyweight gain of males and females from selection (nominal Week 4) was unaffected by treatment of the FO parent females.
During pregnancy and lactation, the pattern of bodyweight change of females that were derived from treated F0 parent females was similar to the controls.


SEXUAL MATURATION (OFFSPRING)

There were no intergroup differences in the mean age of attainment or bodyweight on completion of balano-preputial skinfold cleavage of males or vaginal opening of females that were considered attributable to treatment of the parent females.



GROSS PATHOLOGY (OFFSPRING)
Macroscopic post mortem examination:
The incidence of macroscopic changes noted at autopsy of adult males and females was low in all groups and considered to be unrelated to maternal treatment. No macroscopic changes were apparent amongst the low number of males and females that had unsuccessful matings to indicate any cause of infertility and, in particular. No males or females at the highest dosage appeared infertile.



OTHER FINDINGS (OFFSPRING)

Post weaning behavioural tests:
Treatment of parent females did not appear to impair the performance of their offspring on the
Rotarod, Actimat or Passive Avoidance tests. Results of the individual tests were as follows:

Rotarod:
There were no significant differences in maximum performance times between offspring derived from treated parent females and their respective controls.

Actimat:
Among both males and females, there were no significant differences in the times spent in the
different categories of activity between animals derived from treated parent females and controls.

Passive avoidance test:
Pre-shock entry times (Day 1): For both sexes, although pre-shock entry times were variable, there were no significant differences between animals derived either from treated parent females or controls.
Post shock (Day 2): The majority of animals in each group, regardless of sex or derivation, showed maximum avoidance behaviour.

Mating performance and duration of pregnancy:
Treatment of the parent females had no adverse effect upon the mating performance of their
offspring as indicated by the time taken to successful mating (median pre-coital time),
pregnancy rate and cytology of the vaginal smear on the day of mating. The mean duration of pregnancy was similar in all groups.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Reproductive effects observed:
not specified

No effects on F0 females. No effects on F1 males or females.
No effects on F1 male or female reproductive performance or
on F2 males or females.

Conclusions:
Exposure to test material by gavage to dams had no effects at any dose and exposure to F1 offspring through mother's milk had no effects on behavior or reproductive performance. F2 pups were without adverse effects.
Executive summary:
In a study designed to determine the effects of HHCB (purity not reported in report, confirmed to be >95% pure undiluted material, IFF personal communication with RIFM) on the neonate when exposed through nursing, HHCB was administered at dosages of 0, 2, 6 or 20 mg/kg bw/d once daily by gavage in corn oil to groups of 28 time-mated rats (Crl:CD BR VAF/Plus strain) from Day 14 of pregnancy (end of organogenesis) through to weaning on Day 21 post partum. The females were allowed to litter and rear their young to weaning. From these litters, selected offspring were retained (24 males and females per group) to maturity and assessed for behavioural changes and reproductive capacity. The F1 generation was only exposed to HHCB in utero during the perinatal phase and through transfer in the milk of the lactating dams. The exposure of the F1 foetuses through mother's milk can be estimated based on a pharmacokinetic study in pregnant/lactating rats (Hawkins et al., 1996a reported in section 4.1.2.1.1, under heading: Animal milk studies). HHCB levels in mother’s milk up to 2.28 and 32.8 mg HHCB equivalents (including also metabolites)/l were found at oral doses of 2 and 20 mg 14C-HHCB/kg bw per day, respectively, to the dams (see Table 4.14). Actual intakes cannot be determined because milk consumption during nursing was not measured.

After parturition, the young were counted, sexed, weighed and examined for external abnormalities. On day 4 post partum the pups were weighed and all litters containing more than 8 pups were culled to 8 retaining, where possible 4 males and 4 females. During the preweaning period, all pups were examined to determine the age of reaching certain developmental stages by examining surface righting reflex, startle reflex, air righting reflex and pupil reflex. This F1 generation was also evaluated for behavioural effects by examining changes in motor coordination and balance, activity and avoidance. When the F1 generation reached approximately 84 days of age (having been continuously observed for signs of adverse health) they were mated one male to one female avoiding brother-sister pairings. The females were examined before and after mating to determine time of pregnancy, marked anomalies of the oestrous cycle, median pre-coital time, whether pregnancy had occurred and terminated and duration of pregnancy.

The offspring (F2 generation) were examined for abnormalities at parturition and periodically until day 21 post partum at which time the study was terminated.

There were no effects of treatment in any of the treated parent females during pregnancy or lactation. No effects were apparent on development of the F1 generation during the late prenatal phase, or on postnatal growth, no changes in post weaning behavioural tests or mating performance were seen and post mortem examination of F1 males and females, reproductive capacity, litter data and macroscopic post mortem examination of F2 pups did not reveal abnormalities. The highest dose administered, 20 mg/kg bw/day produced no adverse effects. This study was conducted in accordance with GLP and based on the guidelines endorsed by the ICH Steering Committee on the Detection of Toxicity to reproduction for Medicinal Products (Ford and Bottomley, 1997; Jones et al., 1996).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The quality of the database if high because in different tests fertility has been investigated: a 90-day study in accordance with OECD 408 and in compliance with GLP, a developmental neurotoxicity study in accordance with OECD 426 and in compliance with GLP, a developmental  toxicity study in accordance with OECD 414 and in compliance with GLP, and in vitro and in vivo screening assays on receptor binding. These studies sufficiently adequately fulfill the REACH requirements.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No multigeneration study is available. However, no effects on reproductive organs of male or female rats were seen in a 13-week oral study at doses up to 150 mg/kg bw/day (NOAEL ≥150 mg/kg bw/day). (Hopkins et al., 1996a, Hopkins et al., 1997b, Api and Ford, 1999). HHCB has a very weak estrogenic and anti-estrogenic potency in vitro, dependent on the estrogen receptor type. Marginal repressing effects were also found in vitro on the androgen and progesterone receptor. However, in vivo no estrogenic effects were seen in the uterotrophic assay (Seinen et al., 1999).


Short description of key information:
90-d oral repeated dose toxicity study (OECD 426, GLP): no effects on reproductive organs

Justification for selection of Effect on fertility via oral route:
A two-generation reprotox study is waived based on all available information. The 90-d oral repeated dose toxicity study provides additional information to the developmental toxicity study which is summarized below..

Effects on developmental toxicity

Description of key information
Developmental toxicity (equivalent or similar to OECD414):
- maternal NOAEL: 50 mg/kg bw/d
- developmental NOAEL: 150 mg/kg/d
Developmental neurotoxicity (equivalent or similar to OECD 426):
- maternal and developmental NOAEL: 20 mg/kg/d (highest dose tested)
Link to relevant study records
Reference
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.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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

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
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
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
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.
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
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.
Abnormalities:
not specified
Developmental effects observed:
not specified

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.

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).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The quality of the database if high because in different tests fertility has been investigated: a 90-day study in accordance with OECD 408 and in compliance with GLP, a developmental neurotoxicity study in accordance with OECD 426 and in compliance with GLP, a developmental  toxicity study in accordance with OECD 414 and in compliance with GLP, and in vitro and in vivo screening assays on receptor binding. These studies sufficiently adequately fulfill the REACH requirements.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Key studies:

In a study designed to determine the effects of HHCB (purity not reported in report, confirmed to be >95% pure undiluted material, IFF personal communication with RIFM) on the neonate when exposed through nursing, HHCB was administered at dosages of 0, 2, 6 or 20 mg/kg bw/d once daily by gavage in corn oil to groups of 28 time-mated rats (Crl:CD BR VAF/Plus strain) from Day 14 of pregnancy (end of organogenesis) through to weaning on Day 21 post partum. The females were allowed to litter and rear their young to weaning. From these litters, selected offspring were retained (24 males and females per group) to maturity and assessed for behavioural changes and reproductive capacity. The F1 generation was only exposed to HHCB in utero during the perinatal phase and through transfer in the milk of the lactating dams. The exposure of the F1 foetuses through mother's milk can be estimated based on a pharmacokinetic study in pregnant/lactating rats (Hawkins et al., 1996a reported in section 4.1.2.1.1, under heading: Animal milk studies). HHCB levels in mother’s milk up to 2.28 and 32.8 mg HHCB equivalents (including also metabolites)/l were found at oral doses of 2 and 20 mg 14C-HHCB/kg bw per day, respectively, to the dams (see Table 4.14). Actual intakes cannot be determined because milk consumption during nursing was not measured.

After parturition, the young were counted, sexed, weighed and examined for external abnormalities. On day 4 post partum the pups were weighed and all litters containing more than 8 pups were culled to 8 retaining, where possible 4 males and 4 females. During the preweaning period, all pups were examined to determine the age of reaching certain developmental stages by examining surface righting reflex, startle reflex, air righting reflex and pupil reflex. This F1 generation was also evaluated for behavioural effects by examining changes in motor coordination and balance, activity and avoidance. When the F1 generation reached approximately 84 days of age (having been continuously observed for signs of adverse health) they were mated one male to one female avoiding brother-sister pairings. The females were examined before and after mating to determine time of pregnancy, marked anomalies of the oestrous cycle, median pre-coital time, whether pregnancy had occurred and terminated and duration of pregnancy.

The offspring (F2 generation) were examined for abnormalities at parturition and periodically until day 21 post partum at which time the study was terminated.

There were no effects of treatment in any of the treated parent females during pregnancy or lactation. No effects were apparent on development of the F1 generation during the late prenatal phase, or on postnatal growth, no changes in post weaning behavioural tests or mating performance were seen and post mortem examination of F1 males and females, reproductive capacity, litter data and macroscopic post mortem examination of F2 pups did not reveal abnormalities. The highest dose administered, 20 mg/kg bw/day produced no adverse effects. This study was conducted in accordance with GLP and based on the guidelines endorsed by the ICH Steering Committee on the Detection of Toxicity to reproduction for Medicinal Products (Ford and Bottomley, 1997; Jones et al., 1996).

 

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).

 

Conclusion and discussion:

In an oral peri/postnatal toxicity study (exposure of only the F1-generation to HHCB in utero during the perinatal phase or through any transfer in the milk of the lactating dams), no toxicity in dams or their F1 and F2 offspring (including behavioural and reproductive capacity of the F1 animals) was seen at dose levels of 2, 6, or 20 mg HHCB/kg bw per day. The exposure of F1 foetuses through mother’s milk can be estimated based on a pharmacokinetic study with pregnant/lactating rats given oral doses of 2 and 20 mg 14C-HHCB/kg bw per day.

Levels up to 2.28 and 32.8 mg HHCB equivalents (i.e. HHCB + metabolites)/l of whole milk were reported, for maternal oral doses of 2 and 20 mg/kg bw/d, respectively (see Table 4.14). In an oral developmental study there were signs of maternal toxicity at 150 mg/kg bw/day and higher. There was an increased incidence of skeletal malformations and decreased ossification in foetuses at the highest dose of 500 mg/kg bw/day. The NOAEL for maternal toxicity is 50 mg/kg bw/day and for developmental toxicity the NOAEL is 150 mg/kg bw/day. From the peri/postnatal study described above, a NOAEL of ≥20 mg/kg bw/day can be established for pup weight, pup survival and postnatal death, the highest dose tested. These effects are not included in the oral teratogenicity study. Since this NOAEL is also lower than the NOAEL for teratogenic effects generated during earlier periods of foetal development (150 mg/kg bw/day; see above), this NOAEL (≥20 mg/kg bw/day) will cover also these early teratogenic events. A NOAEL for developmental toxicity of ≥20 mg/kg bw/day will be taken forward to the risk characterization.

 

(Summary in line with EU-RAR, 2008)


Justification for selection of Effect on developmental toxicity: via oral route:
One developmental toxicity study that was performed in accordance with OECD 414 and under GLP conditions is available.

Toxicity to reproduction: other studies

Additional information

Several publications are available in vitro endocrine disruption studies are available in which HHCB was tested, however, the outcome of these studies is overruled by the available in vivo studies.

Justification for classification or non-classification

Based on the available developmental toxicity studies, there is no need to classify HHCB as toxic to the development and reproduction in accordance with the criteria outlined in Annex VI of 67/548/EEC (DSD) and Annex I of 1272/2008/EC (CLP).