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Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): HHCB
- Physical state: colourless liquid
- Analytical purity: Purity not reported in report, confirmed to be >95% pure undiluted material (IFF personal communication with RIFM).
- Lot/batch No.: ES 6302-H
- Expiration date of the lot/batch: 30 December 1996
- Stability under test conditions: 24 hours at ambient temperature

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

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.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).