Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 1998 to 22 December 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ECD Guidelines for Testing of Chemicals, Section 4: Health Effects, Test Guidelines No. 415, 26 May 1983
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Order: 175840-001
- Physical state: amber viscous liquid
- Analytical purity: no data
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent.
- Age at study initiation: 6 ~ 7 weeks old
- Weight at study initiation: At the start of treatment the males weighed 158 to 205g, the females weighed 170 to 210g.
- Fasting period before study: none.
- Housing: Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During the mating period animals were transferred to a similar type cage on a one male to one female per cage basis.
- Diet: A pelleted diet (Rat and Mouse SQC Expanded Diet No. 3, Special Diets Services Limited, Witham, Essex, UK) was provided ad libitum.
- Water: tap-water was made available ad libitum.
- Acclimation period: at least 16 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21± 2
- Humidity (%): 55 ± 15
- Air changes: at least 15 air changes per hour
- Photoperiod: 12h light/12h dark light cycle
IN-LIFE DATES: From 28/07/98 - 22/12/98

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability of the test material formulations was determined by Safepharm Analytical Laboratory. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4C in the dark.
Samples were taken of each test material formulation and were analysed for concentration of the test substance at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within 10% of the nominal concentration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): NA.
- Concentration in vehicle: 0, 25, 125, 500 mg/mL
- Amount of vehicle (if gavage): 2mL/kg
- Lot/batch no. (if required): NA.
- Purity: NA.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC using an external standard.

Samples and standards prepared in methanol to give a concentration of 0.5 mg/mL

HPLC system: Hewlett Packard 1050
Column: Hypersil C18 (150 x 4.6 id)
Mobile phase: Eluent A - water and Eluent B - methanol
At time 0 - 10% B; 14 mins - 100% B and 26 mins - 100% B
Flowrate: 1.5 mL/min
UV detector wavelength: 240 nm
Injection volume: 25 µL
Retention time: 14 to 15 mins

Stability determination: analysed initally and after 14 days in the dark at +4°C.
Stability: 92-96% of initial concentration after 14 days

Formulation concentrations: sampled and analysed within three days of preparation at weekly intervals throughout study period.
Formulation: 93 - 110% of nominal
Details on mating procedure:
- M/F ratio per cage: One to one
- Length of cohabitation: up to 16 days
- Proof of pregnancy: vaginal plug, referred to as day 1 of pregnancy:
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: One female in 1000 mg/kg bw/day group not dosed for one day (Day 17) during gestation due to her clinical condition.
Duration of treatment / exposure:
Males: 10 weeks pre-mating, 16 days mating and through post-mating until weaning (Day 21 of weaning) - Total 16 weeks
Females 2 weeks pre-mating, 16 days mating and through post-mating (23 days) until weaning (Day 21 of weaning) - Total 11 weeks
Frequency of treatment:
The test material was administered once daily
Duration of test:
- Male animals were dosed for seventy four days and female rats were dosed for eighteen days, at their appointed dose levels, prior to pairing.
- Parental males and females were paired within their respective dose groups for up to sixteen days.
- Following evidence of mating, the animals were separated and males returned to their holding cages.
- The pregnant females were allowed to deliver their offspring. The offspring were observed for growth and development during lactation.
- At weaning on Day 21 (or as near to this date as possible) post partum the surviving offspring were killed and examined macroscopically post mortem.
No. of animals per sex per dose:
24 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on previous toxicological test (90day repeat dose oral study)
- Rationale for animal assignment: random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: change immediately before dosing and one and 1h after dosing during the working week wherever possible. All animals were checked twice daily during the normal working week and once daily on weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily, immediately before, immediately after and one hour after dosing, for clinical signs of toxicity.


BODY WEIGHT: Yes
- Time schedule for examinations: During the maturation and mating period the parental generation animals were weighed weekly. Parental generation females showing evidence of mating were weighed on Days 1, 4, 7, 14 and 21 post coiturn. Parental generation females with a live litter were weighed on Days 1, 4, 7, 14 and 21 post parturn.
Individual offspring weights were recorded on Days 1, 4, 7, 14 and 21
post parturn.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. During the maturation periods food consumption was recorded weekly for each cage of parental generation adults. For parental generation females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 21 post coiturn. For parental generation females with live litters, food consumption was recorded for the periods covering Days 1 to 7 and 7 to 14 post parturn.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

FOOD EFFICIENCY: Food Conversion Ratio was calculated weekly during the maturation period of the Parental generation.
Food Conversion Ratio = (Group mean bodyweight gain g/day) during week)/ Group mean food consumption g/rat/day)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice at weaning on Day 21
- Organs examined: All animals were examined macroscopically for both internal and external abnormalities. Selected organs and tissues were retained in fixative
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Imp1ant sites
- Foetal remnants
- Dilatation
- A fragment of plant material
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: No.
- Head examinations: Yes
Statistics:
The following parameters were analysed statistically, where appropriate using the test methods outlined as follows:

Adult male and female bodyweight during the maturation, gestation and lactation periods, adult male food consumption, female food consumption during maturation, gestation and lactation, litter size, litter weight, individual offspring bodyweight, offspring landmarks of physical development.

Values were analysed to establish homogeneity of group variances using Levene’s test followed by one-way analysis of variance. If the variances were unequal subsequent comparisons between control and treated groups were performed using Dunnett’s T3 Multiple Comparison Method. If variances were equal subsequent comparisons between control and treated groups were performed using Dunnett’s Multiple Comparison
Method.

Adult pre-coital intervals, female gestation lengths, offspring reflexological responses and litter sex ratios. Individual values were compared using the Kruskal-Wallis non-parametric rank sum test. Where significant differences were seen, pairwise comparison of control values against treated group values was performed using Mann-Whitney "U" test.

Histopathology
Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed conditions.
Indices:
Mating index:
Mating Index (%) = (No. of animals mated/ No. of animals paired) x 100

Pregnancy Index:
Pregnancy index (%) = (No. of pregnant females/No. of animals mated) x 100

Parturition Index:
Parturition index (%) = (No. of females delivering live pups/No. of pregnant females) x 100

Live Birth index:
Live Birth Index (%) = (No. of pups alive on Day 1/ No. of pups born) x 100

Viability Index:
Viability Index 1 (%) = (No. of pups alive on Day 4/ No. of pups alive on Day 1) x 100
Viability Index 2 (%) = (No. of pups alive on Day 7/ No. of pups alive on Day 4) x 100
Viability Index 3 (%) = (No. of pups alive on Day 14/ No. of pups alive on Day 7) x 100
Viability Index 4 (%) = (No. of pups alive on Day 21/ No. of pups alive on Day 14) x 100
Viability Index 5 (%) = (No. of viable pups at weaning/ No. of pups on Day 1) x 100
Sex ratio:
Sex ratio = (No. of male pups/No. of pups of determined sex) x 100

Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: minor adult toxicities were observed at 1000 mg/kg/d dose level.

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There were no toxicologically significant clinical findings throughout the study.

Mortality:
There were no unscheduled deaths that could be attributed to test material toxicity. There were several deaths that were considered to be due to dosing trauma.

BODY WEIGHT AND WEIGHT GAIN
Maturation
- At 1000 mg/kg/day there was a slight reduction in male bodyweight gain from Week 5 until the end of maturation phase compared to controls. Although male group mean bodyweights were slightly lower than control values the differences were not significant statistically. Female bodyweight gain was not affected by treatment at this dose level.
- At 250 and 50 mg/kg/day there were no significant differences in male and female bodyweight gain throughout the respective maturation phases.

Post Maturation
At 1000 mg/kg/day male group mean bodyweight gain post maturation was slightly lower than that of controls, although the intergroup difference was not significant statistically.
At 250 and 50 mg/kg/day there were no significant differences in male bodyweight throughout this period.

Gestation
There were no significant intergroup differences in bodyweight gain for
females throughout the gestation period.

Lactation
There were no significant intergroup differences in bodyweight gain for females throughout the lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
There were no toxicologically significant effects.

WATER CONSUMPTION
No data.

OPHTHALMOSCOPIC EXAMINATION:
No data.

HAEMATOLOGY
No data.

CLINICAL CHEMISTRY
A statistically significant reduction in plasma cholesterol was detected for both males and females treated with 1000 mg/kg/day when compared with controls. Many values from this and the other dose groups, including controls, were outside the respective normal ranges and, in the absence of any other changes in the blood chemical parameters measured, this finding was considered to be of dubious toxicological significance.
There were no other toxicologically significant changes in the blood chemical parameters measured.
The statistically significant increases in plasma albumin, albumin/globulin ratio and plasma calcium and sodium concentration all involved individual values within the respective normal ranges and, in isolation, all were regarded as incidental. The remaining intergroup differences were confined to a reduction in male 50 and 250 mg/kg/day alkaline phosphatase and female 1000 mg/kg/day alanine aminotransferase but reductions in these parameters cannot be regarded as toxicologically significant.

URINALYSIS
No data.

NEUROBEHAVIOUR
Behavioural Assessments
There were no treatment-related changes in behaviour detected.

Functional Performance Tests
No data.

ORGAN WEIGHTS
At 1000 mg/kg/day there was a slight increase in male liver and kidney weight which, as a percentage of bodyweight, showed a statistically significant (p< 0.01) difference compared to controls. There was also a reduction in absolute prostate weight and prostate weight relative to bodyweight with the intergroup difference again achieving statistical significance compared to controls (p<0.05). Female liver and kidney weights (absolute and relative to bodyweight) were slightly increased compared to control values although only the increased kidney weight achieved statistical significance (p < 0.05).
At 250 and 50 mg/kg/day there were no significant differences compared to controls.

GROSS PATHOLOGY:
At terminal necropsy the incidence and distribution of macroscopic post rnortern findings show no treatment related trends. The gross abnormalities are those commonly observed in this study type.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no significant treatment related findings from the selected reproduction organs examined.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Remarks:
for F1 generation
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Data
Litter Size, Sex and Offspring Viability
There was no adverse effect on litter size, sex or viability that could be convincingly attributed to test material toxicity.
Offspring Clinical Condition
Surviving offspring showed no treatment-related clinical abnormalities.
Offspring Bodyweight and Development
There were no significant differences in the offspring development of animals treated with the test material compared to controls, as indicated by the group mean age of start and completion of the appearance of landmarks of offspring development. Similarly, there were no convincing differences in offspring bodyweight between treated animals and controls during lactation.
Offspring Reflexological Assessment
There were no significant treatment related differences in offspring reflexological responses compared to controls.
Offspring Sex Ratios
There were no significant treatment related differences in litter mean sex ratios on Days 1 and 21 of lactation compared to controls.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant differences in the offspring development of animals treated with the test material compared to controls

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Discussion

 

Maternal Effects:

At 1000 mg/kg/day there was evidence of minor systemic toxicity during the in-life phase of the study. Male rats showed a slight reduction in bodyweight gain and animals of either sex showed slightly elevated liver and kidney weight compared to controls. No associated macroscopic or histological abnormalities were identified however. There were a number of unscheduled deaths at this dose level during the course of the study. These were all attributed to dosing trauma and. were considered not to be indicative of test material toxicity.

 

Reproduction:

 

At 1000 mg/kg/day, mating performance was unaffected by administration of the test material with pregnancy rates and pre-coital intervals comparable to control values. There were a greater number of females that showed a slightly increased gestation length compared to controls. The gestation length observed is within the normal range for this strain of rat however and the intergroup difference was considered not to be toxicologically significant. Offspring individual bodyweight and development were unaffected at this dose level and no toxicologically significant macroscopic or microscopic anomalies were detected amongst offspring at terminal kill.

 

Following the birth of offspring there were five females dosed at 1000 mg/kg/day with total litter loss. Three females dosed at 50 mg/kg/day also showed total litter loss but there were no incidents of total litter loss amongst animals dosed at 250mg/kg/day. The clinical and post mortem findings for offspring from these litters were symptomatic of maternal neglect, possibly due to maternal stress. In particular there were no clinical or pathological findings to suggest that a toxic insult was responsible for these total litter losses. This relatively high level of non-treatment related total litter loss is now a recognised aberration associated with this particular strain of rat under the prevailing study conditions. It was observed for three other Reproduction studies performed at this laboratory during 1998 and was evident at other United Kingdom Contract Research Laboratories during the same period for studies of this type, performed using the same strain of rat and supplier.Studies performed at this laboratory before the introduction of this strain of rat and several studies performed subsequent to a change in animal husbandry (notably a change of diet) have shown a more acceptable, substantially lower background incidence of total litter loss of between 2% and 4%. Although the aetiology of this aberration appears to be inherent within this strain of rat under the conditions of this present study, there does appear to be a slightly higher incidence of total litterlossamongst treated animals (1 3.8%) compared with controls (9.5%). This may indicate that the inherent maternal neglect can be slightly exacerbated by maternal stress, such as might be expected by the administration of an unpleasant tasting test material. In this present study, the finding that the highest incidence of total litterlosswas observed for the 1000 mg/kg/day dose group may be fortuitous or it may indicate that the test material is compounding the inherent trait of a higher degree of maternal neglect. Regardless of the reason for this finding, the historical data presented in Appendix XXXlll provides a strong indication that the total litter losses seen in this study were not associated with a direct toxic effect upon reproduction. In particular, the absence of a true dose response relationship for the total litter losses seen in this study strongly indicates that an increased incidence of maternal neglect is an inherent behavioural trait of the strain of rat used in this study.

At 250 mg/kg/day there werenotoxicologically significant effects on any of the reproduction parameters evaluated and there were no incidents of total litterloss.In addition, there was no evidence of systemic toxicity amongst the adults at this dose level. There was one unscheduled death but again macroscopic and microscopic findings were consistent with dosing trauma and this was considered not to represent test material toxicity.

At 50mg/kg/daythere were no toxicologically significant effects on the reproduction parameters during the course of the study. The three incidents of total litterlossobserved at this dose level were considered to be unrelated to treatment.

The test material is produced by a reaction between boric acid, tall oil fatty acids and an alkanolamine. There are literature reports [2] that boric acid can have an adverse effect on the male reproductive tract of rats and dogs when administered for a chronic period of up to two years, and that rats can become sterile following chronic administration of this substance. There is, however, no epidemiological evidence of an adverse effect in humans to date. Particular attention was therefore focused on the male reproductive tract during this study and additional parameters associated with male fecundity, but not normally investigated as part of a One Generation Reproduction Study, were included in the study design at the request of the United Kingdom Health and Safety Executive.

Male fertility was unaffected by treatment. Organ weight measurement at terminal kill showed a slight reduction in prostate weight but there were no associated morphological abnormalities and there was no reduction in semen functions. Sperm concentration amongst animals treated with the test material were comparable with controls, and sperm morphology and mortality were unaffected by treatment. Histopathological analysis of the stages of testicular spermatogenesis also showed no treatment-related effects. These data demonstrate that there were no adverse effects on the male reproductive tract following sub-chronic repeated administration of the test material at up to 1000 mg/kg/day.


 

 

Applicant's summary and conclusion

Conclusions:
The administration of the test material to adult male and female rats throughout the reproductive cycle resulted in no effects on reproduction that could be attributed to the test material. There was evidence of minor systemic toxicity at a dose level of 1000 mg/kg bw/day. The No Observed Effect Level for reproductive effects was 1000 mg/kg bw/day and the No Observed Effect Level for adult toxicity was 250 mg/kg bw/day.
Executive summary:

TEST GUIDANCE

The study was designed to investigate the effects of the test material on the growth and reproductive performance of the rat and complies with OECD Guidelines for Testing of Chemicals, Section 4: Health Effects, Test Guidelines No. 415, 26 May 1983.

 

METHODS

The test material was administered orally, by gavage, to groups of twenty-four male and twenty-four female rats throughout maturation, mating, gestation and lactation. The dose levels were 50, 250 and 1000 mg/kg bw/day of test material with a similar sized control group receiving vehicle alone.

Following at least ten and two weeks of dosing respectively, male and female rats were paired within their dose groups to produce litters. At weaning of the offspring, all surviving animals were killed and examined macroscopically.

Parental animals were observed daily for clinical signs. Bodyweights and food consumption were recorded weekly during the maturation phase which was continued for males after the mating phase. Mated females were weighed and food consumption recorded on specific days post-coitum and post-partum.

The offspring were observed daily for clinical signs. The litter signs and individual pup bodyweights were recorded on specific days post-partum. During the lactation period the offspring were observed for intra-litter onset and duration of landmarks of physical development. On specific days of lactation, reflexological assessment of offspring was performed.

Postmortem macroscopic examinations were performed on all adults and offspring, including decedents. At necropsy of adult males a semen sample was collected from thevas deferens of the left testis for sperm evaluation. Reproductive and potential target organs and any significant abnormalities from all parental animals were preserved in fixative. Histopathology was carried out on reproductive organs from control and high dose group parental animals. Additional testicular histopathology, involving staging of testicular spermatogenesis, was performed on control and high dose males.

 

RESULTS

Reproduction/development was unaffected by treatment. The incidence of non-treatment related total litter losses seen at 1000 mg/kg bw/day and 50 mg/kg bw/day is a recognised aberration with the particular strain and source of rat used in this study.

At 1000 mg/kg bw/day there was evidence of minor systemic toxicity. Males showed slightly reduced bodyweight gain compared with that of controls during the maturation phase. In addition, there was a slight increase in male and female kidney weight at this dose level, a slight increase in male liver weight and prostrate weight was slightly reduced. There were no associated histopathological changes. There were no toxicologically significant findings at the remaining dose levels, although there were a number of unscheduled deaths at dose levels of 250 and 1000 mg/kg bw/day. These were attributable to dosing trauma and were not related to test material toxicity.

There were no significant differences in the offspring development of animals treated with the test material compared to controls.

 

CONCLUSION

The administration of the test material to adult male and female rats throughout the reproductive cycle resulted in no effects on reproduction that could be attributed to the test material. There was evidence of minor systemic toxicity at a dose level of 1000 mg/kg bw/day. The No Observed Effect Level for reproductive/developmental effects was 1000 mg/kg bw/day and the No Observed Effect Level for adult toxicity was 250 mg/kg bw/day (A NOAEL of 1000 mg/kg bw/d was proposed since only minor systemic effects were observed at this dose level).