Fatty acids, tall-oil, reaction products with boric acid (H3BO3) and diethanolamine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The key study showed that 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 and developmental effects was 1000 mg/kg bw/day (OECD 415).

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

one-generation reproductive 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:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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.
- 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

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.

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.

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

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

Details on study schedule:

Chronological Sequence of Study
- 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.
- The surviving adult Parental animals were killed and examined macroscopically post mortem. Selected reproductive tissues and organs together with potential target organs were retained in fixative. Reproducitve tissues and organs from the high dose and control animals were processed and subsequently examined microscopically by a pathologist.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 animals per sex per 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

Positive control:

Not included

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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. Following mating the parental males were weighed weekly until termination. Parental generation females showing evidence of mating were weighed on Days 1, 4, 7, 14 and 21 post coitum. Parental generation females with a live litter were weighed on Days 1, 4, 7, 14 and 21 post partum.

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

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: No.

CLINICAL CHEMISTRY: No

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: No

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined : sperm motility, sperm morphology, sperm concentration

Litter observations:

STANDARDISATION OF LITTERS: Not applicable

PARAMETERS EXAMINED
The following parameters were examined: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on Day 21 post partum
- Maternal animals: All surviving animals on Day 21 post partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY
Ovaries, uterus, cervix, coagulating gland, pituitary gland, liver, vagina, testes, epididymides, seminal vesicles, prostate, lungs, thyroid gland, mesenteric lymph nodes, kidneys and significant abnormalities.

ORGAN WEIGHTS
Testes with spididymides, prostate, seminal vesicles/coagulating gland, uterus with cervix, ovaries, pituitary, liver, kidneys.

Postmortem examinations (offspring):

SACRIFICE
All animals at Day 21 post partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS : Not examined

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.

Reproductive 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

Offspring viability indices:

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

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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 bw/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 bw/day there were no significant differences in male and female bodyweight gain throughout the respective maturation phases.

Post Maturation
At 1000 mg/kg bw/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 bw/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.

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


ORGAN WEIGHTS
At 1000 mg/kg/d 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/d there were no significant differences compared to controls.

GROSS PATHOLOGY:
At terminal necropsy the incidence and distribution of macroscopic post mortern 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.

Dose descriptor:

NOEL

Remarks:

for reproductive effects

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects

Dose descriptor:

NOEL

Remarks:

for adult toxicity

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

for adult toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

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.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

not specified

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 the vas 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 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.

 

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).

 

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Additional information

The key 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.

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 the vas 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.

Reproduction 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.

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).

Effects on developmental toxicity

Description of key information

The key study reported lower mean maternal body weights with corresponding effects on mean food consumption and lower mean fetal body weights were noted at 1000 mg/kg/day. Based on these results, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when test item was administered orally by gavage to time-mated Crl:CD(SD) rats (OECD 414).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 2020 to 06 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

yes

Remarks:

with no impact on experimental results or study integrity (see below)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2018

Deviations:

yes

Remarks:

with no impact on experimental results or study integrity (see below)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Justification for test system and number of animals: At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist. The number of animals was based on the United States EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, Aug 1998, and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, Jan 2018, which recommended evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 at termination.
- Animal identification: Each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Microchips were implanted following receipt.
- Quarantine: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to the initiation of dosing.
- Selection, assignment, replacement and disposition of animals: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Before the initiation of dosing, any assigned animals considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions. The disposition of all animals was documented in the study records.

HUSBANDRY
- Housing: Animals were individually housed in solid-bottom cages containing appropriate bedding material. Individual (colour-coded) cage cards were affixed to each cage and displayed at least the animal number(s), group number, dose level, study number, and sex of the animal. Housing set-up was as specified in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Where possible, control group animals were housed on a separate rack from the test substance-treated animals.
- Animal enrichment: Animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
- Environmental conditions: Target temperatures of 68 °F to 77 °F (20 °C to 25 °C); target relative humidity 30 % to 70 %; 12-hour light/12-hour dark cycle.
- Water: Municipal tap water treated by reverse osmosis and ultraviolet irradiation was available ad libitum via an automatic watering system. Water bottles were provided if required. Periodic analysis of the water was performed, and results of these analyses held on file at the testing facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Veterinary care: Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Peanut oil NF (Lot 2JA0064; expiry date 31 August 2021; storage conditions 18 to 24 °C protected from light)

Details on exposure:

DOSE FORMULATION AND ANALYSIS
- Preparation of formulations: Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion. The neat test substance was closed and returned to a desiccator immediately after each use to minimize exposure to air or moisture. Any residual volumes from each dosing occasion were discarded unless otherwise requested by the Study Director.
- Preparation details: Dosing formulations (see table below) were prepared at appropriate concentrations to meet dose level requirements. The prepared formulations were not adjusted for purity. Any procedures not covered by SOPs required for formulation were approved by the Study Director and included in the Study Records. Any residual volumes from each dosing occasion were discarded unless otherwise requested by the Study Director.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLE COLLECTION AND ANALYSIS
- Dose formulation samples were collected for analysis as indicated in the table below.
- Dose analysis results were verified prior to dose administration at each sampling interval, if possible.
- All samples to be analyzed were transferred to the Analytical Chemistry Department at the Testing Facility for same day analysis, where possible or stored for analysis within known formulation stability period. Care was taken to minimize exposure of the test substance in the dosing formulations to air or moisture prior to analysis.

ANALYTICAL METHOD
- Analyses were performed by a high-performance liquid chromatography method with ultraviolet detection using a validated analytical procedure. The full experimental procedure was presented in Appendix 1 of the full study report and a summary is given in the table below.
- Concentration acceptance criteria: mean sample concentration within 100 % ± 15 % of theoretical concentration.
- Homogeneity acceptance criteria: relative standard deviation (RSD) of concentrations of ≤ 10 % for each group.

Details on mating procedure:

Female animals were received pre-mated.

Duration of treatment / exposure:

Gestation Days 6 to 20

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

EXPERIMENTAL DESIGN
- Experimental design is summarised in Table 6 (below).
- Administration of test materials: The test substance and vehicle were administered as a single daily oral gavage dose during Gestation Days 6 through 20. All animals were dosed at approximately the same time each day. The plastic feeding tube was thoroughly wiped of residual test substance prior to each dose.
- Justification of route and dose levels: The route of administration was oral (gavage) because this is a potential route of exposure for humans. The dose levels were selected based on data from a dose range-finding study of test item using oral dose levels of 50, 250, 500, and 1000 mg/kg/day. Based on results from the study, a high dosage level of 1000 mg/kg/day was selected. This high-dosage level was not expected to produce an incidence of test substance-related fatalities that would prevent a meaningful evaluation. Lower dosage levels of 50 and 250 mg/kg/day of test item were selected to evaluate any dose-related trends.

COMPUTERIZED SYSTEMS
- Critical computerized systems used in the study were listed in the full study report or presented in the appropriate phase report. All computerized systems used in the conduct of this study have been validated (with the exception of Microsoft Office); when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Maternal examinations:

IN-LIFE PROCEDURES, OBSERVATIONS AND MEASUREMENTS
- General in-life assessments are summarised in Table 7 (below).

THYROID HORMONE SAMPLE COLLECTION
- Blood samples for thyroid hormone analysis were collected as described in Table 8 (below).

THYROID HORMONE SAMPLE PROCESSING
- Samples were allowed to clot at ambient temperature for a minimum of 30 minutes before centrifugation. The samples were centrifuged, and the resultant serum was separated, transferred to duplicate uniquely labelled polypropylene tubes, and transferred to storage.
- Approximately 150 μL of the resultant serum was placed in a tube and was used for T3 and T4 hormone analysis. Approximately 100 μL of the resultant serum was placed in a second tube and was used for thyroid stimulating hormone (TSH) analysis.
- Samples were stored in a freezer set to maintain a target of -70 °C. For T3 and T4 analysis, the samples to be analyzed were transferred to the Bioanalytical Chemistry Department. For TSH analysis, the samples to be analyzed were transferred to the Immunotoxicology Department.

THYROID HORMONE SAMPLE ANALYSIS
- For total T3 and T4 analysis, hormone samples were analyzed using validated ultra high performance liquid chromatography with dual mass spectroscopy (UHPLC/MS/MS) assays.
- Analysis of serum samples to determine TSH concentrations was conducted using a validated Luminex Bead Based (TSH) assay.

TERMINAL PROCEDURES
- Terminal procedures are summarised in Table 9 (below).
- All animals survived until scheduled euthanasia and were weighed before being euthanised by carbon dioxide inhalation.

ORGAN WEIGHTS
- The organs identified in Table 10 (below) were weighed at necropsy for all scheduled euthanasia animals.
- Representative samples of the tissues identified in Table 11 (below) were collected from all animals and preserved in 10% neutral buffered formalin for possible histopathologic examination.

HISTOLOGY
- Tissue trimming was performed at the testing facility. Maternal thyroids and livers from all animals in all groups were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
- Histopathological evaluation was performed by a board-certified veterinary pathologist. Thyroids and livers for all animals in the control and high-dose groups (Groups 1 and 4) were subjected to microscopic examination.

Ovaries and uterine content:

LAPAROHYSTERECTOMY AND MACROSCOPIC EXAMINATION – Gestation Day 21 (embryo/fetal development phase)
- Laparohysterectomies and macroscopic examinations were performed blind to treatment group.
- All surviving females were euthanized and subjected to a gross necropsy. The thoracic, abdominal, and pelvic cavities were opened and the contents examined. Additionally, special attention was paid to the esophagus, stomach, and intestines for signs of irritation. The uterus of each dam was excised and its adnexa trimmed. Corpora lutea were also counted and recorded. Gravid uterine weights were obtained and recorded. The uterus of each dam was opened and the number of viable and nonviable fetuses, early and late resorptions, and total number of implantation sites were recorded, and the placentae were examined.
- The individual uterine distribution was documented using the following procedure: all implantation sites, including early and late resorptions, were numbered in consecutive fashion beginning with the left distal uterine horn, noting the position of the cervix and continuing from the proximal to the distal right uterine horn. Uteri which appear nongravid by macroscopic examination were opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
- Gross lesions were also collected and preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible. The carcasses were discarded.

Fetal examinations:

FETAL EXAMINATIONS
- Fetal examinations were conducted without knowledge of treatment group.
- External, internal, and skeletal fetal findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.).
- Representative photographs of all malformations, as appropriate, were included in the study records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus or normal littermate, were also included in the study records as needed and as appropriate for comparison, when possible.

EXTERNAL
- Each viable fetus was examined in detail, sexed, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region.
- Following euthanasia, anogenital distance was measured for all viable fetuses. The absolute and relative values (to the cube root of fetal body weight) were reported. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.

VISCERAL (INTERNAL)
- The sex of all fetuses was confirmed by internal examination.
- Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (1984). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). The heads from these fetuses were removed and placed in Harrison’s fixative for subsequent processing and soft-tissue examination using the Wilson sectioning technique (Wilson, 1965). Following examination, the carcasses and cephalic slices were discarded.
- The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol for subsequent examination of skeletons.
SKELETAL
- Following fixation in alcohol, each eviscerated fetus was macerated in potassium hydroxide and stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976).
- The skeletal examination was made following this procedure.

Statistics:

See below

Indices:

See below

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Test substance-related clinical observations included abnormal breathing for 2 and 3 females in the 250 and 1000 mg/kg/day groups, respectively, at the daily examinations and/or 1 to 2 hours post-dosing and increased incidences of red fur staining and/or wet fur around the mouth and/or muzzle in the same groups at 1–2 hours postdosing; these findings occurred sporadically throughout the study and were considered non-adverse. Additionally, the abnormal breathing sounds were considered likely related to aspiration of the test substance and not the result of a systemic response to test substance administration. Other observations noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
- All females were determined to be gravid with the exception of Female No. 4525 in the 1000 mg/kg/day group.

Mortality:

no mortality observed

Description (incidence):

- There were no test substance-related effects on survival. All females in the control, 50, 250, and 1000 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 21.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Test substance-related effects on body weight parameters were noted in the 1000 mg/kg/day group. Statistically significantly lower mean body weight gains were noted in this group following initiation of dosing (Gestation Days 6 to 9) compared to the control group. Mean body weight gains in the 1000 mg/kg/day group were generally comparable to the control group during Gestation Days 9 to 12 and 12 to 15 and lower (statistically significant) during Gestation Days 15 to 21 and when the entire treatment period (Gestation Days 6 to 21) was evaluated. As a result, mean absolute body weights in this group were 3.6 % to 6.8 % lower than the control group during Gestation Days 9 to 21; differences were generally statistically significant. Lower mean corrected body weight and body weight gain were also noted in the 1000 mg/kg/day group compared to the control group; differences were statistically significant. Due to the magnitude in difference from the control group, the test substance-related effects on body weight and body weight gains at 1000 mg/kg/day were considered adverse.
- Mean maternal body weights, body weight gains, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 250 mg/kg/day groups were unaffected by test substance administration. Any statistically significantly differences between these groups and the control group were noted in a manner that was not dose-responsive, transient, and/or did not impact mean absolute body weights.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- In the 1000 mg/kg/day group, mean food consumption, evaluated as g/animal/day, was statistically significantly lower than the control group during Gestation Days 6 to 9, 9 to 12, 15 to 21, and when the overall treatment period (Gestation Days 6 to 21) was evaluated. Mean food consumption in this group was comparable to the control group during Gestation Days 12 to 15. The decrements in mean food consumption in the 1000 mg/kg/day group corresponded with the adverse effects on mean body weight and body weight gain and were considered test substance-related and adverse.
- Mean maternal food consumption in the 50 and 250 mg/kg/day groups was unaffected by test substance administration; differences from the control group were slight and not statistically significant.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related liver or thyroid gland weight changes were noted. The organ weight differences observed were considered incidental and unrelated to administration of the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test item.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related microscopic findings on the maternal livers or thyroid glands were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test item.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

OVARIAN AND UTERINE EXAMINATIONS
- Mean fetal body weights (males, females, and combined) in the 1000 mg/kg/day group were statistically significantly lower (6.194 %, 6.712 %, and 6.134 %, respectively) than the concurrent control group and below the minimum mean values in historical control data. The effects on fetal body weight were considered test substance-related and adverse. Intrauterine growth at 50 and 250 mg/kg/day was unaffected by test substance administration.
- Intrauterine survival at 50, 250, and 1000 mg/kg/day were unaffected by test substance administration. Parameters evaluated included mean litter proportions of post-implantation loss, mean number and percentage of live fetuses, and fetal sex ratios. In addition, mean anogenital distances (absolute and relative to the cube root of the fetal weight) were unaffected by test substance administration at all dosage levels. Differences from the control group were slight and not statistically significant.
- Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related external developmental malformations were observed in fetuses in this study. Gastroschisis or omphalocele was noted for 6 fetuses from a single litter (No. 2508) in the 50 mg/kg/day group and consisted of portions of the intestine, stomach, liver, and/or spleen protruding. These malformations were not considered to be test substance-related because they occurred in a single litter in the low-dose group.
- No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Skeletal malformations were noted for 1(1), 2(1), and 1(1) fetuses (litters) in the control, 50, and 1000 mg/kg/day groups, respectively. Fetus No. 4521-07 in the 1000 mg/kg/day group was noted with absent vertebra (only 5 lumbar vertebra). In the 50 mg/kg/day group, Fetus Nos. 2508-07 and 2508-14 were noted with sternoschisis. Both of these fetuses also had an omphalocele noted at the visceral examination. These malformations were not considered test substance-related because they occurred in single fetuses/litters and/or were noted similarly in the control group. In the control group, Fetus No. 1516-08 was noted with sternoschisis.
- No test substance-related skeletal developmental variations were noted. Significantly higher incidence of wavy ribs and short supernumerary cervical ribs were noted at 1000 mg/kg/day and significantly lower incidence of short supernumerary thoracolumbar ribs were noted at 50 and 1000 mg/kg/day relative to the concurrent control group. These rib findings were within range of historical control data and as such were not considered test substance-related. Additional findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of developmental historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- No visceral developmental malformations were observed in fetuses in this study.
- No test substance-related visceral developmental variations were noted. Variations observed in the test substance-treated groups were noted infrequently, similarly in the control group, and/or were not observed in a dose-related manner.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

FETAL MORPHOLOGICAL DATA
- The numbers of fetuses (litters) available for morphological evaluation were 299(25), 275(25), 289(25), and 274(24) in the control, 50, 250, and 1000 mg/kg/day groups, respectively.
- Malformations were observed in 1(1), 8(2), 0(0), and 1(1) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

ANALYSES OF DOSING FORMULATIONS

- The analyzed dosing formulations contained 93.8% to 109% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

- Results of the analyses of dosing formulations are summarized in Tables 15 and 16 (below).

Table 15 – Results of homogeneity analysis

Homogeneity Assessment of the 13 Aug 2020 formulations	Group 2 (12.5 mg/L)	Group 4 (250 mg/L)
Mean Concentration (mg/mL)	13.6	263
RSD (%)	1.8	3.3
Mean Concentration % of Target	109	105

 

Table 16 – Results of concentration analysis

Date of preparation	Group 2 (12.5 mg/L)	Group 3 (62.5 mg/L)	Group 4 (250 mg/L)
13 Aug 2020	13.6 mg/mL (109 % of target)	64.8 mg/mL (104 % of target)	263 mg/mL (105 % of target)
27 Aug 2020	12.5 mg/mL (100 % of target)	58.6 mg/mL (93.8 % of target)	254 mg/mL (102 % of target)

THYROID HORMONE ANALYSIS                 

- In the 1000 mg/kg/day group, test substance-related lower total maternal T3 hormone levels were noted relative to the concurrent control group and historical control minimum values. However, in the absence of effects on T4 and TSH levels and without corresponding microscopic findings on the thyroid gland or effects on thyroid weights, these effects were considered non-adverse.

- The total T3, and T4, and TSH hormone levels were unaffected by test item administration at 50 and 250 mg/kg/day.

Conclusions:

Lower mean maternal body weights with corresponding effects on mean food consumption and lower mean fetal body weights were noted at 1000 mg/kg/day. Based on these results, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when test item was administered orally by gavage to time-mated Crl:CD(SD) rats.

Executive summary:

GUIDELINE

The design of the study was based on United States EPA Guideline OPPTS 870.3700 and OECD Test Guideline 414. The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

 

METHODS

Female Crl:CD(SD) rats (25 per group) were dosed via oral gavage at 0, 50, 250 or 1000 mg/kg bw/day once daily during Gestation Days 6 to 20. Parameters and end points evaluated during the study were clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormone assessments, gross necropsy findings, organ weights, histopathologic examinations, intrauterine growth and survival, anogenital distance and fetal morphology.

 

RESULTS

All maternal animals survived to scheduled necropsy on Gestation Day 21. Test substance-related clinical observations included abnormal breathing, red fur staining and wet fur around the mouth and/or muzzle at the daily examinations and/or 1–2 hours postdosing in the 250 and 1000 mg/kg/day groups. These clinical observations were considered non-adverse due to their sporadic occurrence.

 

Test substance-related maternal effects in the 1000 mg/kg/day group, included lower mean body weight gains with correspondingly lower mean food consumption when the entire dosing period was evaluated (Gestation Days 6 to 21), resulting in a mean absolute body weight that was 6.8 % lower than the control group on Gestation Day 21. In addition, lower mean corrected body weight and corrected body weight gain were noted in this group compared to the control group. Maternal food consumption in the 1000 mg/kg/day group was lower than the control group generally throughout the entire treatment period. Due to the magnitude of these differences from the control group, the test substance-related effects on mean maternal body weight, body weight gains, and food consumption were considered adverse. Mean body weights, body weight gains, food consumption, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 250 mg/kg/day groups were unaffected by test substance administration.

 

In the 1000 mg/kg/day group, lower maternal T3 hormone levels were noted relative to the control group in the absence of effects on TSH and T4 levels. Due to the magnitude of the difference from the control group, the effect on T3 levels was considered test substance-related. However, due to the lack of corresponding microscopic findings or effects on the thyroid gland weight, the lower mean T3 level was not considered adverse. Thyroid hormone levels (T3, T4, and TSH) were unaffected by test item administration at 50 and 250 mg/kg/day.

 

No remarkable maternal macroscopic or microscopic (thyroid gland and liver) findings were noted at any dosage level at the scheduled necropsy. There were no test substance-related effects on mean thyroid gland or liver weights. Test substance-related lower (up to 6.712%) mean fetal body weights (males, females, and combined) were noted in the 1000 mg/kg/day group compared to the control group and correlated with lower mean gravid uterine weight and adverse maternal toxicity in this group.

 

Due to the magnitude of difference from the controls, the test substance-related effects on fetal weights were considered adverse. There were no test substance-related effects on intrauterine growth at 50 and 250 mg/kg/day or intrauterine survival, anogenital distance, and fetal morphology (external, visceral, and skeletal) at 50, 250, and 1000 mg/kg/day.

 

CONCLUSION

Lower mean maternal body weights with corresponding effects on mean food consumption and lower mean fetal body weights were noted at 1000 mg/kg/day. Based on these results, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when test item was administered orally by gavage to time-mated Crl:CD(SD) rats.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 July 1998 to 22 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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.
- 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

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

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

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.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

no effects observed

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.

Key result

Dose descriptor:

NOEL

Remarks:

maternal dose

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

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

no

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.

 

 

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).

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Species:

rat

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 study

The design of the key study was based on United States EPA Guideline OPPTS 870.3700 and OECD Test Guideline 414. The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

Female Crl:CD(SD) rats (25 per group) were dosed via oral gavage at 0, 50, 250 or 1000 mg/kg bw/day once daily during Gestation Days 6 to 20. Parameters and end points evaluated during the study were clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormone assessments, gross necropsy findings, organ weights, histopathologic examinations, intrauterine growth and survival, anogenital distance and fetal morphology.

All maternal animals survived to scheduled necropsy on Gestation Day 21. Test substance-related clinical observations included abnormal breathing, red fur staining and wet fur around the mouth and/or muzzle at the daily examinations and/or 1–2 hours postdosing in the 250 and 1000 mg/kg/day groups. These clinical observations were considered non-adverse due to their sporadic occurrence.

Test substance-related maternal effects in the 1000 mg/kg/day group, included lower mean body weight gains with correspondingly lower mean food consumption when the entire dosing period was evaluated (Gestation Days 6 to 21), resulting in a mean absolute body weight that was 6.8 % lower than the control group on Gestation Day 21. In addition, lower mean corrected body weight and corrected body weight gain were noted in this group compared to the control group. Maternal food consumption in the 1000 mg/kg/day group was lower than the control group generally throughout the entire treatment period. Due to the magnitude of these differences from the control group, the test substance-related effects on mean maternal body weight, body weight gains, and food consumption were considered adverse. Mean body weights, body weight gains, food consumption, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50 and 250 mg/kg/day groups were unaffected by test substance administration.

In the 1000 mg/kg/day group, lower maternal T3 hormone levels were noted relative to the control group in the absence of effects on TSH and T4 levels. Due to the magnitude of the difference from the control group, the effect on T3 levels was considered test substance-related. However, due to the lack of corresponding microscopic findings or effects on the thyroid gland weight, the lower mean T3 level was not considered adverse. Thyroid hormone levels (T3, T4, and TSH) were unaffected by test item administration at 50 and 250 mg/kg/day.

No remarkable maternal macroscopic or microscopic (thyroid gland and liver) findings were noted at any dosage level at the scheduled necropsy. There were no test substance-related effects on mean thyroid gland or liver weights. Test substance-related lower (up to 6.712%) mean fetal body weights (males, females, and combined) were noted in the 1000 mg/kg/day group compared to the control group and correlated with lower mean gravid uterine weight and adverse maternal toxicity in this group.

Due to the magnitude of difference from the controls, the test substance-related effects on fetal weights were considered adverse. There were no test substance-related effects on intrauterine growth at 50 and 250 mg/kg/day or intrauterine survival, anogenital distance, and fetal morphology (external, visceral, and skeletal) at 50, 250, and 1000 mg/kg/day.

 

Lower mean maternal body weights with corresponding effects on mean food consumption and lower mean fetal body weights were noted at 1000 mg/kg/day. Based on these results, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity when test item was administered orally by gavage to time-mated Crl:CD(SD) rats.

Supporting study

The supporting 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.

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 the vas 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.

Reproduction 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.

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 (NOAEL of 1000 mg/kg bw/d was proposed since only minor systemic effects were observed at this dose level).

Justification for classification or non-classification

Classification in accordance withEU Regulation (EC) No. 1272/2008 is not required because any adverse effects reported in the available OECD 415 and OECD 414 studies were considered secondary to maternal toxicity.