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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 March 2014 - 13 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD TG 422
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Details on test material:
- Physical state: solid
- Analytical purity: 100%
- Expiration date of the lot/batch: not supplied (retest after 2 years)
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: males 264 to 321g; females 198 to 237g
- Fasting period before study: no
- Housing: Initially, all non-recovery animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Recovery group animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.
- Diet: ad libitum. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used.
- Water: ad libitum. Mains water.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): >=15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 05 June 2014 (first day of treatment) and 28 July 2014 (final day of necropsy)

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 item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. Since the ICP-MS method used for formulation analysis was considered not to indicate stability, test item formulation stability was not determined and therefore, fresh formulations were prepared each day and dosed within three hours of preparation. It is assumed that the formulation was stable for this duration. As stability was not
determined, this is an exception with regards to GLP and has been reflected in the GLP compliance statement. Homogeneity of the test item formulations was determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil used as test item is not soluble in water
- Concentration in vehicle: 16.7, 50 or 167 mg/mL
- Amount of vehicle (if gavage): 6 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) using an external standard technique.
Analytical procedure
Preparation of standard solutions
Stock solutions of test item in tetrahydrofuran were prepared for external standard calibration. An aliquot, 100 mg, of test item was accurately weighed into a 100 mL volumetric flask and brought to volume with tetrahydrofuran to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in 2% nitric acid with a concentration of 0.05, 0.1 and 0.15 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Analysis of samples
The formulations received were extraced with tetrahydrofuran. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with tetrahydrfuran. This was then ultra-sonicated for 30 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with 2% nitric acid to achieve the working concentration.
Preparation of accuracy samples
Samples of arachis oil BP were acurately fortified with known amounts of the test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as th etest samples described above.
Preparation of linearity standards
A range of standard solutions were prepared in tetrahydrofuran from a stock solution of 1.003 mg/mL by serial dilution covering the concentration range 0 to 0.15350 mg/mL.
Instrumental setup
The standards and samples were analysed by ICP-MS using the following conditions:
ICP-MS: Agilent 7500 cx
Acquisition mode: Spectrum (multi-tune)
Element and Mass: Zn, 66
Peak Pattern: Full quant (3)
Repetitions: 5
Interference Equation: None
Detector Setting: Auto
Integration Time: 0.3 second

Results
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time fo the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with the known concentration; hence the specificity of the method by retention time was confirmed.
Linearity
The linearity of the analytical system used for sample analyses was found to have a quadratic correlation within the calibration range of 0 to 0.15 mg/mL. The R2 fit of the calibration curve to the data was 0.997 and was considered to be acceptable.
Accuracy
The fortified samples of Arachis oil BP were found to have arecovery value of +/-10% of the fortification.
Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 86% to 143 %.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: not applicable
Duration of treatment / exposure:
Day 1 to day 42
Frequency of treatment:
Daily
No. of animals per sex per dose:
Non-recovery dose groups: 12 males/12 females per dose
Recovery dose groups: 5 males/5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses selected based on the results of the 14-day range-finding study
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately after dosing, 30 minutes and one hour after dosing. During the treatment-free period recovery animals were observed daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately after dosing, 30 minutes and one hour after dosing. During the treatment-free period recovery animals were observed daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 5 post partum
- Organs examined: uterus

OTHER:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 42 for males; Day 4 post partum for females (non-recovery/control groups). Day 56 for recovery group animals (i.e. after 14 day recovery period).
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: No recovery/control groups - 5 males/5 females; Recovery group - all animals
- Parameters included in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 42 for males; Day 4 post partum for females (non-recovery/control groups). Day 56 for recovery group animals (i.e. after 14 day recovery period).
- Animals fasted: No
- How many animals: No recovery/control groups - 5 males/5 females; Recovery group - all animals
- Parameters included in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: final week of treatment/final week of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters included in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to start of treatment and at weekly intervals thereafter.
- Dose groups that were examined: All non-recovery groups
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other:
Fetal examinations:
- External examinations: No
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
see below
Indices:
Live Birth index:
Live Birth Index (%) = (No. of pups alive on Day 1/ No. of pups born) x 100

Viability Index:
Viability Index (%) = (No. of pups alive on Day 4/ No. of pups alive on Day 1) x 100

Sex ratio (% males):
Sex ratio = (No. of male pups/No. of pups) x 100
Historical control data:
not applicable

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
not applicable

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEC
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
No statistically significant differences were detected for corpora lutea, implantation counts or pre- and post-implantation losses for females given the test item at all dose levels when compared with controls.

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum from treated animals were comparable with controls. A total litter loss was observed for one female treated with 1000 mg/kg bw/day (Female 86). It is worth noting that the litter size at birth for this
animal was lower than the control background data ranges and clinical signs for the offspring included cold, pale, weak and no milk in the stomach indicating neglecting behavior by the dam. Due to the isolated nature of this incident, it was considered unlikely to be of any toxicological significance.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups when compared with controls.

Offspring body weight gain and litter weights on Day 1 and 4 post partum from the treated animals were comparable with controls.

Statistical analysis of surface righting reflex data did not reveal any significant intergroup differences when compared with controls.

All surviving pups in the litter from Female 90 given the test material at 1000 mg/kg bw/day were noted to be small on Days 4 and 5 post partum. There were no other notable observations for these pups and due to the isolated nature of this finding, it was considered to be of no toxicological relevance.

Clinical signs detected in the remaining pups throughout the treated groups included small size, cold, no milk in stomach, physical injury, found dead or missing and, were considered to be low incidence findings observed in offspring in studies of this type and as such unrelated to test item
toxicity.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be
1000 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development), to assess the ability of the animals to recover from any toxicity following the withdrawal of treatment and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 or 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study.

Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected non-recovery males from each dose group during the final week of treatment, and for five selected parental females from each dose group on Day 4post partum. Urinalysis was performed on five non-recovery males per dose group during the final week of treatment and five non-recovery males and females from each dose group were selected for hematology and blood chemistry assessments prior to termination.

Adult non-recovery males were terminated on Days 43 or 44, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on Day 26post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Following forty-two days of treatment, recovery group animals were maintained without treatment for a further fourteen days. Urinalysis was performed on all recovery group males during the final week of the treatment period. In addition, hematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy, but as there were no treatment-related histopathology findings, histopathological examinations of tissues from these animals was not performed.

Results…….

Adult Responses

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

Throughout the study, there were no clinical signs considered to be related to the toxicity of the test item.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests

There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex.

Sensory Reactivity Assessments

Sensory reactivity scores across all treated groups were similar to controls.

Body Weight

Body weight development for animals of either sex remained unaffected throughout the treatment and recovery period.

Food Consumption

No adverse effects on dietary intake were noted for males during the study or for females during the pre-pairing, gestation or lactation phases of the study. Dietary intake and food efficiency in recovery animals of both sexes was also unaffected.

Water Consumption

Visual inspection of water bottles did not indicate any differences in water intake for treated animals of either sex in comparison with controls.

Reproductive Performance

Mating

There was no effect of treatment with the test item on mating performance.

Fertility

There were no treatment-related effects in conception rates for treated animals.

Gestation Lengths

There were no differences in gestation lengths in treated animals when compared with controls.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4post partumwere comparable with controls. A total litter loss was observed for one female treated with 1000 mg/kg bw/day, however, this was considered unlikely to be of any toxicological significance. There were no inter-group differences in sex ratio (percentage male offspring) or viability indices for litters from treated groups when compared with controls.

Offspring Growth and Development

Clinical signs for all pups from one litter from the high dose group included small size; due to the isolated nature of this incidence, however, this finding was considered to be of no toxicological significance. Offspring growth and development from the remaining litters from treated groups was similar to controls.

Laboratory Investigations

Hematology

No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.

Blood Chemistry

No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.

Urinalysis

No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.

Pathology

Necropsy

No treatment-related macroscopic observations were detected at any dose level.

Organ Weights

At the end of the dosing period, group mean absolute and body weight-related liver weights in males given the test item at 1000 mg/kg bw/day were slightly but statistically significantly higher than controls. Group mean absolute and body weight-related thyroid weights in the females from this dose group were slightly but statistically significantly lower than controls at the end of the treatment and recovery periods. There were no histopathology correlates, and these observations along with any other statistically significant differences were considered to be of no toxicological relevance.

Histopathology

Microscopic examination of tissues from the control and high dose groups did not reveal any treatment-related findings.

Conclusion

The oral administration of the test item to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day was well tolerated. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day, based on the absence of toxicologically significant or treatment-related findings.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.