9-Octadecenoic acid (Z)-, zinc salt, basic

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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

Cross-reference

Reason / purpose for cross-reference:

reference to same 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:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

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

Sex:

male/female

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

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

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 %.

Duration of treatment / exposure:

Day 1 to day 42

Frequency of treatment:

Daily

Details on study schedule:

animals 10-11 weeks old at mating

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

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

Positive control:

not applicable

Examinations

Parental animals: Observations and 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

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/a
- Dose groups that were examined: N/a

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
- Battery of functions tested: sensory activity / grip strength / motor activity:

OTHER:

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Adult non-recovery males were sacrificed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Days 43/44.
- Maternal animals: Adult non-recovery females were sacrificed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were sacrificed on Day 26 post coitum. Female 86 from the high dose group, which showed a total litter loss on Day 4 post partum, was sacrificed and sent to necropsy on this day.

GROSS NECROPSY
- All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

See below

Reproductive indices:

Pre-coital interval:
Pre-coital interval calculated as the time elapsing between initial pairing and observation of positive evidence of mating

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

Gestation length:
Gestation length calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

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 (%) = (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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths on the study. There were no clinical signs considered to be related to the toxicity of the test item. During the treatment period, sporadic instances of increased post-dose salivation were seen in animals of either sex given the test item at a dose level of 1000 mg/kg bw/day from Day 5 of dosing. Increased post-dose salivation was also seen in one male given the test item at 300 mg/kg bw/day on Day 28 and some control animals on Day 29 of dosing. Such observations are common in this type of study and are normally associated with the taste of the formulation. Male 5 from the control group was observed with a scab from Day 27, which may have resulted from infighting and was considered unrelated to dosing.
There were no clinical signs for any of the recovery animals during the treatment-free period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was no effect of treatment at any dose level on male body weight development during the treatment or treatment-free periods.
There was no effect of treatment on body weight development in females during the pre-pairing, gestation or lactation phases of the study. Group mean body weight gains in treated females allocated to the recovery groups were generally similar to the respective controls throughout the study.
Food intake and food efficiency across all groups of test item treated males remained similar to controls throughout the study.
There was no effect of treatment with the test item at any dose level on food consumption or food efficiency in females during the pre-pairing phase of the study. Food intake for treated females at all dose levels during gestation and lactation remained similar to controls. Food intake and food efficiency in treated recovery females were also similar to the respective controls throughout the study.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not applicable

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects on reproductive organ weights

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating: There was no effect of treatment with the test item on mating performance. All females mated within the first four days of pairing.
- Fertility: Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.
Two control females and one female given the test item at 100 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in these females or their partner males’ reproductive organs which could have been considered to be the cause of the infertility.
One female treated with 100 mg/kg bw/day did not give birth to a live litter and was observed with one dead foetus in the right horn of uterus at necropsy. As there were no similar incidents in the intermediate or high dose groups, it was considered unrelated to treatment with the test item.
- Gestation Length: Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
At the end of the treatment period, males given the test item at a dose level of 1000 mg/kg bw/day showed statistically significantly higher absolute and body weight-related liver weights when compared with controls (p<0.05); the corresponding weights in high dose females were similar to controls. There was no dose-relationship, the majority of individual values from the treated males were within the background data ranges and there were no associated histopathology
abnormalities. This finding, was therefore, considered unlikely to be of any toxicological significance.
Group mean absolute and body weight-related thyroid/parathyroid weights in 1000 mg/kg bw/day females at the end of the treatment and treatment-free periods were statistically significantly lower than controls (p<0.01 and p<0.05, respectively). The majority of individual values were, however, within the background data ranges and in the absence of any histopathology correlates, this finding was considered unlikely to be of any toxicological significance. Any other intergroup differences in females achieving statistical significance were not dose-related and considered unrelated to treatment with the test item.
At the end of the recovery period, other intergroup differences achieving statistical significance in animals previously given the test item at 1000 mg/kg bw/day included higher absolute and body weight-related prostate and adrenal weights in males (p<0.05) and higher absolute and body weight-related spleen weights in females. The majority of individual values were within the background data ranges and these differences were considered to be of no toxicological significance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no macroscopic abnormalities considered to be related to treatment with the test item at any dose level.
Incidental abnormalities included reddened lungs (one female given 100 mg/kg bw/day and one recovery female previously given the high dose) and sloughing of the glandular region in the stomach (one female given 300 mg/kg bw/day).

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic examination of tissues from the control and high dose groups did not reveal any treatment-related findings. Any histopathological findings were considered to have occurred spontaneously or post-mortem. In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment-related abnormalities were noted.

OTHER FINDINGS (PARENTAL ANIMALS)
HAEMATOLOGY
No toxicologically significant effects were detected in animals of either sex at any dose level.
At the end of the treatment period, there were no statistically significant differences for hematology parameters in treated animals of either sex when compared with controls.
At the end of the recovery period, males previously given the test item at 1000 mg/kg bw/day showed marginally but statistically significantly higher group mean values of hemoglobin (p<0.05), red blood cells (p<0.05) and hematocrit (p<0.01) in comparison with controls. Group mean lymphocyte counts in these males were also slightly but statistically significantly lower than controls (p<0.05). The corresponding values in females previously given the high dose were similar to controls, however group mean neutrophil count in these females was statistically significantly higher than controls (p<0.05). The majority of individual values were within the background data ranges and these observations were considered unlikely to be of any toxicological significance.
CLINICAL CHEMISTRY
No toxicologically significant effects were detected in animals of either sex at any dose level.
At the end of the dosing period, males given test item at dose of levels of 1000 or 300 mg/kg bw/day showed statistically significantly higher values of bilirubin when compared with controls (p<0.05). The mean bilirubin value in males receiving the test item at 100 mg/kg bw/day was also slightly higher than controls, albeit without achieving statistical significance or showing clear dose-relationship. Group mean plasma concentration of glucose in females from the high dose group was also statistically significantly higher than controls (p<0.05) but without a dose relationship. The majority of individual values were within the background data ranges and the corresponding values from the recovery animals at the end of the treatment-free period were similar to controls. In the absence of any associated histopathology findings, these observations were considered unlikely to be of any toxicological significance. Any other intergroup differences achieving statistical significance were not dose-related and considered unrelated to treatment with the test item.
At the end of the treatment-free period, group mean albumin and alkaline phosphatase plasma concentrations in males previously given the high dose were statistically significantly higher than controls (p<0.05 and p<0.01, respectively). The corresponding values in females from this dose group were similar to controls and with all individual values from the males being within the background data ranges, these observations were considered unlikely to be of any toxicological significance.
URINALYSIS
No toxicologically significant effects were detected in males at any dose level.
At end of the treatment and treatment-free periods, urine samples from some treated males from all dose groups tested positive for the presence of protein. Since urine samples from one control non-recovery male and one control recovery male also tested positive for the presence of protein and there were no correlated histopathology observations, this finding was considered unlikely to be of any toxicological significance. The remaining urinalysis parameters appeared to be similar across all groups including controls.
NEUROBEHAVIOUR
Behavioral Assessments - No treatment-related changes in the behavioural parameters were detected at any dose level.
Functional Performance Tests - There was considered to be no effect of treatment with the test item at any dose level on functional performance in animals of either sex.
Any intergroup differences achieving statistical significance were not dose-related and considered to be unrelated to treatment with the test item.
Sensory Reactivity Assessments - Sensory reactivity scores across all test item-treated groups were similar to controls.
ORGAN WEIGHTS
At the end of the treatment period, males given the test item at a dose level of 1000 mg/kg bw/day showed statistically significantly higher absolute and body weight-related liver weights when compared with controls (p<0.05); the corresponding weights in high dose females were similar to controls. There was no dose-relationship, the majority of individual values from the treated males were within the background data ranges and there were no associated histopathology
abnormalities. This finding, was therefore, considered unlikely to be of any toxicological significance.
Group mean absolute and body weight-related thyroid/parathyroid weights in 1000 mg/kg bw/day females at the end of the treatment and treatment-free periods were statistically significantly lower than controls (p<0.01 and p<0.05, respectively). The majority of individual values were, however, within the background data ranges and in the absence of any histopathology correlates, this finding was considered unlikely to be of any toxicological significance. Any other intergroup differences in females achieving statistical significance were not dose-related and considered unrelated to treatment with the test item.
At the end of the recovery period, other intergroup differences achieving statistical significance in animals previously given the test item at 1000 mg/kg bw/day included higher absolute and body weight-related prostate and adrenal weights in males (p<0.05) and higher absolute and body weight-related spleen weights in females. The majority of individual values were within the background data ranges and these differences were considered to be of no toxicological significance.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

for adult toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

2

Dose descriptor:

NOEL

Remarks:

for reproductive effects

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
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.

CLINICAL SIGNS (OFFSPRING)
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.

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

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic abnormalities were detected in interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to the toxicity of the test item.

OTHER FINDINGS (OFFSPRING)
Statistical analysis of surface righting reflex data did not reveal any significant intergroup differences when compared with controls.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Results of formulation analysis

Week No.	Nominal concentration (mg/mL)	Concentration found
		(mg/mL)	% of nominal
1	0	ND	-
	16.7	16.2	97
	50	51.7	103
	167	161	97
2	0	ND	-
	16.7	18.8	112
	50	56.1	112
	167	143	86
3	0	ND	-
	16.7	19.0	114
	50	44	88
	167	238	143
4	0	ND	-
	16.7	19.2	115
	50	52.8	106
	167	164	98
5	0	ND	-
	16.7	17.0	102
	50	58.2	116
	167	161	97
6	0	ND	-
	16.7	22.2	133
	50	50.3	101
	167	160	96

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.