Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-epoxypropyl o-tolyl ether
EC Number:
218-645-3
EC Name:
2,3-epoxypropyl o-tolyl ether
Cas Number:
2210-79-9
Molecular formula:
C10H12O2
IUPAC Name:
2-[(2-methylphenoxy)methyl]oxirane

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Species: Rat
Strain: Sprague-Dawley
Sub-strain: Crl:CD®(SD) lOS BR
Source of supply: Charles River (UK) Limited, Kent, UK
Number: 96
Sex: Time-mated females
Age: Young adult animals
Temperature: 22 ± 3 °C
Humidity: 30 to 70%
Lighting: Twelve hours of continuous artificial light in each twenty-four hour period
Ventilation: At least fifteen air changes per hour
Animals are housed in accordance with the 'Code of Practice for the Housing and Care of Animals Used in Scientific Procedures' and in alignment with the United Kingdom Home Office recommendations and requirements.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
Time-mated females were dosed once daily by gavage, from Day 5 (post-implantation) to Day 19 of gestation (the day prior to necropsy). The volume of test and control item administered to each animal was based on the most recent scheduled body weight and adjusted accordingly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dosing formulations were sampled on two occasions during the study and analyzed for concentration by Envigo Research Ltd., Shardlow, UK Analytical Services. Stability.
Details on mating procedure:
Females were time-mated with sexually mature proven males of the same strain by the animal supplier. The day of observation of positive evidence of mating was designated
Day 0 of gestation. The animal supplier identified mated females and provided identification details of their respective mating partners.
Duration of treatment / exposure:
Day 5 to Day 19 of gestation.
Frequency of treatment:
Once per day
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
Serial Observations
1 GeneralObservationslMeasurements

1.1 Morbidity/Mortality Inspection
Twice daily, early and late during the working period.

1.2 Clinical Observations
Once daily during the gestation period. During the dosing period, individual clinical observations were performed immediately before dosing, up to 30 minutes after dosing and one hour after dosing.

1.3 Body Weights
Individual body weights were recorded on Day 3, 5, 6, 7, 8, 11, 14, 17 and 20 of gestation.

1.4 Food Consumption
Dietary intake was recorded for individual animals on Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

1.5 Water Consumption
Monitored daily by visual inspection of water bottles.

Examinations

Maternal examinations:
Terminal investigations
1 Necropsy
Post mortem procedures were performed on all animals including any animal found dead or killed in extremis during the study. Animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of presumed gestation.

1.1 Gross Examination
Full external and internal macroscopic examination of adult females.
Ovaries and uterine content:
The uterus of any apparently non-pregnant female was immersed in 0.5% ammonium polysulfide solution to reveal evidence of implantation.
The uterus of each adult female was examined and the following recorded;
i. Pregnancy status
ii. Number of corpora lutea
iii. Gravid uterus weight
iv. Number, status (Live fetus, dead fetus, late intra-uterine death or early intra-uterine death) and intra-uterine position of implantations
Fetal examinations:
Fetuses will be removed and live fetuses were weighed before subcutaneous overdose of a barbiturate agent.
For fetuses from decedent females prior to Day 20 of gestation, examination of the fetuses was restricted to external appearance. Each individual fetus from all litters at Day 20 of gestation were examined and the following was recorded:
i. External fetal findings
ii. Fetal weight
iii. Fetal sex
iv. Placental weight
v. Skeletal and visceral fetal findings
After external examination, approximately half of each litter was fixed in 70% Industrial Methylated Spirit (lMS), processed for skeletal evaluation and finally stored in 50% glycerol. The remainder of each litter was fixed in bouins fluid for visceral evaluation by microdissection and finally stored in 10% neutral buffered formalin.
Statistics:
Quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Analysis was performed on the following parameters:
Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and fetal litter and placental weights.
The data were first assessed using Shapiro-Wilk test for normality and Bartlett's test for homogeneity of variance. If the data are considered to be normal and homogeneous, parametric assessment, one way analysis of variance and, if significant, Dunnett's multiple comparison test was employed. Where the data are considered not to be normal or non homogeneous, non-parametric assessment, Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney 'U' test wsd used.
Indices:
All scheduled data was summarized in tabular form; additionally calculated data (i.e. body weight gain, adjusted body weights) was presented. Unscheduled data (i.e. arrival body weight, additional health monitoring body weights) was reported where this information is of assistance in the interpretation of the scientific outcome of the study. As the litter is the standard unit of assessment, mean values and incidences were calculated first within each litter and then group mean values calculated from these litter values.
Group mean values were calculated from all females with a live litter at Day 20 of gestation. Reproductive indices were calculated for all females/litters at Day 20 of gestation. For decedent animals, calculation of reproductive indices was restricted to pre-implantation loss. Non-pregnant animals are defined as having no implantations; corpora lutea for these animals are considered not to be associated with mating/pregnancy and therefore are not reported as part of the litter data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Description (incidence and severity):
Treatment at 600 mg/kg bw/day was associated with a number of notable clinical signs with ataxia and hunched posture being observed for all animals and a much lower incidence of prostration and noisy respiration also being apparent. Increased post-dosing salivation was also observed for approximately half the animals at this dosage.
Treatment at 300 mg/kg bw/day was associated with noisy respiration in ten females during the study; the incidence of this finding was higher than that observed at 600 mg/kg bw/day. Increased post-dosing salivation was also observed for two animals at 300 mg/kg bw/day.
Treatment at 100 mg/kg bw/day was associated with noisy respiration in two females during the study and hunched posture in one animal. Noisy respiration was also apparent for one control animal.
Description (incidence):
There were two unscheduled deaths on the study.
Female 75, receiving 600 mg/kg bw/day, showed noisy respiration on Day 5 of gestation and noisy respiration, ataxia and hunched posture on Day 6. Decreased, laboured and noisy respiration, ataxia and hunched posture and staining of the snout was apparent on Day 7 and the animal was killed for animal welfare considerations. Necropsy revealed a clear fluid-filled mass between the left side of the chest cavity and skin (under the left forelimb). This is considered to have been a result of accidental trauma during the dosing procedure and, as such, this death was considered to be unrelated to treatment.
Female 66, receiving 300 mg/kg bw/day, was found dead on Day 19 of gestation. No clinical signs had been apparent prior to this death and, other than the uterus only containing dead implantations, there was no findings apparent at macroscopic necropsy examination. Although the cause of this death is unknown, in view of the absence of any treatment deaths at 600 mg/kg bw/day, it was considered to be incidental and unrelated to treatment.
Description (incidence and severity):
At 600 mg/kg bw/day, initial mean body weight gain appeared lower than control until Day 8 of gestation, thereafter there was no obvious effect on body weight gain during gestation. Overall body weight gain during gestation, including when adjusted for the contribution of the gravid uterus, was similar to control
At 100 and 300 mg/kg bw/day, there was no obvious effect on body weight gain during gestation, including overall body weight gain when adjusted for the contribution of the gravid uterus.
Description (incidence and severity):
At 600 mg/kg bw/day, initial mean food consumption appeared lower than control until Day 8 of gestation, thereafter there was no obvious effect on food intake during gestation. The lower initial food intake was consistent with the lower initial body weight at this dosage.
At 100 and 300 mg/kg bw/day, there was no obvious effect on food consumption during gestation.
Description (incidence and severity):
At 600 mg/kg bw/day, necropsy examination of the adult females revealed a number of macroscopic stomach findings, including thickened non-glandular region, sloughing of the non-glandular region and raised white patches on the non-glandular region. There was also a low incidence of raised white patches on the glandular region and raised limiting ridge.
At 100 and 300 mg/kg bw/day, the incidence of stomach findings at necropsy was much lower and restricted to low incidences of yellow stomach contents, sloughing of the non-glandular region, raised white patches on the glandular region and raised limiting ridge. Additionally one female also showed gaseous distension of the caecum.
At 100, a low incidence of stomach findings persisted but was restricted to yellow stomach contents, thickened non-glandular region and raised white patches on the glandular region. Additionally one female also showed gaseous distension of the caecum.
Collectively these findings for treated animals suggest that the test item is irritant in nature, although sloughing of the glandular region of the stomach was also observed for one control female.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
gross pathology

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
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:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Within the confines of this study, 600 mg/kg bw/day delivered the Maximum Tolerated Dose (MTD) as demonstrated by the clinical observations and effects on body weight and the gross pathology observations at necropsy. The 300 mg/kg bw/day dose was considered to be the No Observed Adverse Effect Level (NOAEL) for the pregnant female rat based on clinical signs, intermittent reduced body weight gain and food consumption. The NOAEL for the in utero growth, survival and morphological development of the conceptus was considered >600 mg/kg bw/day.
Executive summary:

Introduction

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis. The study was designed to comply with the following guidelines:

US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (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 twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 600 mg/kg bw/day (employing a dose volume of 4 mL/kg bw). A further group of twenty-four time mated females was exposed to the vehicle (Polyethylene glycol 400) only over the same treatment period to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

There were two unscheduled deaths on the study.

At 600 mg/kg bw/day, Female 75 showed adverse clinical signs on Days 6 and 7 and was killed for animal welfare considerations. Necropsy findings indicated that this death was a result of accidental trauma during the dosing procedure and this death was considered to be unrelated to treatment.

At 300 mg/kg bw/day, Female 66 was found dead on Day 19 of gestation. The only clinical sign apparent for this animal was increased salivation post-dosing and necropsy findings did not reveal any obvious cause of this death. Whilst the aetiology of this death is unknown, the absence of any treatment-related deaths at 600 mg/kg bw/day indicated that this death was incidental and unrelated to treatment.

Clinical Observations

Treatment at 600 mg/kg bw/day was associated with a number of notable clinical signs with ataxia and hunched posture being observed for all animals and prostration, noisy respiration, tiptoe gait, piloerection, generalized fur loss and increased pre-dosing salivation also being apparent at a lower incidence. Increased post-dosing salivation was also observed for approximately half the animals at this dosage.

At 100 and 300 mg/kg bw/day, noisy respiration was observed for two and ten females respectively and increased salivation was evident at a lower incidence but these were considered to reflect difficulties in dosing particular animals with the test item formulation rather than any treatment-related effect.

Body Weight

At 600 mg/kg bw/day, lower body weight gains were apparent during Days 3 to 4 and 5 to 8 of gestation, but thereafter mean body weight gains were similar, or slightly superior to control to termination. Cumulative body weight gain from the start of treatment was lower than control until Day 11 of gestation, but overall body weight gain during gestation, including when adjusted for the contribution of the gravid uterus, was similar to control.

At 100 and 300 mg/kg bw/day, there was no obvious effect on body weight gain during gestation.

Food Consumption

At 600 mg/kg bw/day, lower mean food consumption, compared to control, was apparent to Day 8 of gestation, thereafter food intake was similar to control.

At 100 and 300 mg/kg bw/day, there was no obvious effect on food consumption during gestation.

Water Consumption

Visual inspection of water bottles during gestation did not indicate any obvious effect of treatment at 100, 300 or 600 mg/kg bw/day.

Post Mortem Studies

At 600 mg/kg bw/day, necropsy revealed a number of macroscopic stomach findings, including thickened non-glandular region, sloughing of the non-glandular region, raised white patches/white patches on the non-glandular region and, at a lower incidence, raised white patches on the glandular region and raised limiting ridge.

At 300 mg/kg bw/day, stomach findings at necropsy was restricted to low incidences of yellow stomach contents, sloughing of the non-glandular region, raised white patches/white patches on the glandular region and raised limiting ridge. One female also showed gaseous distension of the cecum.

At 100 mg/kg bw/day, stomach findings at necropsy was restricted to yellow stomach contents, thickened non-glandular region, sloughing of the glandular region and raised white patches/white patches on the glandular region. One female also showed gaseous distension of the cecum.

Litter Responses

Litter Data

There was no effect of maternal treatment on litter data, as assessed by preimplantation loss, number of implantations, subsequent embryofetal survival, live litter size and sex ratio on Day 20 of gestation at 100, 300 or 600 mg/kg bw/day.

Litter, Placental and Fetal Weights

There was no effect of maternal treatment on mean fetal, placental and litter weight on Day 20 of gestation at 100, 300 or 600 mg/kg bw/day.

External Fetal Examination

The incidence and distribution of external findings on Day 20 of gestation did not indicate effects on fetal development at 100, 300 or 600 mg/kg bw/day.

Detailed Visceral Examinations

Neither the type, incidence nor distribution of findings apparent during detailed visceral examination of fetuses indicated effects of maternal treatment on fetal development at 100, 300 or 600 mg/kg bw/day.

Detailed Skeletal Examinations

Neither the type, incidence nor distribution of findings apparent during detailed skeletal examination of fetuses indicated effects of maternal treatment on fetal development at 100, 300 or 600 mg/kg bw/day.

Conclusion

Within the confines of this study, 600 mg/kg bw/day delivered the Maximum Tolerated Dose (MTD) as demonstrated by the clinical observations and effects on body weight and the gross pathology observations at necropsy.  The 300 mg/kg bw/day dose was considered to be the No Observed Adverse Effect Level (NOAEL) for the pregnant female rat based on clinical signs, intermittent reduced body weight gain and food consumption. The NOAEL for the in utero growth, survival and morphological development of the conceptus was considered >600 mg/kg bw/day.