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Toxicological information

Developmental toxicity / teratogenicity

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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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
As per IUCLID5 Sections 1.1 - 1.4.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Animal Information
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in three batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 199 to 316g.

Animal Care and Husbandry
The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. A certificate of analysis of the batch of diet used is given in Annex 2. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Animals were allocated to treatment groups as follows:
Treatment Dose Level Treatment Concentration Animal Numbers
Group (mg/kg bw/day) Volume (mL/kg) (mg/mL)
Control 0 4 0 24 (1-24)
Low 30 4 7.5 24 (25-48)
Intermediate 100 4 25 24 (49-72)
High 300 4 75 24 (73-96)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

The test item was administered daily, from Day 3 to Day 19 of gestation, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with the vehicle alone. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted on Days 4, 5, 8, 11, 14 and 17.
Duration of treatment / exposure:
Day 3 to Day 19
Frequency of treatment:
Daily
Duration of test:
20 Days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 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)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
Serial Observations

General Observations/Measurements

Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before dosing, up to thirty minutes after dosing and one hour after dosing. All observations were recorded, see deviations from Study Plan.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Terminal Investigation

Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn
L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus

The fetuses were killed by subcutaneous injection of a suitable barbiturate agent. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.

Examinations

Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.

All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
All data was summarized in tabular form, including reproductive indices. Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation. Values given in appendices may represent rounded values for presentation purposes. Group mean values were generally calculated using unrounded values therefore it is not always possible to calculate the exact group mean values from values presented in the appendices.

As the litter was standard unit of assessment, values were first calculated within the litter and group mean values represent the mean of these individual litter values.

Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:

((number of corpora lutea - number of implantations)/number of corpora lutea) x 100

Percentage post-implantation loss was calculated as:

((number of implantations - number of live fetuses)/number of implantations) x 100

Sex Ratio
Sex ratio was calculated as:

% male fetuses (sex ratio) = (Number of male fetuses / Total number of fetuses) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
A summary incidence of daily clinical observations is given in Table 2. Individual data are presented in Appendix 1.

There was considered to be no obvious effect of treatment at any dose level.

Increased salivation was noted (intermittently) in nine animals treated with 300 mg/kg bw/day from Days 7 to 18. Such observations are commonly observed in this type of study and are generally considered to be due to an irritant/unpalatable nature of the test item and/or formulation and as such are considered not to be specifically related to systemic toxicity of the test item.

Generalized fur loss was noted in two female animals treated with 30 mg/kg bw/day from Days 9 to 20 and chromodacryorrhoea was noted in one female treated with 100 mg/kg bw/day over two days (Days 9 to 10). As similar findings were not apparent in the high dose animals these are considered to be incidental and unrelated to treatment with the test item.

One control female animal exhibited a scab on Days 17 and 18 and a further control female exhibited noisy respiration on Day 17, as none of these animals were treated with the test item these findings are considered to be incidental.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Group mean body weights and standard deviations are given in Table 3 and group mean values are presented graphically in Figure 1. Group mean body weight gains and adjusted body weights and standard deviations are given in Table 4 and Table 5. Individual data are given in Appendix 2, Appendix 3 and Appendix 4.

There was considered to be no adverse effect of treatment on body weight performance at dosages of 30, 100 or 300 mg/kg bw/day.

Females treated with 300 mg/kg bw/day exhibited body weight losses between Days 3 and 4 of gestation. Signs of recovery were evident thereafter as gains in body weight were apparent, however, a marginal reduction in body weight gain when compared to control was observed between Days 8 to 11 but without achieving statistical significance. Cumulative body weight gain was generally lower throughout the treatment period achieving statistical significance (p<0.05) between Days 3 and 11. This ultimately resulted in an overall reduction in body weight gain of approximately 8% when compared to control, however, the overall change was still within the historical control data ranges. Body weight gain when adjusted for gravid uterus weight was statistically significantly (p<0.05) reduced when compared to controls. Adjusted body weight cumulative change when adjusted for gravid uterus weight was also reduced when compared to controls but without achieving statistical significance. Both of these adjusted values were also within the historical control data ranges. Due to the marginal reductions in body weight gains and the fact that signs of recovery were noted after the first two days of treatment these findings are considered not to be adverse in nature.

Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be generally unaffected by treatment at 100 or 30 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Group mean food consumptions are given in Table 6 and presented graphically in Figure 2. Individual data are given in Appendix 5.

There was considered to be no adverse effect of treatment on food consumption at dosages of 30, 100 or 300 mg/kg bw/day.

Females treated with 300 mg/kg bw/day showed marginal reductions in food consumption throughout the treatment period which achieved statistical significance (p<0.05-0.01) between Days 5 and 14.

No significant differences as compared to the control group were detected on food consumption in females treated with 100 or 30 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A summary incidence of female necropsy findings is given in Table 7. Individual data are given in Appendix 6.

Three females treated with 300 mg/kg bw/day exhibited raised white patches on the nonglandular region of the stomach.

No macroscopic abnormalities considered to be related to treatment with the test item were detected in females treated with 100 or 30 mg/kg bw/day. Two female animals treated with 30 mg/kg bw/day exhibited generalized fur loss at necropsy.

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
Details on maternal toxic effects:
Summary fetal data are given in Table 8. Individual data are given in Appendix 7 and Appendix 8.

There was no obvious effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio or pre- or post-implantation losses at 30, 100 or 300 mg/kg bw/day.

Intergroup differences for mean fetal, litter or placental weights did not indicate any obvious effects of maternal treatment at 30, 100 or 300 mg/kg bw/day.

Statistical analysis of the data did not reveal any significant intergroup differences.

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:
other: no effects

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
Details on embryotoxic / teratogenic effects:
Summary fetal external findings, visceral findings, skeletal findings and skeletal development are given in Table 9 to Table 11. Individual data are given in Appendix 9 to Appendix 11.

Neither the type, incidence nor the distribution of findings observed during external examination of the fetuses at necropsy on gestation Day 20 and subsequent detailed visceral and skeletal examination indicated any adverse effect of maternal exposure on fetal development.

Statistical analysis did not reveal any significant intergroup differences.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of 1,6-hexanediol, reaction product with chloromethyloxirane to pregnant rats during gestation at dose levels of 30, 100 and 300 mg/kg bw/day, resulted in marginal effects on body weight performance and food consumptions at 300 mg/kg bw/day. However, due to the small differences in comparison to control and the fact that no significant effects were noted in the clinical condition of these animals, the effects were considered not to be adverse in nature. The stomach findings noted in three female animals were also considered to be non-adverse as they are considered not to be relevant to humans; the unique structure of the rodent’s stomach is considered to have led to a prolonged exposure to the test item and the corresponding anatomical area is not present in man. As such, in terms of risk assessment, 300 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.

No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 300 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 30, 100, and 300 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) 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 weights, 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 no unscheduled deaths during the study.

Clinical Observations

There were no clinical signs apparent that were considered to be related to the toxicity of the test item.

Body Weight

There was considered to be no significant adverse effect of treatment on body weight performance at dosages of 30, 100 or 300 mg/kg bw/day.

Food Consumption

There was considered to be no adverse effect of treatment on food consumption at dosages of 30, 100 or 300 mg/kg bw/day.

Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies

Three female animals treated with 300 mg/kg bw/day exhibited raised white patches on the non-glandular region of the stomach.

No further macroscopic abnormalities apparent were considered to be related to treatment with the test item amongst other treated animals.

Litter Data and Litter Placental and Fetal Weights

The number of implantations, subsequent embryofetal survival, live litter size and sex ratio on Day 20 of gestation were considered to be unaffected by maternal treatment at 30, 100 or 300 mg/kg bw/day. Mean fetal, placental and litter weights were also considered to have been unaffected by maternal treatment at 30, 100 or 300 mg/kg bw/day.

Fetal Examination

External examination of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 30, 100 or 300 mg/kg bw/day. Findings at detailed skeletal and visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 30, 100 or 300 mg/kg bw/day.

Conclusion

The oral (gavage) administration of 1,6-hexanediol reaction product chloromethyloxirane to pregnant rats during gestation at dose levels of 30, 100 and 300 mg/kg bw/day, resulted in marginal effects on body weight performance and food consumptions at 300 mg/kg bw/day. However, due to the small differences in comparison to control and the fact that no significant effects were noted in the clinical condition of these animals, the effects were considered not to be adverse in nature. The stomach findings noted in three female animals were also considered to be non-adverse as they are considered not to be relevant to humans; the unique structure of the rodent’s stomach is considered to have led to a prolonged exposure to the test item and the corresponding anatomical area is not present in man.

As such, in terms of risk assessment, 300 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.

No treatment-related changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 300 mg/kg bw/day.